CN100475976C - A kit for simultaneous amplification and detection of hepatitis and HIV nucleic acid - Google Patents

Abstract

A method for inspecting hepatitis and AIDS nucleic acid by synchronized amplification and its reagent knit are disclosed. The process is carried out by taking magnetic ball as automatic medium, specific synchronized capturing HBV, CHV and HIV nucleic acid, accelerating biotin primer construction and purification by RNA external label and internal label, real-time synchronized inspecting and T-PCR amplifying based on Tagman probe. The reagent knit consists of dis-inhibitor, cracking liquid, magnetic ball suspension, washing liquor, internal check, RT-PCR reactive liquor, enzyme mixture, fluorescent mixture, positive check and negative check. It's accurate and automatic, has single-tube operation, closed inspection AND synchronized extraction, and it has better sensitivity and specific performance and can be used for large-scale blood screening and large-capacity clinical inspection.

Description

A kit for simultaneous amplification and detection of hepatitis and HIV nucleic acid

technical field

The invention belongs to the technical field of nucleic acid diagnosis of diagnostic reagents, in particular to a highly sensitive, specific and suitable for synchronous extraction and synchronous real-time detection of hepatitis (including hepatitis B and hepatitis C) and HIV nucleic acid for routine blood nucleic acid screening methods and kits.

Background technique

According to the statistics of the World Health Organization (WHO), more than half of the countries do not conduct thorough testing on the blood donated by blood donors, resulting in the spread of diseases such as AIDS, hepatitis, malaria and syphilis in many places. Among the 560 HIV-positive people who increase every year in the world, 5% to 10% of them are infected through blood transfusion or blood products. Among African children and women, the proportion is as high as 20%-25%. Since the 1980s, France, Japan, Romania, the United States, Germany, and my country have all had serious consequences due to lack of blood screening. Once a blood-borne disease occurs in one place, it will often cause explosive spread. Therefore, the safety of blood transfusion has extremely special significance for the safety of the people and the stability of the society. Clinical blood testing is also facing increasing testing pressure. According to WHO estimates, the number of AIDS infections in my country will reach 10 million by 2010, and will continue to grow rapidly. More than 60% of people in our country have been infected by hepatitis B virus, and finally about 1/10 people become HBsAg positive. In addition, hepatitis C, which has the highest degree of chronicity among all hepatitis, has an incidence of about 40 million.

Compared with the extreme importance of blood safety, the current detection methods are still far behind the needs of society. At present, hepatitis B virus (HBV), hepatitis C virus (HCV), and AIDS (HIV), which pose a serious threat to blood transfusion safety, are mainly detected by enzyme immunoassay (EIA). Although after several generations of improvement, great progress has been made in terms of sensitivity, specificity, precision and stability, but as an indirect lagging detection method, what it detects is the human antibody elicited by the virus instead of the virus itself. Therefore, it cannot eliminate the missed detection of specimens in the "window period". The so-called "window period" refers to the early stage of infection, when antibodies have not been produced in the body or the antibodies are below the detection limit. Serological tests at this time are generally negative. And the window period of HBV, HCV, HIV virus infection to produce antibody is particularly long again, wherein the window period of hepatitis C virus (HCV) is about 66-82 days, hepatitis B virus (HBV) and human immunodeficiency syndrome virus ( That is, AIDS) (HIV) window period is about 59 days and 22 days. In addition, due to the occurrence of events such as virus mutations, some immunological methods may miss detection. Therefore, there are inherent defects in immunological diagnostic methods, and missed detection cannot be eliminated. The lag of testing methods has brought serious threats to blood transfusion safety and clinical testing.

In recent years, the development and gradual maturity of nucleic acid technology has provided the most powerful tool for people to directly detect the presence of viruses. Nucleic acid amplification technology (Nucleic acid Amplification Technology, NAT) is a general term for this type of technology. The detection of nucleic acid is the virus gene itself, which is the detection of the virus at the most fundamental level. Nucleic acid detection greatly shortens the window period for virus detection, and has extremely high sensitivity, specificity, precision and stability. Facts have proved that NAT detection can shorten the average "window period" of HBV, HCV and HIV infection by 25 days, 59 days and 11 days, respectively, shortening the "window period" by 45%, 89% and 50%, respectively. Because of this, more and more countries have realized the importance of blood nucleic acid testing, which is implemented as a mandatory indicator in clinical blood transfusion and blood products in countries such as Western Europe. At the same time, the United States, Japan, and the European Union have included NAT testing in blood donors routine screening program.

A number of single nucleic acid detection products have been put on the market abroad. The United States, Australia, Canada and other countries have products coming out one after another. Roche has also developed nucleic acid diagnostic kits. However, these kits are either an open PCR-ELISA detection mode, or are cumbersome to operate, prone to detection contamination, and are all single-indicator single detection. These defects make it difficult to adapt to the needs of the current market situation. Analyzing the current situation of these nucleic acid diagnostic reagents, there are several serious deficiencies as follows: 1) The reagents are open, and the possibility of contamination is high; the degree of automation is low, and automation and full sealing are the most critical technical bottlenecks for nucleic acid detection. 2) Reagents are single-item detection. No blood screening reagent can simultaneously extract and detect multiple viral nucleic acids (such as HBV, HCV, HIV) in real time. For example, Roche blood screening reagents approved by the FDA are all single-item operations. The amplification procedures are different, and the detection process is also different; the TMA technology used by another GEN-PROBE company approved by the FDA can amplify HCV and HIV at the same time, but cannot amplify HBV at the same time; Each test is different; either it is operated in the same tube, but once the test result is positive, it is impossible to distinguish whether it is HCV or HIV. So far, the US FAD has only approved the above two products for blood nucleic acid screening. 3) The operation is cumbersome, the repeatability is poor, and the throughput is low, so it is not suitable for clinical testing and large-scale blood screening. 4) Poor compatibility, most manufacturers that provide nucleic acid extraction reagents often do not provide amplification detection reagents or the two cannot be well matched, requiring user adjustment or even optimization; some can provide matching reagents at the same time, but they are not necessarily applicable to specific Even if the above conditions are met, the user should choose a designated instrument such as COBAS AMPLICOR according to the requirements of the amplification detection reagent. 5) Reagents are often not ready-to-use. For example, it is necessary to dissolve dry powder before use, and add a specified amount of organic reagents such as ethanol or isopropanol to some bottles and cans. Some reagents still have complicated preparation work before use. Carelessness will cause the complete failure of the experiment. The operation of different personnel also introduces operational errors in the step of reagent preparation; and once opened or reconstituted, some reagents are only valid for a few days even if they are stored at low temperature, which is especially not suitable for multiple batches of repeated use when the amount of specimens is small . The aforementioned conditions have seriously hindered the large-scale use of blood nucleic acid screening in blood stations and inspection and quarantine institutions, and are also not conducive to the wide application of nucleic acid testing in laboratory departments of large and medium-sized hospitals. One of the bottlenecks.

Therefore, there is an urgent need in this field to develop an automated, high-throughput, multi-item simultaneous nucleic acid extraction and real-time amplification detection technology platform suitable for routine blood nucleic acid screening and a stable performance, strong operability, Kits for ready-to-use reagents, with related instruments and consumables matching them.

Contents of the invention

The object of the present invention is to provide a kind of kit that can synchronously extract the nucleic acid of HBV, HCV, HIV virus, synchronous real-time amplification detection HIV, HBV, HCV virus nucleic acid, to overcome the inherent defects and deficiencies of traditional protein detection, while making up for the existing There are many shortcomings of similar products at home and abroad, and even fatal flaws that cannot be solved in methodology.

The invention proposes a method and a kit capable of synchronously extracting and synchronously measuring the presence of HIV (AIDS), HBV (hepatitis B), and HCV (hepatitis C) viral nucleic acids in serum or plasma specimens in real time. The method according to the present invention includes the following four relatively independent and interrelated contents, and optional combinations are provided for the complete realization of the kit of the present invention. ① The present invention discloses a method for selecting magnetic beads as an automated medium based on the principle of hybridization, and using a magnetic bead separator to achieve specific and simultaneous capture of various viral nucleic acids, as well as its improved method and reagents. A biotin-modified oligonucleotide with the same sequence as one of the primers is used as the capture probe of the nucleic acid extraction reagent, which has the advantage of avoiding missed detection and not interfering with subsequent PCR detection. ②The present invention also discloses a new type of RNA external standard (i.e. positive control) and RNA internal control (competitive internal standard) rapid PCR construction and purification method, which has not been seen in any literature reports or example applications, and is characterized by fast, Efficient, the advantage is that there is no template DNA pollution in RNA products or its pollution is too low to be detected, especially suitable as a quality control to monitor the effectiveness of nucleic acid extraction, RT reaction, and PCR amplification detection. 3. The present invention also proposes the basis of designing a green environment-friendly kit---the genetic engineering construction method or the PCR construction method of the positive control and the internal control, so that the positive control and the internal control in the kit are all non-infectious. ④The last and most critical thing is that the present invention discloses a TaqMan PCR amplification method suitable for one-step single-tube double-enzyme combined detection of multiple detections for blood screening. The system contains UNG-dUTP "built-in" anti-pollution Components, the detected items are HBV DNA, HCV RNA, and HIV RNA three viral nucleic acids, both DNA and RNA, and the system has internal control components to detect whether false negatives occur, the most prominent of this invention The advantage lies in the excellent sensitivity and specificity of the detection reagent and the coverage of each subtype, and the amplification efficiency of different subtypes is approximately equivalent, so that it can meet the extremely demanding requirements of blood nucleic acid screening; its notable feature is the use of Inexpensive dual-enzyme (ie, containing reverse transcriptase and thermostable DNA polymerase) system, which is different from single-enzyme systems such as Roche's rTth or ZO5 enzymes. Obviously, apart from the expensive price factor of single-enzyme system raw materials, the optimization difficulty of single-enzyme system is not the same as that of double-enzyme system. In the past, it was considered unfeasible to use a relatively cheap dual-enzyme system in routine blood screening that required sensitivity, specificity, anti-pollution, and multiple items.

1. The present invention provides a method based on the principle of hybridization fishing, specific synchronous capture and extraction of HBV, HCV, and HIV nucleic acids, which is characterized in that a biotin-modified oligonucleotide consistent with a primer sequence in subsequent amplification detection is selected. Nucleotides are used as capture probes for nucleic acid extraction reagents, which have the advantage of avoiding missed detection and not interfering with PCR detection: in theory, all fragments that can be amplified by PCR will not be detected in the sample extraction step due to the design of the capture probe sequence. Not only because the conservation and specificity of the primer sequences used for amplification have undergone strict screening and clinical practice tests, but more importantly, any positive samples that can be amplified by PCR must be able to be used for the probe. On the other hand, the final step of nucleic acid purification based on hybrid capture method is high temperature elution, it is difficult to avoid the separation of biotin probe and streptavidin-coated magnetic beads into the eluate, there may be μM concentration The probe enters the eluate, and if the sequence of the probe is not part of the primer, it may act as a redundant oligonucleotide and interfere with PCR amplification. On the contrary, at best, the ratio of primers in the PCR reaction is not equal. The PCR in this specific case is basically the same as the PCR in the same ratio. In addition, this invention also provides a flexible universal capture method, that is, to design an intermediate probe, an unmodified common oligonucleotide (consistent with a primer sequence in the subsequent fluorescent PCR system) whose 3' There is a series of A at the end, such as Oligo(d A) _18-25 , as the backbone probe for intermediate hybridization, and biotin-modified Oligo(d T) ₂₅ is used as a general hybridization capture probe; the 5' end nucleic acid of the intermediate probe The sequence serves as the "recognition arm" that specifically recognizes the viral nucleic acid in the specimen, hybridizes and captures the viral nucleic acid, and the Oligo(d A) _18-25 at its 3' end serves as the recognition site for the biotinylated Oligo(d T) ₂₅ , captured by it, and the complex formed by the final hybridization is adsorbed by streptavidin-coated magnetic beads, and washed in sequence with washing solutions A, B, and C to remove non-specific hybrids and PCR/RT-PCR inhibitors such as Impurities such as sugars, denatured proteins, fats, high salt or heavy metals, etc. Finally, the magnetic beads enter the eluent and are heated, and the nucleic acid template is eluted from the hybridization complex and enters the eluent. However, biotinylated Oligo (d T ) ₂₅ Waste magnetic beads are automatically removed by the instrument, and the eluate can be used as a template for fluorescent PCR detection. The advantage of this method is that the biotin-labeled Oligo(dT) ₂₅ is consistent and the binding efficiency depends only on the hybridization efficiency of the designed intermediate probe. The present invention also provides another flexible method, synthesizing an oligonucleotide comprising a linear arrangement of the above-mentioned HBV, HCV, HIV specific probe sequences, as a triplet common probe, which can be separated by connecting arms such as 3 -8 T or A, the modification of biotin is still located at the 5' end of the common probe, which can save some amount of streptavidin-coated magnetic beads, and correspondingly increase the amount of magnetic beads to excess, which is conducive to detection sensitivity It is especially suitable for tests where the amount of magnetic beads used has been saturated (such as multi-item extraction), because the magnetic beads adsorbed by the magnet have a degree of saturation, and more than a certain amount cannot be adsorbed cleanly. The present invention does not exclude the combination or composite combination of the above methods, such as synthesizing an oligonucleotide comprising the linear arrangement of the above-mentioned HBV, HCV, HIV specific probe sequences, and its 3' end is connected with a series of A such as Oligo(d A) _18-25 , as a backbone probe for intermediate hybridization, using biotin-modified Oligo(dT) ₂₅ as a universal hybridization capture probe, etc.

The most significant advantage of specific extraction and purification using the above-mentioned hybridization principle is that the specificity of detection is improved and false positives of PCR are avoided. Although PCR is a highly specific detection method, there are still a certain proportion of false positive reports caused by non-contamination factors. After excluding the factors of unreasonable primer sequence design, it is considered that the extracted template contains a large number of related to other heteronucleic acids. The specific extraction method only extracts the nucleic acid under study, thereby avoiding interference factors. In addition, this method is based on the principle of hybridization and is used to extract substances rich in PCR inhibitors, such as hyperlipidemia, high hemolysis, jaundice, and even heparinized blood samples without interference; while the traditional silica gel membrane extraction method is rich in heparin and nucleic acid Negatively charged linear macromolecules are easily adsorbed to the silica gel membrane and finally eluted into the eluent as a template to inhibit PCR/RT-PCR. In routine blood screening, due to the large number of daily testing samples and heavy workload, even if the PCR false positive is as low as 1 in 10,000, the probability of a sample rich in inhibitors is 1 in 10,000, and one will be encountered within a few days. Time is wasted for retesting, blood is wasted, and false negatives may occur due to the presence of PCR inhibitors. The specific synchronous extraction method disclosed in this invention combined with the following synchronous amplification detection reagents provides the double specificity guarantee of hybridization extraction and TaqMan probe method PCR for blood screening, and effectively removes the PCR inhibitors to avoid false negatives.

2. The present invention also provides a method for PCR construction and purification of a novel RNA external standard (i.e. positive control) and RNA internal control (competitive internal standard) quickly adding biotin-modified primers, which has not yet been seen in any literature reports or examples application. It is characterized by rapidity and high efficiency, and there is no contamination of template DNA in the RNA preparation or its contamination is so low that it cannot be detected. The primers used to construct the RNA internal control have their 5'-added ends modified with biotin, and the 5'-added end of one primer is the transcriptional promoter recognition site sequence appended with T7 RNA polymerase. The RNA internal control can be quickly prepared. When the DNA containing the target sequence is obtained by PCR/RT-PCR, add the streptavidin-coated magnetic beads directly, combine with the biotinylated PCR product, discard the liquid, and use PCR buffer After washing twice with buffer solution, add transcription buffer and T7 RNA polymerase and react at 37°C for 50-60 minutes, then dilute the transcribed RNA and the original DNA template complex with PCR buffer, heat-denatured and freeze on ice Cold, the biotin label on the DNA template is firmly bound to the streptavidin-coated magnetic beads, while the transcribed RNA does not have a biotin label and cannot be bound to the magnetic beads, and it is denatured to make it with the DNA template Completely separate, centrifuge to take the supernatant or use a magnet to separate the magnetic beads, and the remaining pure RNA product can be further concentrated and purified by ethanol precipitation. This method is used to construct the RNA external standard and RNA internal control. It only needs one positive sample extract, one PCR/RT-PCR or two PCR/RT-PCR, plus two hours of purification work to obtain pure RNA preparations were used as quality controls. The traditional method is to amplify the fragments by PCR, insert them into vectors such as T vectors after purification, pick positive clones to extract plasmids and digest the recombinant plasmids to linearize them, then purify them, perform in vitro transcription, digest them with DNase, and then purify the RNA. The whole process requires a lot of equipment in the genetic engineering laboratory, including carriers, competent cells, restriction enzymes, and the cultivation of bacteria, and the whole process takes a long time. In contrast, the method disclosed in the present invention is much simpler and faster, and the biggest advantage of this method is that the RNA preparation provided is far purer than the traditional method. Because the digestion of short fragments of DNA templates is always incomplete, multiple digestions and purifications and even column purifications are of no avail, and a considerable amount of DNA can still be detected in the no-RT control PCR test. However, the RNA control containing DNA is difficult to truly serve as an external control or internal control for RNA virus detection, because it cannot truly reflect the RT efficiency; if a quantitative control is established on this basis, there is a risk of inaccurate quantification, because RNA virus detection The most important thing is its RNA, and the quantitative control contains DNA/RNA, which will inevitably cause the detection to be out of sync and tend to be distorted, and it is difficult to use it as a quantitative quality control of viral load.

3. The present invention also proposes the basis of designing a green environment-friendly kit --- the genetic engineering construction method or PCR construction method of positive control and internal control: the preparation of positive control and the preparation of internal control, the independent control of positive control and internal control Use or mixed use ----- design according to the requirements of customer groups, equipment and other hardware conditions. The present invention has significant advantages in biological safety.

According to the nucleic acid type of the virus detected, the present invention prepares non-infectious nucleic acid fragments in vitro as the positive control (RNA external standard) and internal control (RNA internal standard) of the kit; The fragment (such as HBV is a plasmid containing HBV C region, HCV is a plasmid containing HCV 5'NCR region, HIV is a plasmid containing HIV GAG region) is linearized by enzyme digestion, and then transcribed into RNA in vitro, and digested with DNase RNA preparations were obtained after purification. Of course, the method described in the present invention can be used to quickly construct. Here, only the use of the general traditional method is described to emphasize the basis of the green kit: infectious factors cannot be introduced into the kit, although its operators must follow the procedures. Infectious specimens to operate. The above-mentioned HBV, HCV, HIV positive controls can be used alone or in combination. The above-mentioned three fragments can also be assembled into a vector and then linearized using genetic engineering methods. After in vitro transcription, use its DNA or RNA, or a combination of the two.

The internal control uses a competitive internal standard, that is, it has the same primer binding region as the positive control, but the nucleic acid sequence or arrangement of the primer spacer is different, so that it cannot be combined with the detection probe (positive control, which can be called an external standard probe). ) binding, but can bind to the internal standard probe. The internal control can be constructed by site-directed mutagenesis of the positive control template, or by block PCR. Similarly, the internal control is constructed into corresponding DNA or RNA according to the nucleic acid type of the detected virus, which can be used alone or in combination. Three internal control fragments can also be assembled into one fragment by genetic engineering methods or end-added PCR methods middle. In short, to choose the most suitable solution according to the needs, not only pay attention to the effective monitoring of the internal control for the whole process of nucleic acid extraction, RT process, and PCR amplification detection process, but also pay attention to its introduction without affecting the sensitivity of the detection; at the same time, pay attention to The introduction amount of the internal control in each test: if it is too high, it will not be able to truly reflect the inhibition of the specimen, such as slight inhibition cannot be seen; if it is too low, the failure rate of the internal control will be high and the repeatability will be poor; generally, the detection sensitivity is 2-20 times higher The amount of the internal control is used as a monitoring, and there may be changes in practice. As for the specific application plan, adding an appropriate amount of internal control to the lysate to participate in the nucleic acid extraction step is a standard implementation, but it does not exclude some users who directly add the internal control when configuring the PCR reaction solution. , and no internal control is introduced in the specimen extraction process, this practice can also realize the effective monitoring of whether the specimen extract contains PCR/RT-PCR inhibitors, so it should also be affirmed. In addition, some users cannot use the internal standard probe to detect the occurrence of false negatives due to the single-channel detection wavelength of the fluorescent PCR instrument. Although the kit provided by the present invention can also be used, the judgment of the result should be cautious.

4. The present invention also proposes a TaqMan probe-based RT-PCR amplification method for simultaneous multiple detections in real time, which is characterized in that it uses a single-tube one-step RT-PCR dual-enzyme system reagent, and multiple detection items contain both DNA also contains RNA, and the anti-pollution system selected is the classic UNG-dUTP system. The one-step RT-PCR reagent used is a dual-enzyme system, which contains reverse transcriptase and heat-resistant DNA polymerase. The reverse transcriptase is not heat-resistant, and the RT temperature should not be higher than 60°C, preferably 45-55°C, especially It is 50°C. However, the UNG prepared by conventional genetic engineering at this temperature (such as the vast majority of UNG currently available on the market) has degraded reverse-transcribed U-containing DNA (called U-DNA), so this UNG cannot be used. The preferred solution is Heat-labile UNG such as from cold-loving marine bacteria BMTU3346 (heat-labile UNG/heat-labile UNG: EC3.2.2.3Roche-derived from cold-loving bacteria BMTU3346, USB--derived from Gadus morhau, Epicenter-- Derived from Gadus morhau, etc., these companies have products for sale). It is characterized by long-term stability when stored in a certain matrix, and its half-life is only two minutes when the temperature is 40°C in PCR buffer, and no specific program settings are required for application, because it is in the preparation of RT-PCR reagents and RNA templates The enzyme works when it is in the middle (mixing), and quickly degrades the last RT-PCR pollution such as residual U-DNA. The best reaction is 5-20 minutes at 20--30°C. When RT-PCR is started, this UNG is rapidly inactivated without affecting RT or PCR. The most prominent advantages of the multi-synchronous detection reagent of the present invention lie in the excellent sensitivity and specificity of the detection reagent, the coverage rate of each subtype, and the approximately equivalent amplification efficiency of different subtypes. At present, there is no report using a dual-enzyme single-tube RT-PCR system to introduce an anti-pollution solution and can be successfully used for multiple simultaneous detections that require sensitivity (that is, DNA and RNA). The company reported on the preliminary application of the automated multi-detection ZO5 enzyme single-enzyme RT-PCR TaqMan system for blood screening, but it uses a single-enzyme system, its shortcomings are obvious, the system is expensive and patented by the company, Difficult to popularize and apply; due to the use of two cheap enzymes in the dual-enzyme system, the optimal reaction conditions are different because the two enzymes are independent systems, and the dual-enzyme single-tube RT-PCR system with high sensitivity has been recognized in the industry. It is a difficult topic, and its relatively cheap cost has made it one of the hotspots of relevant scholars' research in recent years. Multiple simultaneous inspections are also considered challenging puzzles.

The excellent quality of the present invention largely depends on the selected HBV primer, HBV TaqMan probe; HCV primer, HCV TaqMan probe; HIV primer, HIV TaqMan probe sequence. Therefore, the primers and probe sequences disclosed in the present invention should undoubtedly be protected by the claims of the application.

In summary, the present invention is to propose a test kit for measuring HIV (AIDS), HBV (hepatitis B), HCV (hepatitis C) viral nucleic acid, including the magnetic bead extraction reagent for synchronously extracting HBV-HCV-HIV nucleic acid And disposable consumables, which contain de-inhibitor, lysate, magnetic bead suspension, washing solution A, washing solution B, washing solution C, eluent, etc., and HBV-HCV-HIV nucleic acid amplification reagents, which contain RT -PCR reaction solution, probe, enzyme mixture, positive control, negative control, and internal control, etc. The matching instrument can use KHB DP-1000, KHB DP-2000, etc. manufactured by Shanghai Kehua Test Instrument System, but not limited to these, there may be MagNA pure LC instrument of ROCHE, equivalent instrument of TECAN, ThermoLabsystem company Equivalent instruments, Beckman-Coulter equivalent instruments, ABI equivalent instruments, etc.

The lysate mentioned above is a guanidine thiocyanate solution for lysing virus particles in body fluid samples such as serum and plasma, specifically 4-6M guanidine thiocyanate, 20-50mM Tris, pH6.0-8.0, 1-20% NP-40, 1-20% TritonX-100, 0.1-10% SDS, 0.01-1 μM biotinylated probe (capture probe), 1-10% polymer, etc. Wherein the capture probe is: 5' BIOTIN-acc acc aaa tgc ccc tat-3' (to HBV) (SEQ.ID.NO.28), 5' BIOTIN-agtacc aca agg cct ttcg-3' (to HCV) ( SEQ.ID.NO.29), 5'BIOTIN-cta tgt cac ttcccc ttg gtt ctc tc-3' (to HIV) (SEQ.ID.NO.30), can entrust Shanghai Sangong or Dalian Bao biosynthesis, purity It is PAGE or HPLC; the magnetic bead suspension is streptavidin-coated magnetic beads, specifically, magnetic beads of superparamagnetic material containing TWEEN20, and the surface is coated with a uniform layer of streptavidin, with a diameter of 0.1-10 μM , containing components such as BSA and NaN3, such as magnetic beads produced by E.Merck Company, Roche Company, Invitrogen Company, Toyabo Company, Agowa Company, Chemogen Company, Promega Company, and Seradyn Company; Buffer solution containing lithium chloride, specifically washing solution A: LiCI, Tris PH7.0, lithium dodecylsulfonate, preservatives and dispersants such as Tween 20, pigments such as magenta or other dyes, among which LiCI The concentration can be from 0.15M-3M, the Tris concentration can be 10-100mM, the pH can be 6.0-8.0, the concentration of lithium dodecylsulfonate can be from 1%, the preservative can be sodium azide or PROCLIN300, PROCLIN150, thimerosal , antibiotics, etc., the concentration of Tween 20 can be from 0.001-0.1%; washing solution B: the composition is similar to that of washing solution A but does not contain lithium dodecylsulfonate, the pigment is different or does not contain pigment; washing solution C: the composition and washing Liquid B is similar but the concentration of LiCI reduces (as being 0.1-1M) and does not contain pigment etc.; Described de-inhibitor is the alcoholic solution that contains protease, de-inhibitor: contains protease and glycerin etc.; Described eluent is low Salt buffer or water, eluent: Tris buffer prepared by DEPC-treated purified water, added with dispersant such as Tween 20 and EDTA, NaN3, DTT, etc.

In the present invention, the intermediate probe used for sample extraction (containing in the lysate) is

5’-acc acc aaa tgc ccc tat ttt ttt agt acc aca agg cct ttc g aaa

aaa cta tgt cac ttc ccc ttg gtt ctc tc aaa aaa aaa aaa aaa aaa

aaa aaa-3' (SEQ. ID. NO. 15) and 5' Biotin-Oligo(d T) ₂₅ (SEQ. ID. NO. 16).

The primers: the hepatitis B virus detection primers are aimed at a gene fragment in the coding gene of the hepatitis B virus core region (region C), BF: 5'-acc acc aaa tgc ccc tat-3' (SEQ.ID.NO. 1), BR: 5'-ttc tgc gacgcg gcg a-3' (SEQ.ID.NO.2). The hepatitis C virus detection primers are aimed at the 5'untranslated region of the hepatitis C virus gene sequence, and the two primer sequences are: CF: 5'-cgg gag agc cat agt gg-3'(SEQ.ID.NO.4), CR: 5'-agtacc aca agg cct ttc g-3' (SEQ. ID. NO. 5). The HIV detection primers are aimed at the gene sequence of the HIV gag region. The two primer sequences are: IF: 5'-gac atc aag cag cca tgc aaa t--3'(SEQ.ID.NO.7), IR: 5'- cta tgt cac ttc ccc ttg gtt ctc tc-3' (SEQ. ID. NO. 8).

The probes are Taqman probes, including internal standard (i.e. internal control) probes and detection probes of three viruses, respectively marked by different fluorescent groups or the same fluorescent group, but at least the internal standard probe The fluorophore is different from the fluorophore of the detection probe, and the preferred TaqMan probe is a TaqMan-MGB probe or a non-fluorescent quencher TaqMan probe, such as BHQ-1 or BHQ-2. Since the conventional TAMRA quenching group is a fluorescent quenching group, which has fluorescence itself, in multiple reactions, this fluorescence interference will be superimposed on each other, and it itself is a fluorescent group occupying a channel of the instrument, making multiple Detect one less detection; the probe sequence used in the present invention is: HBV probe is: 5'FAM(Ned)-cgacga ggc agg acc cct aga aga a-NFQ3'(SEQ.ID.NO.3); HCV The probe is: 5'FAM(Vic)-ctg cggaac cgg tga gta cac-NFQ-3'(SEQ.ID.NO.6); the HIV probe is: 5'FAM(Fam)-cca tca atg aggaag ctg cag aat ggg ata-NFQ3'(SEQ.ID.NO.9); internal standard probe is ICP: 5'Vic(Rox)-aac cttgga acc ttg gaa ctg gag aga gaa-NFQ3'(SEQ.ID.NO.14 ).

The RT-PCR reaction solution contains dATP, dCTP, dGTP, dUTP, buffer, KCI, preservatives and stabilizers, the above primers, etc.; the enzyme mixture is a multi-enzyme component system, specifically containing reverse Recording enzymes, DNA polymerases, RNasin, heat-labile UNG, RT-PCR promoters (such as heat shock proteins, gp32 proteins, etc.) and enzyme stabilizers (such as various sugar molecules and surfactants, DTT, protease inhibitors Specifically, 1.8Mm d NTPs (including dUTP), 3.6mM DTT, 3.5mM MgCl ₂ , 0.4μM each primer (BF, BR, CF, CR, IF, IR), etc.; enzyme mixture: 0.6U/μl AMV reverse transcriptase, 0.6U/μl hot-start Taq DNA polymerase, 1U/μl RNasin; 0.1U/μl UNG (heat-labile), etc.; probe: 10μM probe HBV Probe, HCV Probe, HIV Probe, 5μM internal standard probe, stabilizer and buffer.

The positive control is: a mixture containing part of HBV DNA, HCV RNA, and HIV RNA fragments, all of which are gene DNA or RNA outside the amplified target gene, wherein the HBV positive control consists of only a piece of DNA without HBV RNA, HCV positive Only one piece of RNA does not contain HCV DNA, and the HIV positive fragment has only one piece of RNA that does not contain HIV DNA. The mixture of the three is properly diluted and stored in a nucleic acid stabilization solution as a positive control for the kit; the internal control is a competitive internal standard RNA contains the same primer-binding region as the detection item, but is different from the probe-binding region of the detection item, and cannot bind the detection probe, but can bind the internal standard probe. For example, the internal control can be an in vitro transcribed RNA containing a HIV primer binding region, but the probe region cannot recognize the HIV probe through mutation (PCR mutation method or other genetic engineering means), but can recognize the internal standard probe in the kit, The internal control is RNA without DNA; the internal standard probe is an artificially synthesized oligonucleotide that has nothing to do with the detection item, and its 5' TaqMan modified fluorescent group is different from the detection of any positive control The fluorescent group of the probe is convenient for distinguishing the test result of the same sample from the internal control result, and it is required that the internal control result must be positive when the test result of the sample is negative. The negative control is the plasma of normal blood donors.

The sample processing of the kit of the present invention adopts the magnetic bead extraction method. This technology uses streptavidin magnetic beads (based on the principle of nucleic acid hybridization). Since both DNA and RNA have the same hybridization performance, it is generally believed that this method has the same or similar yields for DNA and RNA. Impurities, and finally the nucleic acid is eluted in low salt or water as a template for nucleic acid amplification detection. The KHB DP-1000 or 2000 instruments can be used, while the use on other automated instruments is not excluded. After this technology is applied to this diagnostic kit, on the one hand, it avoids the use of toxic reagents such as phenol, chloroform, and isoamyl alcohol in traditional methods; The influence of operational factors on the outcome. Those skilled in the art can quickly grasp its operation process and understand its technical essentials through short-term training, and can also add a little modification to form new technical processes and test kits according to the content provided by the present invention, but these all fall into the Within the scope of protection claimed by the present invention.

The one-step RT-PCR TaqMan dual-enzyme system used for amplification detection in the kit of the present invention uses reverse transcriptase such as AMV or MMLV or Superscript I/Superscript II/SuperscriptIII or Ominiscript, heat-resistant DNA The polymerase uses Taq enzyme or gold Taq enzyme or so-called hot-start Taq enzyme (such as Taq enzyme with its monoclonal antibody or chemically modified or ligand oligonucleotide pre-inactivated), and other heat-resistant Taq enzymes can also be selected Polymerase such as Tfl enzyme etc. The selected PCR buffer is TRIS-HCI or TRIS-H ₂ SO ₄ or TRIS-HOAc or Good's buffer Bicine, Tricine, containing potassium chloride, and can also contain ammonium sulfate, dimethyl sulfoxide, formamide, glycerol, TMAC, Urea, gp32 protein, betaine, bovine serum albumin, nonionic surfactant, TWEEN-20, TRITON X-100, NP-40, Brij-35, polyethylene glycol, PVP, DTT, Aptamer, protamine , HSA, SSB, Alpha Casine, Topoisomerase 1 and 2, Sucrose, Mannose, Sorbitol, Trehalose, Arabinose, Ficoll, Glycogen, Gelatin, Salmon Sperm DNA, Calf Thymus DNA, rRNA, tRNA , PolyA RNA, OLIGO (dT), self-synthesized agent MP and other additives.

The TaqMan probe-based RT-PCR amplification method designed by the present invention for real-time simultaneous multi-detection is characterized in that the sequence characteristics of the designed primers and probes are unique to HIV, hepatitis B virus, and hepatitis C virus. Among them, the two primer sequences of hepatitis B virus are: BF: 5'-acc acc aaa tgc ccc tat-3' (SEQ.ID.NO.1), BR: 5'-ttctgc gac gcg gcg a-3' (SEQ .ID.NO.2), the probe adopts the Taqman probe type, that is, the fluorescent reporter group and the quencher group are respectively labeled at both ends of the probe base, and the HBV probe sequence is: 5′FAM(Ned)-cga cga ggc agg accccct aga aga a-NFQ3' (SEQ. ID. NO. 3).

The two primer sequences of hepatitis C virus are: CF: 5'-cgg gag agc cat agt gg-3' (SEQ.ID.NO.4), CR: 5'-agt acc aca agg cct ttc g-3' (SEQ.ID.NO.5), the probe adopts the Taqman probe type, that is, the fluorescent reporter group and the quencher group are respectively labeled at both ends of the probe base, and the HCV probe sequence is: 5'FAM (Vic) -ctg cgg aac cgg tga gta cac-NFQ-3' (SEQ. ID. NO. 6).

The two primer sequences of HIV are: IF: 5'-gac atc aag cag cca tgc aaat--3' (SEQ.ID.NO.7), IR: 5'-cta tgt cac ttc ccc ttg gtt ctc tc- 3' (SEQ. ID. NO. 8). The probe uses a Taqman probe, that is, a fluorescent reporter group and a quencher group are respectively labeled at both ends of the probe base. The Taqman probe sequence is: 5'FAM(Fam)-cca tca atg agg aag ctg cag aat ggg ata-NFQ3'(SEQ.ID.NO.9). The internal standard probe is ICP: 5'Vic(Rox)-aac ctt gga acc ttg gaa ctg gag agagaa-NFQ3'(SEQ.ID.NO.14).

During PCR amplification, the fluorescent PCR instrument detects the fluorescent signal generated by each reaction tube, and the report is positive if the signal intensity exceeds the detector threshold. At this time, the number of PCR amplification cycles (called CT) is related to the template in the sample. The content has a negative logarithmic relationship, which is automatically recorded by the instrument. The real-time fluorescent PCR instrument can realize the fluorescence signal change diagram of the whole process of specimen template amplification, which can very vividly judge the negative and positive of the specimen and distinguish its strength.

Due to the complexity of the nucleic acid detection operation, the entire process is susceptible to various factors, which may cause the amplification to fail, so that the operator of the kit may draw a wrong conclusion that the test specimen is negative. In order to avoid this situation, it is necessary to set up a control, that is, an internal control, in each test tube, so that even if a very rare error such as a certain sample tube is forgotten to add a template, the internal control can detect it, or a certain error in the PCR instrument If there is a problem with the temperature efficiency of the well, the internal control can also monitor it.

In the optimal composition of the kit of the present invention, an internal control quality control system is used, that is, an internal control RNA of a specific sequence is added to the lysate, and the samples go through the steps of lysis, purification, reverse transcription, amplification, and TaqMan detection together. The internal control sequence contains a nucleic acid fragment that has nothing to do with the specimen to be tested (such as a completely artificially designed random sequence fragment or a rearrangement of the detection item probe sequence or a plasmid such as PUC18 sequence, etc.), and the two ends contain the primers used in the kit sequence, so it can be amplified with the same pair of primers as the sample to be tested. The internal control probe used was a Taqman probe specific for this irrelevant sequence. If the test result is that both the specimen and the internal control are negative, it indicates that the test has failed and the test result is invalid; other circumstances of the test result can occur and the test result is valid. Although the kit without internal control lacks a quality control standard, it does not affect normal use.

The kit of the present invention is used as a reagent for qualitative detection, but it does not exclude that it can be used as a reagent for quantitative detection. Because the method adopted in the present invention is TaqMan PCR, only need to introduce the external standard product and can become the kit of quantitative analysis, although the meaning of blood nucleic acid screening is qualitative analysis, lays particular emphasis on the sensitivity and specificity of reagent; Efficacy monitoring focuses on quantification, and loading tests pay attention to the accuracy of quantification.

The kit of the present invention needs to be used in conjunction with a nucleic acid magnetic bead extraction instrument and a fluorescent PCR instrument.

The present invention has the following advantages: (1) sealing detection, high degree of automation, reduced manual operations, avoiding possible pollution and human errors; (2) synchronous extraction of multiple nucleic acid viruses, synchronous real-time detection; (3) High sensitivity, good specificity, high precision, good stability, and easy operation; (4) Suitable for large-scale high-throughput blood screening, such as nucleic acid screening in blood centers and blood product manufacturers, and also It can be applied to large-capacity and high-efficiency clinical nucleic acid testing. (5) The whole process takes a short time and has a high energy efficiency ratio. 96 tests completed quickly and reported results with high confidence in just a few hours.

Description of drawings:

Figure 1 is a schematic diagram of Streptavidin magnetic beads capturing nucleic acids.

Wherein (A) is the structure diagram of the magnetic beads combined with the biotin probe;

(B) is the schematic diagram that the hybridization complex is captured by the Streptavidin magnetic beads, and the hybridization complex-magnetic beads are enriched by the magnet;

(C) Schematic diagram of the entire hybrid capture magnetic bead.

Figure 2 is a schematic diagram of the rapid DNA/RNA control substance construction

Figure 3 is a schematic diagram of the rapid internal control DNA/RNA quality control product construction

Detailed ways

Example 1 Biotin-modified primer plus terminal PCR method to quickly prepare internal control and positive control (the basis of the green environmental protection kit),

1. The preparation of HIV internal control and positive control is as follows

1.1 Rapid construction of positive control: commission Shanghai Sangon to synthesize the following primers, the sequence is:

5’BIOTIN-AAT TCT AAT ACG ACT CAC TAT AGG GAG gac atc aag cag cca tgc aaa t-3’

(The italic part at the 5' end is the T7 RNA pol promoter region, and the 3' end is the upstream primer of the HIV GAG region)

(SEQ.ID.NO.10)

5'BIOTIN-cta tgt cac ttc ccc ttg gtt ctc tc-3' (downstream primer of HIV GAG region)

(SEQ.ID.NO.11)

Take a specimen that is clinically strongly positive for HIV nucleic acid (or a plasmid containing the HIV GAG gene), use QIAamp ViralRNA Mini Kit (QIAGEN) to extract viral nucleic acid, and operate strictly according to its instruction manual. The extraction product is amplified with the above two primers through the one-step RT-PCR reagent established by the present invention, and operates according to the following cycle parameters:

50°C for 30 minutes --- 94°C for 4 minutes --- 94°C for 20 seconds,

      55°C for 20 seconds, cycle 30-40 times, and finally 72°C for 5 minutes at the end of extension.

                                                       

Part of the reaction product can be detected by electrophoresis and observed as a 160bp band. Add 10 times the volume of 1XPCR Buffer to the amplification product, add 100-500 μg of streptavidin-coated magnetic beads, mix well at room temperature for 10 minutes, centrifuge at low speed to remove the supernatant, add 400 μl of 1XPCR Buffer to wash the magnetic beads twice, and collect Magnetic beads, use the transcription reagents in the RiboMAXTM Large ScaleRAN Production Systems-T7 Kit (Promega Company), such as NTPs, Rnasin, DTT, transcription buffer, T7 RNA Pol. After adding the sample according to the instructions, transcribe at 37°C for 1 hour, and take it out, the same Dilute with 1XPCRBuffer, but do not wash and need denaturation treatment, you can use 90°C heat denaturation or NaOH denaturation, you can also choose guanidine thiocyanate denaturation, and finally centrifuge to remove the template DNA bound to the magnetic beads, take the supernatant in a new In a clean centrifuge tube, add absolute ethanol to precipitate RNA, remove transcription substrates such as NTPs, and dissolve in RNA protection agent. Of course further digestion with DNase is also possible. The obtained RNA is the HIV RNA positive control mother solution, which can be used as the biosafety green positive control of the kit for the whole process of nucleic acid extraction, RT reaction, and PCR TaqMan amplification detection by gradiently diluting it in a certain liquid.

1.2 Construction of internal control: Based on the positive control DNA constructed above (or a clinically positive HIV nucleic acid specimen), Shanghai Sangong was commissioned to synthesize the following primers, the sequence of which is:

5'-AAC CTT GGA ACC TTG GAA CTG GAG AGA GAA gagtgcatccagtgcatgcag-3' (SEQ.ID.NO.12, whose 5' end is rearranged from the HIV detection probe sequence, has the same GC% as the detection probe, Similar Tm values; the 3' end is the nucleic acid sequence adjacent to the 3' end position of the detection probe on the HIV positive control)

5'-TTC TCT CTC CAG TTC CAA GGT TCC AAG GTT tct ctt taa tta aca ttt gc-3' (the 5' end of SEQ.ID.NO.13 is complementary to the 5' end of SEQ.ID.NO.12; 3 ' end is complementary to the nucleic acid sequence adjacent to the 5' end of the detection probe on the HIV positive control)

Use positive control DNA as PCR template, use SEQ.ID.NO.10, SEQ.ID.NO.13 as a pair of primers and SEQ.ID.NO.11, SEQ.ID.NO.12 as another pair of primers To amplify, the cycle parameters used are:

94°C for 2 minutes---94°C for 10 seconds,

  45°C for 15 seconds, cycle 20 times, and finally extend at 72°C for 5 minutes.

  72°C for 10 seconds,

The above two amplification products were purified, mixed and serially diluted, and used as templates for the next round of PCR respectively. This round of PCR uses SEQ.ID.NO.10 and SEQ.ID.NO.11 as a primer pair for amplification, the procedure is the same as above, and the number of cycles is increased to 30-40. The product of this round of amplification is the internal control DNA, and a part of the product can be taken for electrophoresis detection and observed as a 160bp band (containing HIVDNA134BP and TTRNA promoter sequence 27BP). Similarly, its magnetic bead purification, transcription, and preservation are all the same as the above-mentioned positive control (RNA), and finally the pure HIV internal control RNA is prepared. The HIV sequence that can be referred to is as follows, and the schematic diagram that can be referred to is shown in Figure 3.

gaca tcaagcagcc atgcaaatgt taattaaagagac catcaatgag gaagctgcag aatgggatag

agtgcatcca gtgcatgcag ggcctattgc accaggccag atgagagaac caaggggaag tgacatag

(SEQ. ID. NO. 25)

2. The preparation of HCV internal control and positive control is as follows

2.1 Rapid construction of positive control: commission Shanghai Sangon to synthesize the following primers, the sequence is:

5’BIOTIN-AAT TCT AAT ACG ACT CAC TAT AGG GAG cgg gag agc cat agt gg-3’

(The italic part at the 5' end is the T7 RNA pol promoter region, and the 3' end is the upstream primer of the HCV NCR region)

(SEQ.ID.NO.17)

5'BIOTIN-agt acc aca agg cct ttc g-3' (downstream primer of HCV NCR region)

(SEQ.ID.NO.18)

Take a specimen that is clinically strongly positive for HCV nucleic acid (or a plasmid containing HCV 5'NCR), use QIAamp ViralRNA Mini Kit (QIAGEN) to extract viral nucleic acid, and operate strictly according to its instruction manual. The extracted product is amplified by the one-step RT-PCR reagent with the above two primers, and operated according to the following cycle parameters:

50°C for 30 minutes --- 94°C for 4 minutes --- 94°C for 20 seconds,

      50°C for 20 seconds, cycle 30-40 times, and finally 72°C for 5 minutes at the end of extension.

                                                     

Part of the reaction product can be detected by electrophoresis and observed as a 187bp bright band. Prepare a biosafety green positive control with the same operation as 1.1.

2.2 Construction of internal control: Based on the positive control DNA constructed above, Shanghai Sangong was commissioned to synthesize the following primers, the sequence of which is:

5'-AAC CTT GGA ACC TGG ACC GGG CGGAATTGCCAGGACGACCGGG-3' (SEQ.ID.NO.19, whose 5' end is rearranged from the HCV detection probe sequence, has the same GC% and similar Tm as the detection probe value; the 3' end is the nucleic acid sequence adjacent to the 3' end position of the detection probe on the HCV positive control)

5'-CCC GGT CCA GGT TCC AAG GTT accactatggctctcc-3' (SEQ.ID.NO.20 its 5' end is complementary to the 5' end of SEQ.ID.NO.19; 3' end is adjacent to the HCV positive control for detection The nucleic acid sequence at the 5' end position of the probe is complementary) with positive control DNA as the PCR template, with SEQ.ID.NO.17, SEQ.ID.NO.20 as a pair of primers and SEQ.ID.NO.18, SEQ.ID.NO.18, SEQ.ID.NO.20 respectively .ID.NO.19 is another pair of primers to amplify, and the cycle parameters used are:

94°C for 2 minutes---94°C for 10 seconds,

  45°C for 15 seconds, cycle 20 times, and finally extend at 72°C for 5 minutes.

  72°C for 10 seconds,

The above two amplification products were purified, mixed and serially diluted, and used as templates for the next round of PCR respectively. This round of PCR uses SEQ.ID.NO.17 and SEQ.ID.NO.18 as a primer pair for amplification, the procedure is the same as above, and the number of cycles is increased to 30-40. The amplification product of this round is the internal control DNA, and a part of the product can be detected by electrophoresis and observed as a 187bp band. Similarly, its magnetic bead purification, transcription, and storage are all the same as the above-mentioned positive control (RNA), and finally the pure HCV internal control RNA is prepared. The HCV sequence that can be referred to is as follows, and the schematic diagram that can be referred to is shown in Figure 3.

C GGGAGAGCCA TAGTGGTCTG CGGAACCGGT GAGTACACCG GAATTGCCAG

GACGACCGGG TCCTTTCTTG GATCAACCCG CTCAATGCCT GGAGATTTGG GCGTGCCCCC

GCGAGACTGC TAGCCGAGTA GTGTTGGGTC GCGAAAGGCC TTGTGGTACT

(SEQ.ID.NO.26)

3. The preparation of HBV internal control and positive control is as follows

3.1 Rapid construction of positive control: commission Shanghai Sangon to synthesize the following primers, the sequence is:

5' AGG GAG acc acc aaa tgc ccc tat-3' (SEQ.ID.NO.21)

(The part in italics at the 5' end is an irrelevant sequence, preventing DNA degradation from 5' to cause degradation of the primer-binding region of the positive control, and the 3' end is the upstream primer of HBV)

5' AGG GAG ttc tgc gac gcg gcg a-3' (SEQ.ID.NO.22)

(The italic part at the 5' end is an irrelevant sequence, and the 3' end is the downstream primer of HBV)

Take a specimen that is clinically strongly positive for HBV nucleic acid (or a plasmid containing HBV DNA), use QIAamp ViralMini Kit (QIAGEN Company) to extract viral nucleic acid, and operate strictly according to its instruction manual. The extracted product was amplified with the above two primers through the PCR reagent established by our laboratory, and operated according to the following cycle parameters:

94°C for 2 minutes---94°C for 20 seconds,

  50°C for 20 seconds, cycle 30-40 times, and finally extend at 72°C for 5 minutes.

  72°C for 30 seconds,

Part of the reaction product can be detected by electrophoresis and observed as a 131bp bright band. Use the Gel Recovery and Purification Kit to recover and purify the electrophoresis bands to become the HBV positive control mother solution, and prepare a biosafe green stable positive control with an appropriate concentration by gradient dilution with DNA/RNA preservation solution.

3.2 Construction of internal control: Based on the positive control DNA constructed above, Shanghai Sangong was commissioned to synthesize the following primers, the sequence of which is:

5'-TACGACGACGACGACGACGACGAGA gaactccc tcgcctcgc-3' (SEQ.ID.NO.23, whose 5' end is rearranged from the HBV detection probe sequence, has the same GC% and similar Tm value as the detection probe;

The 3' end is the nucleic acid sequence adjacent to the 3' end of the detection probe on the HBV positive control)

5'-TCTCGTCGTCGTCGTCGTCGTCGTA tctaacaacagtagtttc-3' (SEQ.ID.NO.24 its 5' end is complementary to the 5' end of SEQ.ID.NO.23; the 3' end is adjacent to the detection probe 5' end position on the HBV positive control Complementary nucleic acid sequence)

Use positive control DNA as PCR template, use SEQ.ID.NO.21, SEQ.ID.NO.24 as a pair of primers and SEQ.ID.NO.23, SEQ.ID.NO.22 as another pair of primers To amplify, the cycle parameters used are:

94°C for 2 minutes---94°C for 10 seconds,

  45°C for 15 seconds, cycle 20 times, and finally extend at 72°C for 5 minutes.

  72°C for 10 seconds,

The above two amplification products were purified, mixed and serially diluted, and used as templates for the next round of PCR respectively. This round of PCR uses SEQ.ID.NO.21 and SEQ.ID.NO.22 as a primer pair for amplification, the procedure is the same as above, and the number of cycles is increased to 30-40. The amplification product of this round is the internal control DNA, and a part of the product can be detected by electrophoresis and observed as a 131bp band. Use the gel recovery and purification kit to recover and purify the electrophoresis bands to obtain the HBV internal control DNA mother solution, and use the company's DNA/RNA preservation solution to prepare a biosafe green stable HBV internal control DNA with an appropriate concentration. The HBV sequence that can be referred to is as follows, and the schematic diagram that can be referred to is shown in Figure 3.

tatagaccac caaatgcccc tatcttatca acacttccgg aaactactgt

tgttagacga cgaggcagga cccctagaag aagaactccc tcgcctcgca gacgaaggtc

tcaatcgccg cgtcgcagaa gatctcaatc

(SEQ. ID. NO. 27)

Example 2 Specimen processing unit: magnetic bead method for nucleic acid extraction and reagent preparation

The reagent composition of the specimen processing unit based on the principle of hybridization includes:

Lysis solution: 4.8M guanidine thiocyanate, 50mM Tris, PH7.0, NP-40, Triton X-100, SDS, biotinylated probes, polymers, etc. where the capture probes are:

5'BIOTIN-acc acc aaa tgc ccc tat-3'(HBV)

5'BIOTIN-agt acc aca agg cct ttc g-3'(HCV)

5'BIOTIN-cta tgt cac ttc ccc ttg gtt ctc tc-3'(HIV)

Magnetic bead suspension: 1mg/ml streptavidin-coated magnetic beads, containing preservatives and dispersants such as Tween 20

Detergent A: LiCI, Tris PH7.0, lithium dodecylsulfonate, preservatives and dispersants such as Tween 20, pigments, etc.

Washing liquid B: LiCI, Tris PH7.0, preservatives and dispersants such as Tween 20, pigments, etc.

Washing liquid C: LiCI, Tris PH7.0, preservatives and dispersants such as Tween 20, pigments, etc.

De-inhibitor: Contains protease and glycerin, etc.

Eluent: Tris buffer prepared by DEPC-treated purified water, with dispersants such as Tween 20 and EDTA, NaN3, DTT, etc.

Internal reference: a solution containing about 40,000 copies/ml of internal reference RNA, which is added to the lysate at a ratio of 1:20 when used, and the concentration of the internal control in the lysate is about 2,000 copies/ml, each test Add 0.1ml of lysate, that is, 200 copies of the internal control to participate in the extraction. Amplification Since only 1/5 of the template amount is used for RT-PCR TaqMan detection, the amount of internal control RNA in each detection tube is less than 40 copies.

Operating procedures:

Take six dedicated 96-well plates (provided by the kit), numbered ①②③④⑤⑥.

Add 20 μl of de-inhibitor to the plate ① with a lance or a common pipette, add 100 μl of serum or plasma of the sample to be tested, and repeat the pipetting for about 1 time, and change a tip for each sample added. Note that the tip with a filter element should be used ; Finally, 100 μl of lysate was added.

Add 100 μl of magnetic bead suspension to the plate ② with a row gun or a common pipette. Pay attention to inverting the magnetic bead suspension several times a minute or two before using it until the magnetic beads are evenly dispersed and no visible precipitation is formed.

Add 200 μl of washing solution A to plate ③ with a row gun or a common pipette; add 200 μl of washing solution B to plate ④; add 200 μl of washing solution C to plate ⑤; add 80 μl of eluent to plate ⑥.

Move the above six boards into positions 1-6 of the KHB DP-2000 instrument operating cabin in sequence of numbers, and move the fourth board ④ into the deep hole Tip Comb of the special board.

Activate the "Start" button on the KHB DP-2000 instrument to make it execute the program.

It takes about 70 minutes to run, and after the end, take out the plates ①-⑥, the liquid in the plate ⑥ is the nucleic acid template, and the plates ①-⑤ are waste plates, which are discarded after disinfection.

Example 3 Nucleic Acid Amplification Detection Unit: Mode and Reagent Preparation of Single Tube Dual Enzyme One-step RT-PCR TaqMan Probe Multiple Detection System

The nucleic acid amplification detection unit adopts fluorescent TaqMan PCR, which is required to be used with a fluorescent PCR instrument. Its components include the following:

RT-PCR reaction solution: buffer containing 1.8Mm d NTPs (including dUTP), 3.6mM DTT, 3.5mM MgCl ₂ , primers BF, BR, CF, CR, IF, IR 0.4μM each.

Enzyme mixture: 0.6U/μl AMV reverse transcriptase, 0.6U/μl hot-start Taq DNA polymerase, 1U/μl RNasin; 0.1U/μl UNG (heat-labile), etc.

Probe: 10μM probe HBV Probe, HCV Probe, HIV Probe, 5μM internal standard probe, stabilizer and buffer.

Reagent ratio and preparation: According to the ratio of RT-PCR reaction liquid:enzyme mixture:probe=8:6:1 for each test, 15 μl/test is prepared, and 15 μl of nucleic acid template is added to the machine for amplification and real-time detection.

Taking ABI7500 as an example, the amplification parameters of the instrument are set as follows:

Cycle parameter setting: (can be set by referring to the operating software of various instruments)

Application of one of the detection modes of embodiment 4

According to the actual experimental hardware conditions, the fluorescent PCR instrument used by the user in this example is a dual-channel MJ Opticon2 fluorescent PCR instrument. The preferred detection probes are all labeled with FAM fluorescence, the internal standard probe is labeled with HEX, and the internal control is preferably added to RT-PCR reagent preparation but not added in the lysate. The RT-PCR/PCR reaction solution only contains primers for each detection item, and the probes are a mixture of detection probes and internal standard probes for each item. The internal control selection includes three detection item primer binding regions, The intermediate probe binding region is the common internal control RNA for the internal standard probe binding sequence.

The specimens to be tested are numbered P1, P2, P3..PN, and two control substances (negative control and positive control) are involved in parallel experiments. The sample to be tested contains a very low copy number of HBV DNA or HCV RNA or HIV RNA. Among them, the weak positive sample P1 is HBV DNA 1000 copies/ML, P2 is HCV RNA 1000 copies/ML, P3 is HIV RNA 1000 copies/ML, P4 is P1 diluted ten times with negative plasma, P5 is P2 with negative plasma ten times P6 is derived from ten-fold dilution of P3 with negative plasma; P7 is derived from twenty-fold dilution of P1 with negative plasma; P8 is derived from twenty-fold dilution of P2 with negative plasma; P9 is P3 with two-fold dilution of negative plasma. P10 is obtained by forty-fold dilution of P1 with negative plasma, P11 is obtained by forty-fold dilution of P2 with negative plasma, and P12 is obtained by forty-fold dilution of P3 with negative plasma.

serial number specimen P1 Specimen to be tested 1 P2 Specimen to be tested 2 P3 Specimen to be tested 3 ... ... PN Side specimen N NC negative control PC positive control x diluted plasma

Results The internal controls of the tested samples were all positive, while the HBV samples were all positive when the load was 1000 copies/ML, 100 copies/ML, 50 copies/ML, and 25 copies/ML, while the test results of the negative control and diluted plasma is negative. HCV samples were all positive when the load was 1000 copies/ML, 100 copies/ML, and 50 copies/ML, while the test results of 25 copies/ML, negative control, and diluted plasma were negative. The HIV samples were all positive when the load was 1000 copies/ML, 100 copies/ML, 50 copies/ML, and 25 copies/ML, while the test results of negative control and diluted plasma were negative.

Application of the second detection mode of embodiment 5

This detection mode is the best embodiment of the present invention: use a four-color or even five-color fluorescent PCR instrument such as ABI7500, the detection probes are respectively labeled with FAM, VIC, and NED, and the internal standard is labeled with ROX. An internal control was added to the lysate. The T-PCR reaction solution contains primers for each detection item, and the probes contain detection probes and internal standard probes for each item. As the internal control, the internal control RNA comprising the binding region of the HIV primer and the binding region of the intermediate probe as the binding sequence of the internal standard probe is selected. The sample is the same as in Example 3, and the experimental results are also the same as in Example 3, but the quality control is stricter, the operation is more convenient, the detection mode is more compatible with the sample extraction, and the detection throughput is higher.

Application of the third detection mode of embodiment 6

This detection mode is a compromise between Examples 3 and 4: the instrument can use a two-color fluorescent PCR instrument, and the detection throughput and rigorous quality control are the same as in Example 4, but once a positive amplification result is obtained, it is impossible to distinguish which test item is positive . The preferred point is that the detection probes are all labeled with FAM fluorescence, and the internal standard is labeled with HEX, VIC, NED, JOE, or TET. An internal control was added to the lysate. The RT-PCR reaction solution contains primers for each detection item, and the probes contain detection probes and internal standard probes for each item. As the internal control, the internal control RNA comprising the binding region of the HIV primer and the binding region of the intermediate probe as the binding sequence of the internal standard probe is selected.

The sample is the same as in Example 3, and the experimental results are also the same as in Example 3. The quality control is strict, the operation is convenient, the detection mode matches the sample extraction, and the detection throughput is high, but HBV positive or HCV positive or HIV positive or both positive or even None of the three co-positives can be known. However, this is not considered a disadvantage in blood screening, because the purpose of blood screening is to screen out unqualified blood, no matter which virus nucleic acid is positive or combined positive, it is eliminated blood.

Application of the fourth detection mode of embodiment 7

The following nucleic acids were synthesized as common intermediate (capture) probes for HBV, HCV, HIV, internal control RNA:

5’-acc acc aaa tgc ccc tat ttt ttt agt acc aca agg cct ttc g aaa

aaa cta tgt cac ttc ccc ttg gtt ctc tc aaa aaa aaa aaa aaa aaa

aaa aaa-3′(99bp) purity: PAGE or HPLC (SEQ.ID.NO.15)

Biotinylated probe: 5'Biotin-Oligo(dT) ₂₅ (SEQ.ID.NO.16).

The above two probes are prepared in the lysate at the same time, and have a fixed ratio, and the optimization of the amount of the common intermediate (capture) probe has a decisive effect on the extraction sensitivity.

Reagent is all the same as embodiment 2.1 of the present invention

The detection process is the same as in Example 2 (specimen extraction), the detection mode is in accordance with Example 4, and the result is exactly the same as that in Example 4.

According to the disclosure content of the present invention, those skilled in the art can implement the kit of the blood screening item claimed in the present invention without too many experiments, and achieve the expected effect. The embodiments disclosed in the present invention are only for describing the present invention, but are not construed as limiting the present invention. Those skilled in the art use obvious similar substitutions or modifications, or replace the preparations described here with some chemically or biologically structurally functionally related preparations, or change the relevant content of the present invention, but do not exceed the present invention. The spirit, scope and ideas of the invention all fall within the scope of protection claimed by the present invention.

sequence listing

SEQ.ID.NO.1:

5'-acc acc aaa tgc ccc tat-3',

SEQ.ID.NO.2:

5'-ttc tgc gac gcg gcg a-3',

SEQ.ID.NO.3:

5′FAM-cga cga ggc agg acc cct aga aga a-NFQ3′,

SEQ.ID.NO.4:

5'-cgg gag agc cat agt gg-3',

SEQ.ID.NO.5:

5'-agt acc aca agg cct ttc g-3',

SEQ.ID.NO.6:

5'FAM-ctg cgg aac cgg tga gta cac-NFQ3',

SEQ.ID.NO.7:

5'-gac atc aag cag cca tgc aaa t-3',

SEQ.ID.NO.8:

5'-cta tgt cac ttc ccc ttg gtt ctc tc-3',

SEQ.ID.NO.9:

5'Vic(Rox)-aac ctt gga acc ttg gaa ctg gag aga gaa-NFQ3',

SEQ.ID.NO.10:

5'BIOTIN-AAT TCT AAT ACG ACT CAC TAT AGG GAG gac atc aag cag cca tgc aaa t-3',

SEQ.ID.NO.11:

5'BIOTIN-cta tgt cac ttc ccc ttg gtt ctc tc-3',

SEQ.ID.NO.12:

5'-AAC CTT GGA ACC TTG GAA CTG GAG AGA GAA gagtgcatccagtgcatgcag-3',

SEQ.ID.NO.13:

5'-TTC TCT CTC CAG TTC CAA GGT TCC AAG GTT tct ctt taa tta aca ttt gc-3',

SEQ.ID.NO.14:

5'Vic(Rox)-aac ctt gga acc ttg gaa ctg gag aga gaa-NFQ3',

SEQ.ID.NO.15:

5’-acc acc aaa tgc ccc tat ttt ttt agt acc aca agg cct ttc g aaa

aaa cta tgt cac ttc ccc ttg gtt ctc tc aaa aaa aaa aaa aaa aaa

aaa aaa-3′,

SEQ.ID.NO.16:

5'Biotin-Oligo(d T) ₂₅ ,

SEQ.ID.NO.17:

5’BIOTIN-AAT TCT AAT ACG ACT CAC TAT AGG GAG cgg gag agc cat agt gg-3’

SEQ.ID.NO.18:

5'BIOTIN-agt acc aca agg cct ttc g-3'

SEQ.ID.NO.19:

5'-AAC CTT GGA ACC TGG ACC GGG CGGAATTGCCAGGACGACCGGG-3',

SEQ.ID.NO.20:

5'-CCC GGT CCA GGT TCC AAG GTT accactatggctctcc-3',

SEQ.ID.NO.21:

5'AGG GAG acc acc aaa tgc ccc tat-3',

SEQ.ID.NO.22:

5'AGG GAG ttc tgc gac gcg gcg a-3',

SEQ.ID.NO.23:

5'-TACGACGACGACGACGACGACGAGA gaactccc tcgcctcgc-3',

SEQ.ID.NO.24:

5'-TCTCGTCGTCGTCGTCGTCGTCGTA tctaacaacagtagtttc-3',

SEQ.ID.NO.25:

gaca tcaagcagcc atgcaaatgt taattaaagagac catcaatgag gaagctgcag aatgggatag

agtgcatcca gtgcatgcag ggcctattgc accaggccag atgagagaac caaggggaag tgacatag

SEQ.ID.NO.26:

C GGGAGAGCCA TAGTGGTCTG CGGAACCGGT GAGTACACCG GAATTGCCAG

GACGACCGGG TCCTTTCTTG GATCAACCCG CTCAATGCCT GGAGATTTGG GCGTGCCCCC

GCGAGACTGC TAGCCGAGTA GTGTTGGGTC GCGAAAGGCC TTGTGGTACT

SEQ.ID.NO.27:

5'FAM-cca tca atg agg aag ctg cag aat ggg ata-N FQ3',

SEQ.ID.NO.28:

5'BIOTIN-acc acc aaa tgc ccc tat-3',

SEQ.ID.NO.29:

5'BIOTIN-agt acc aca agg cct ttcg-3',

SEQ.ID.NO.30:

5'BIOTIN-cta tgt cac ttc ccc ttg gtt ctc tc-3'.

Claims (8)

1. A test kit for nucleic acid detection of AIDS, hepatitis B, and hepatitis C, characterized in that it comprises de-inhibitor, lysate, magnetic bead suspension, washing liquid, eluent, internal control, and RT-PCR reaction liquid , an enzyme mixture, a fluorescent probe, a positive control, and a negative control, wherein the de-inhibitor is an alcohol solution containing protease; the lysate is a guanidine thiocyanate solution for cleavage of virus particles in serum and plasma samples, and Containing biotin-modified oligonucleotides as capture probes extracted from nucleic acids; the washing solution is a solution containing sodium chloride; the eluent is low-salt buffer or water; the magnetic bead suspension contains streptavidin-wrapped magnetic beads; the RT-PCR reaction solution contains HBV, HCV, HIV primers and dNTP and buffer salt solution; the fluorescent probe is a Taqman probe; the enzyme mixture is a multi-enzyme component system; The positive control is a section of DNA or RNA containing HBV, HCV, and HIV amplified outside the target gene; the negative control is the plasma of normal blood donors; the internal control is a competitive internal standard RNA, which contains the same detection items The primer binding region, but different from the probe binding region of the detection item, it cannot bind the detection probe but can bind the internal standard probe; where: The HBV primers in the PT-PCR reaction solution are: 5'-acc acc aaa tgc ccc tat-3', 5'-ttc tgc gac gcg gcg a-3', The Taqman probe for HBV is: 5′FAM-cga cga ggc agg acc cct aga aga a-NFQ3′, HCV primers are: 5'-cgg gag agc cat agt gg-3', 5'-agt acc aca agg cct ttc g-3', The Taqman probes for HCV are: 5'FAM-ctg cgg aac cgg tga gta cac-NFQ3', HIV primers are: 5'-gac atc aag cag cca tgc aaa t-3', 5'-cta tgt cac ttc ccc ttg gtt ctc tc-3', Taqman probes for HIV are: 5'FAM-cca tca atg agg aag ctg cag aat ggg ata-NFQ3', The internal standard probe is SEQ.ID.NO.14. 2. The kit according to claim 1, characterized in that it uses reverse transcriptase, heat-resistant DNA polymerase dual-enzyme single-tube one-step RT-PCR TaqMan multiple simultaneous inspection system, and the detection items include DNA and RNA viruses; Contains UNG-dUTP anti-pollution components; also contains an internal control as a quality control for the entire monitoring of each test. 3. The kit according to claim 1, characterized in that biotin-modified primers are used to quickly construct and purify internal control RNA and positive control RNA pure RNA. 4. The kit according to claim 1, characterized in that said lysate contains capture probes used for sample extraction: For HBV: 5'BIOTIN-acc acc aaa tgc ccc tat-3', For HCV: 5'BIOTIN-agt acc aca agg cct ttc g-3', For HIV: 5'BIOTIN-cta tgt cac ttc ccc ttg gtt ctc tc-3'. 5. The kit according to claim 1, characterized in that said lysate contains intermediate probes used for sample extraction: 5’-acc acc aaa tgc ccc tat ttt ttt agt acc aca agg cct ttc g aaa aaa cta tgt cac ttc ccc ttg gtt ctc tc aaa aaa aaa aaa aaa aaa aaa aaa-3' and 5' Biotin-Oligo(d T) ₂₅ . 6. The kit according to claim 1, characterized in that the lysate consists of: 4-6M guanidine thiocyanate, 20-50mM Tris, PH6.0-8.0, 1-20% NP-40, 1-20 %Triton X-100, 0.1-10% SDS, 0.01-1 μM capture probe, 1-10% polymer; the capture probe for HBV is: 5'BIOTIN-acc acc aaa tgc ccc tat-3', for HCV It is: 5'BIOTIN-agt acc aca agg cct ttcg-3', for HIV: 5'BIOTIN-cta tgt cac ttcccc ttg gtt ctc tc-3', the purity is PAGE or HPLC; the magnetic bead suspension contains TWEEN20 magnetic beads of superparamagnetic material, the surface is coated with a uniform layer of streptavidin, with a diameter of 0.1-10μM, containing BSA and NaN3 components, and the eluent is Tris buffer prepared by DEPC-treated purified water , with Tween 20 dispersant and EDTA, NaN3 and DTT. 7. The kit according to claim 1, characterized in that the RT-PCR reaction solution is buffer containing 1.8Mmd NTPs, 3.6mM DTT, 3.5mM MgCl ₂ , primers BF, BR, CF, CR, IF , IR 0.4 μM each. 8. The kit according to claim 1, characterized in that said enzyme mixture contains reverse transcriptase, DNA polymerase, RNasin, heat-labile UNG, RT-PCR promoter and enzyme stabilizer.

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