CN112662815B - Primer-probe combination, kit and method for detecting Takara virus and Tamiami virus - Google Patents

Abstract

The invention relates to the technical field of virus detection, in particular to a combination of primers and probes, a kit and a method for detecting Takaribe virus and Taimiami virus. The present invention provides a combination of primers and probes for respectively or simultaneous real-time fluorescent quantitative RT-PCR detection of Takaribe virus and Taimi virus, which contains two pairs of specific primers and two specific probes, wherein, The sequence of the specific primer is shown in SEQ ID NO.1-4, and the sequence of the specific probe is shown in SEQ ID NO.5-6. The primer-probe combination and detection method provided by the invention have high specificity and sensitivity, good repeatability, simple and rapid detection, and have practical application value. Under the situation that new zoonotic diseases continuously appear, the present invention is particularly important in early detection and diagnosis of diseases.

Description

Primer-probe combination, kit and method for detecting Takaribe virus and Taimi virus

technical field

The invention relates to the technical field of virus detection, in particular to a combination of primers and probes, a kit and a method for detecting Takaribe virus and Taimiami virus.

Background technique

Arenaviruses consist of an outer membrane and a nucleocapsid, are round in shape, contain a lipid (outer) membrane, and have a diameter of about 50-300nm (average 110-130nm). The surface of the outer membrane is covered with a protein-like filament with a length of about 10nm, and different numbers of ribosomes from the host cell can be seen inside, which looks like sand grains under the electron microscope, hence the name arenavirus. The viral genome is composed of bidirectional segmented single-stranded negative-sense RNA, the larger one is the L segment, about 7400 nucleotides in length, encoding the RNA-dependent RNA polymerase L protein and the Z protein with transcription and replication regulation functions; The smaller one is the S segment, about 3400 nucleotides in length, encoding nucleoprotein NP and glycoprotein GP. L and S fragments are not present in equal amounts in the virion but in a 2:1 molar ratio. The genes on each fragment are separated by a non-coding region containing a hairpin structure, there is no cap structure at the 5' end, and the sequence at the 3' end is conserved. Since the S segment is more conservative than the L segment, the S segment is often used for virus evolution research and classification identification.

According to the latest virus classification issued by ICTV (International Committee on Virological Classification) in 2018 (https://talk.ictvonline.org/taxonomy/), Arenaviridae is divided into 4 genera: Antennavirus, Hartmanivirus, Mammarenavirus and Reptarenavirus, including 43 species. There are 35 species in the genus Mammarenavirus, which can be divided into Old World arenaviruses and New World arenaviruses. Among the Old World arenaviruses, the representative virus is lymphocytic choriomeningitis virus (LCMV). New world arenavirus mainly includes 4 clades, clade A has five viruses of Pirital, Pichinde, Flexal, Parana and Alloahuayo virus; clade B has eight viruses of Junin, Machupo, Guanarito, Amapari, Tacaribe, Sabia, Cupixi and Chapare viruses ; clade C has two viruses, Oliveros and Latino; clade A/Rec includes three viruses, Whitewater Arroyo, Tamiami and Bear Canyon; phylogenetic tree shows that Lujo virus belongs to the New World arenavirus, but it is not in the four clades. The arenaviruses that are pathogenic to humans mainly include Lassa virus causing Lassa fever, Junin virus causing Argentine hemorrhagic fever, Machupo virus and Chaparevirus causing Bolivian hemorrhagic fever, Lujo virus causes Lujo hemorrhagic fever, Guanarito virus causes Venezuelan hemorrhagic fever, Sabid virus causes Brazilian hemorrhagic fever, lymphocytic choriomeningitis Virus (LCMV) causes lymphocytic choriomeningitis.

Tacaribe virus (Tacaribe virus) was first discovered in Trinidad fruit bats (Oryzomys gaeldi) in 1956, and laboratory workers have been infected by contact with laboratory aerosols. The virus is mainly in Circulated in bats in the Trinidad area; Tamiami virus was first identified in Florida cotton rats (Sigmodon hispidus) in 1970, and the virus is mainly confined to rodents in the Florida area of the United States spread.

Laboratory testing of viral diseases mainly relies on virus isolation, serological testing, and viral nucleic acid testing. Viral nucleic acid detection is mainly based on PCR technology. Real-time fluorescent quantitative RT-PCR has the advantages of rapidity, high sensitivity and specificity, and is currently the most widely used laboratory detection method. The method is based on the principle of fluorescence resonance energy transfer, monitors the change of the product amount through the change of the fluorescence intensity, and draws a curve for the change of the monitored fluorescence signal as the reaction time progresses. This method is operated in a closed tube throughout the process, which reduces the risk of contamination; in addition to qualitative analysis of PCR products, quantitative analysis can also be performed by drawing a standard curve.

Contents of the invention

The purpose of the present invention is to provide a sensitive and specific real-time fluorescence quantitative RT-PCR primer-probe combination, kit and method that can detect Takaribe virus and Taimiami virus respectively.

To achieve the above object, the technical scheme of the present invention is as follows: search and download the genome sequences of Takaribe virus and Taimi virus in databases such as NCBI (https://www.ncbi.nlm.nih.gov/taxonomy) , select a more complete viral genome sequence with a clearer isolation date and region, and a viral genome sequence that indicates the genome sequence of the standard strain. Evaluate whether the direction of the viral genome sequence needs to be corrected, remove individual N-base sequences of poor quality, consult relevant entry information, and determine the sequence inclusion criteria as having clear virus isolation age, region and other information. The overall and partial comparison analysis is performed on the sequence files to determine the classification of each gene sequence. According to the results of sequence comparison analysis, combined with the analysis of the preferential binding site of DNA polymerase in the viral genome sequence and the optimal denaturation temperature, screen and determine the target sequence that is highly conserved in each virus genotype, and design specific primers according to the target sequence And probes, after a large number of screening and artificial optimization, the sequences are respectively shown in SEQ ID NO.1-4 and SEQ ID NO.5-6, and two pairs of specificities for Takaribe virus and Taimi virus respectively Primers and two specific probes are used to obtain a combination of primers and probes for real-time fluorescent quantitative RT-PCR for the detection of Takaribe virus and Taimi Ami virus.

Specifically, the technical scheme of the present invention is as follows:

The present invention provides a target sequence combination for the real-time fluorescence quantitative RT-PCR detection of Takaribe virus and Taimi virus, which contains two target sequences, and the target sequences have the following characteristics as shown in SEQ ID NO.7-8 The nucleotide sequence shown.

Among the above target sequence combinations, the target sequence of Takaribe virus is shown in SEQ ID NO.7, and the target sequence of Taimiamivirus is shown in SEQ ID NO.8. The above sequences are highly conserved among different genotypes of each virus and are easy to perform PCR amplification, and are applicable to efficient detection of different genotypes of each virus.

The present invention provides a primer probe combination for real-time fluorescent quantitative RT-PCR detection of Takaribe virus and Taimi virus, which contains two pairs of specific primers and two specific probes, wherein the specific The sequence of the specific primer is shown in SEQ ID NO.1-4, and the sequence of the specific probe is shown in SEQ ID NO.5-6.

Among the above-mentioned specific primers and specific probes, the sequence of specific primers for Takaribe virus is shown in SEQ ID NO.1-2, and the specific probe sequence is shown in SEQ ID NO.5; The sequence of the specific primer of Amida virus is shown in SEQ ID NO.3-4, and the specific probe sequence is shown in SEQ ID NO.6.

Preferably, the specific probes are respectively labeled with fluorescent groups that produce different fluorescent colors, and the fluorescent groups include a fluorescent reporter group and a fluorescent quencher group, and the fluorescent reporter group is selected from FAM, HEX, Any one in Texas Red, CY5, TET, JOE, CY3, TAMRA, ROX, LC RED640, LC RED705; Described fluorescence quenching group is any one selected from BHQ1, BHQ2, BHQ3, Dabcyl.

Further preferably, the sequence is as shown in SEQ ID NO.5. The 5' end of the specific probe is marked with a FAM fluorescent reporter group, and the 3' end is marked with a BHQ1 fluorescence quencher group; the sequence is as shown in SEQ ID NO.6. The 5' end of the indicated specific probe is labeled with the HEX fluorescent reporter group, and the 3' end is labeled with the BHQ1 fluorescent quencher group.

Preferably, the kit also includes positive control standards for Takaribe virus and Taimiami virus.

The positive control of the Takaribe virus is preferably the RNA corresponding to the DNA sequence shown in SEQ ID NO.7 or the Takaribe virus RNA. The positive control of the tamiamivirus is preferably the RNA corresponding to the DNA sequence shown in SEQ ID NO.8 or the tamiamivirus RNA.

Further preferably, the kit also contains other reagents for real-time fluorescent quantitative RT-PCR detection, including but not limited to reaction buffer, enzyme and water.

The reaction program of the primer-probe combination or the kit in the present invention when performing real-time fluorescent quantitative RT-PCR detection can be determined according to the characteristics of the reverse transcriptase and polymerase used.

Preferably, the reaction program of the primer-probe combination or the kit when performing real-time fluorescent quantitative RT-PCR detection is: reverse transcription at 50°C for 30 minutes; pre-denaturation at 95°C for 10 minutes, denaturation at 95°C for 15 seconds, and annealing at 60°C for 60 seconds Run 40 cycles.

By adopting the above-mentioned reaction procedure, the high-efficiency amplification detection of Takaribe virus and Taimiami virus can be better ensured.

In the reaction system of the primer-probe combination or the kit of the present invention when performing real-time fluorescence quantitative RT-PCR detection, the molar ratio of the upstream primer, the downstream primer and the probe is 2:2:1. More preferably, the 25 μL reaction system when performing real-time fluorescent quantitative RT-PCR detection is: 12.5 μL of 2× buffer, 0.5 μL of 10 μM upstream primer, 0.5 μL of 10 μM downstream primer, 0.25 μL of 10 μM probe, 1 μL of enzyme, and 5 μL of template , make up to 25 μL with RNA hydrolase-free deionized water.

The combination of primers and probes provided by the present invention can be used to detect Takaribe virus and Taimiamivirus respectively in different reaction systems, and the above-mentioned upstream primers are those in the primers shown in SEQ ID NO. Any one, the downstream primer is any one of the primers shown in SEQ ID NO.2,4, and the probe is any one of the probes shown in SEQ ID NO.5,6.

The present invention also provides the application of the primer-probe combination or the kit in the detection of Takaribe virus and Taimi virus.

For example, in some embodiments, it is possible to determine whether a human or animal is infected with Takaribe virus and/or Tamiami virus by testing an isolated sample. In some embodiments, food (especially food of animal origin) or other samples that may be infected with the above-mentioned viruses can also be tested, so as to facilitate the monitoring work and epidemiological research of Takaribe virus and Tamiami virus.

The present invention also provides a method for using real-time fluorescent quantitative RT-PCR to detect Takaribe virus and Taimiami virus respectively, which uses the RNA of the sample to be tested as a template, and utilizes the method shown in SEQ ID NO.1-4 The specific primers and the specific probes shown in SEQID NO.5-6 are used for real-time fluorescent quantitative RT-PCR detection, and the amplification curve is used to determine whether the sample to be tested contains Takaribe virus and Taimiami virus .

The above-mentioned method for extracting total viral RNA can adopt conventional methods in the art, such as spin column extraction.

Preferably, the reaction program of the real-time fluorescent quantitative RT-PCR is: reverse transcription at 50°C for 30 minutes; pre-denaturation at 95°C for 10 minutes, denaturation at 95°C for 15 seconds, and annealing at 60°C for 60 seconds for 40 cycles. .

Preferably, in the reaction system when performing real-time fluorescent quantitative RT-PCR detection, the molar ratio of the upstream primer, downstream primer and probe is 2:2:1. More preferably, the 25 μL reaction system of the real-time fluorescence quantitative RT-PCR is: 12.5 μL of 2× buffer, 0.5 μL of 10 μM upstream primer, 0.5 μL of 10 μM downstream primer, 0.25 μL of 10 μM probe, 1 μL of enzyme, 5 μL of template, no Make up to 25 μL of deionized water for RNA hydrolase.

As preferably, the above-mentioned method for judging whether the sample to be tested contains Takaribe virus and Taimi Ami virus according to the amplification curve is as follows:

Judgment of valid results: Positive control (or internal standard of detection tube) has Ct≤35 and has an amplification curve, negative control tube has no amplification curve or CT value>38;

If there is an amplification curve corresponding to the fluorescence channel of the pathogen measured in the detection sample tube and Ct<35, it can be judged that the corresponding pathogen is detected positive; if the detection sample has no amplification curve or Ct>38 in the detection channel, it can be judged that the sample is the The virus test is negative; if the test sample is suspicious in the detection channel 35<Ct<38, it is recommended to retest. If the retest result Ct<38, the sample can be judged as positive. If the retest result has no amplification curve or Ct≥38, The sample can be judged as negative;

Judgment of invalid results: If the test sample is negative, the positive control of the reaction system (or the internal standard of the test tube) must be positive, otherwise the result of the test tube is invalid and a retest is required.

In the determination of the above results, the positive control of Takaribe virus is preferably RNA corresponding to the DNA sequence shown in SEQ ID NO.7 or Takaribe virus RNA. The positive control of the tamiamivirus is preferably the RNA corresponding to the DNA sequence shown in SEQ ID NO.8 or the tamiamivirus RNA.

The real-time fluorescent quantitative RT-PCR detection reaction system of Takaribe virus and Taimiami virus described in the present invention is applicable to all real-time fluorescent quantitative PCR instruments.

The beneficial effects of the present invention are:

The present invention combines the probability of occurrence of relevant pathogens, the risk of prevalence and the operability of the laboratory detection process to form a new combined detection scheme with Takaribe virus and Taimiami virus as detection targets.

The present invention has determined the highly conserved target sequences of Takaribe virus and Taimiamivirus through a large number of sequence comparison analysis, and obtained target sequences suitable for real-time fluorescent quantitative RT-PCR detection respectively through a large number of screening and optimization for the determined target sequences. The specific primers and probes have good universality in each genotype of Takaribe virus and Taimi virus.

The invention establishes a separate or simultaneous real-time fluorescent quantitative RT-PCR detection method for detecting Takaribe virus and Taimiami virus. In this method, two pairs of specific primers and differently labeled probes are introduced into a real-time fluorescence quantitative RT-PCR reaction system respectively or simultaneously for Takaribe virus and Taimiami virus, and the dynamic changes of real-time fluorescent signals , perform qualitative analysis on unknown samples; it can also draw a standard curve through the detection of standard reference products to achieve quantitative analysis on unknown templates. Compared with other detection methods, the detection method provided by the present invention has the advantages of high sensitivity and specificity, simple operation, short detection time, less required sample volume, low pollution, etc., and can directly detect RNA extracted from unknown samples , which has high application value in the rapid detection of viruses. Under the situation that the incidence of zoonotic diseases is constantly increasing, the present invention has great significance in early detection and diagnosis of diseases.

Description of drawings

FIG. 1 is a graph showing the amplification curves of RNA templates transcribed in vitro by Takaribe virus at different concentrations detected by real-time fluorescent quantitative RT-PCR in Example 4 of the present invention.

Fig. 2 is a graph showing the amplification curves of RNA templates transcribed in vitro by real-time fluorescent quantitative RT-PCR detected with different concentrations of Tamiamivirus in Example 4 of the present invention.

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field, or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that can be purchased through formal channels.

The design of specific primers and probes of Takaribe virus and Taimi Ami virus of embodiment 1

1. Design and screening of primers and probes

Search and download the genome sequences of Takaribe virus and Taimi virus in public databases such as NCBI (https://www.ncbi.nlm.nih.gov/taxonomy). Select a more complete viral genome sequence with a clearer isolation date and region, and a viral genome sequence that indicates the genome sequence of the standard strain. Evaluate whether the direction of the viral genome sequence needs to be corrected, remove individual N-base sequences of poor quality, consult relevant entry information, and determine the sequence inclusion criteria as having clear virus isolation age, region and other information. The overall and partial comparison analysis of the sequence files was carried out to determine the classification of each gene sequence, and the highly conserved target sequences in each genotype of Takaribe virus and Taimiami virus were screened and determined according to the results of sequence comparison analysis.

Design specific primers and probes for the above target sequences, and simultaneously design and screen specific primers and probes in combination with the analysis of the DNA polymerase preferential binding sites in the viral genome sequence and the optimal denaturation temperature. After a lot of design, screening and comparison experiments, the present invention finds that it is not only the primers that meet the general primer design principles or the primers designed by the primer design software can realize efficient, specific and sensitive real-time fluorescent quantitative RT-PCR detection of Takaribe virus and miami virus. The present invention is aimed at the affinity and mismatch rate of primers, probes and target sequences, the secondary structure between primers and the secondary structure between primers and target sequences, and the GC content, Tm value, length and length of amplified fragments of primers conducted a lot of manual optimization design and screening, and finally obtained the following specific primer pairs and specific probes for Takaribe virus and Taimi virus respectively:

Takaribe virus:

Specific primers:

Tacaribe-F: 5'-AGGGTGAGCAGTGTCTTATC (SEQ ID NO.1)-3'

Tacaribe-R: 5'-TGCCTTGAGAGTCCTTCCTAC (SEQ ID NO.2)-3'

Specific probes:

Tacaribe-P: 5'-ATTCAACCTGAAGACTGGCCGACTGACTG(SEQ ID NO.5)-3'

Taimiamivirus:

Specific primers:

Tamiami-F: 5'-ATAACTCTCCAATATCCACACCC-3' (SEQ ID NO.3)

Tamiami-R: 5'-TGTCGTTTCCCACAGCATC-3' (SEQ ID NO.4)

Specific probes:

Tamiami-P: 5'-TTGTGAGAGCCAGGAGGATTCTCAGTTTCTTC-3' (SEQ ID NO. 6).

2. Synthesis of primers and probes

The above primers and probes were synthesized by Shanghai Sangon Bioengineering Co., Ltd. Among them, the 5' end of the specific probe shown in SEQ ID NO.5 is labeled with a FAM fluorescent reporter group, and the 3' end is marked with a Black Hole Quencher 1 fluorescence quencher; the sequence is shown in SEQ ID NO.6 The 5' end of the specific probe is labeled with the HEX fluorescent reporter group, and the 3' end is labeled with the Black Hole Quencher 1 fluorescence quencher group.

Example 2 Establishment of a detection method for real-time fluorescent quantitative RT-PCR detection of Takaribe virus and Taimi virus

1. Extraction of viral nucleic acid The cell culture supernatant of the virus to be tested was collected, and the viral nucleic acid was extracted using the QIAamp Viral RNA MiniKit.

2. Preparation of positive standard

For the two viruses, by synthesizing the conserved region of the two virus genes (as shown in SEQ ID NO.7-8) as a positive standard, the synthetic target gene is cloned into the pET-28a (+) vector, and the restriction site The points are NdeI and XhoI, and gene synthesis and plasmid cloning were completed by Beijing Tianyi Huiyuan Biological Company. The target gene contained in the cloned plasmid was amplified by common PCR with 2×PCR Mix reagent from Thermo Company, and the amplification primers were general primers T7 (TAATACGACTCACTATAGGG, SEQ ID NO.9) and T7t (ACATCCACTTTGCCTTTCTC, SEQ ID NO.10). It is transcribed into RNA in vitro, purified, recovered and quantified. The Gel Extraction Kit kit from Qian Company was used to recover and purify the PCR products, and the recovered DNA template was quantified by a Nanodrop UV-Vis spectrophotometer. The Ribomaxtm large scale RNA productionsystem-sf6 and T7 kits from Promega Company were used for RNA in vitro transcription of target genes, and the RNeasy mini kit of Qian Company was used to recover RNA in vitro transcription products. After analyzing the purity of the RNA template (SFTSV-NP) obtained by in vitro transcription by agarose gel electrophoresis, after diluting with DEPC-treated water, use a Nanodrop spectrophotometer to perform three measurements to ensure that the measured value is between 100-300ng/μL After taking the measured average value to calculate the mass concentration of each purified RNA product, the following formula was used to calculate the respective copy number concentration:

Y (copies/μL)=[X (g/μL)/transcription length in nucleotides×340]×6.02×10 ²³ .

The above operations were carried out according to the kit instructions.

3. Real-time fluorescent quantitative RT-PCR detection

Adopt AgPath-ID One Step RT-PCR Kit (Ambion) kit to carry out real-time fluorescent quantitative RT-PCR reaction, utilize the specific primer (sequence as shown in SEQ ID NO.1-4) and probe that embodiment 1 finally obtains (Sequence shown in SEQID NO.5-6) carry out real-time fluorescent quantitative RT-PCR detection.

In the present invention, by optimizing the reaction conditions of the specific primers and probes for the detection of the two viruses obtained in Example 1, it can achieve efficient combined detection, and the specific reaction conditions are as follows:

The 25 μL reaction system is: 12.5 μL of 2× buffer, 0.5 μL of 10 μM upstream primer, 0.5 μL of 10 μM downstream primer, 0.25 μL of 10 μM probe, 1 μL of enzyme, 5 μL of template, and deionized water without RNA hydrolase to make up to 25 μL;

The reaction program was: reverse transcription at 50°C for 30 minutes, pre-denaturation at 95°C for 10 minutes, denaturation at 95°C for 15 seconds, and annealing at 60°C for 60 seconds for 40 cycles.

4. Result analysis and judgment

The test results need to be judged according to the positive control (or internal standard in the detection tube) set in the reaction system, the amplification result and the amplification curve using deionized water without RNA hydrolase as the negative control.

Judgment of valid results: Positive control (or internal standard of detection tube) has Ct ≤ 35 and has an amplification curve, negative control tube has no amplification curve or CT value > 38.

If there is an amplification curve corresponding to the fluorescence channel of the pathogen measured in the detection sample tube and Ct<35, it can be judged that the corresponding pathogen is detected positive; if the detection sample has no amplification curve or Ct>38 in the detection channel, it can be judged that the sample is the The virus test is negative; if the test sample is suspicious in the detection channel 35<Ct<38, it is recommended to retest. If the retest result Ct<38, the sample can be judged as positive. If the retest result has no amplification curve or Ct≥38, The sample can be judged as negative.

Judgment of invalid results: If the test sample is negative, the positive control of the reaction system (or the internal standard of the test tube) must be positive, otherwise the result of the test tube is invalid and a retest is required.

The specificity analysis of embodiment 3 real-time fluorescent quantitative RT-PCR detection method

With Takaribe virus and Taimiami virus in vitro transcribed and synthesized RNA standards as positive controls, Seoul virus, Hantaan virus, fever with thrombocytopenia syndrome bunya virus, and dengue virus type 1-4 were used as positive controls. , a total of 8 arenavirus RNAs of Junin virus, Sabia virus, Lassa fever virus, Maqiupo virus, Guanarito virus, Chapare virus, Ruyo virus and lymphocytic choriomeningitis virus, and 100 copies Serum RNA extracts from healthy people were used as a control virus, and specificity analysis was performed on the detection method provided in Example 2.

The detection method of Example 2 was used for real-time fluorescent quantitative RT-PCR detection, and the design of the specific treatment group was as follows: RNA standard products (concentration: 1×10 ⁸ copies/ μL) was used as positive control; Seoul virus, Hantaan virus, fever with thrombocytopenia syndrome bunya virus, dengue virus type 1-4 virus, Jiuning virus, Sabia virus, Lassa fever virus, horse Qiubo virus, Eight arenavirus RNAs including Guanarito virus, Chapare virus, Ruyo virus and lymphocytic choriomeningitis virus, and 100 healthy human serum RNA extracts were used for specificity verification.

The results are shown in Table 1 below.

Table 1. Specificity analysis of three real-time fluorescent quantitative RT-PCR detection methods for arenaviruses

The sensitivity (detection limit) analysis of embodiment 4 real-time fluorescent quantitative RT-PCR detection method

Quantify the in vitro transcribed RNA of Takaribe virus and Taimiami virus extracted above, and calculate the RNA copy number according to the following formula: Y(copies/μl)=X(g/μl)×6.02×10 ²³ / Transcript length innucleotides×340, where Y is the RNA copy number and X is the concentration of RNA. Perform 10-fold serial dilutions of the above-mentioned known concentrations of RNA, and dilute the in vitro transcribed Takaribe virus and Taimiami virus standard RNA to 1×10 ⁸ copies/μL to 1×10 ¹ copies/μL for a total of 8 a concentration gradient.

Using the above gradiently diluted RNA with different concentrations as a template, the detection method in Example 2 was used to perform real-time fluorescent quantitative RT-PCR detection, and the detection limit of the detection method in Example 2 was determined.

The amplification curves of each virus and each concentration are shown in Figure 1 and Figure 2. In Figure 1-2, the curves from left to right are 8 concentrations from 1×10 ⁸ copies/μL to 1×10 ¹ copies/μL Amplification results of the gradient. Take the copy number of samples with different concentrations as the abscissa, and the cycle threshold (CT) as the ordinate to make a standard curve, and obtain the respective single-fold real-time fluorescent RT-PCR of the two viruses through the resulting standard curve. The results are shown in Table 2 below. The detection method of Example 2 has good sensitivity.

Table 2 Single-plex real-time fluorescent quantitative RT-PCR results of Takaribe virus and Taimiami virus

The repeatability analysis of embodiment 5 real-time fluorescent quantitative RT-PCR detection method

In vitro transcribed RNA standards for Takaribe virus and Tamiamivirus were selected at three different concentration gradients with dilutions of 1×10 ⁶ copies/μL, 1×10 ⁴ copies/μL, and 1×10 ² copies/μL The product was used as a template, and 5 parallel repeated experiments were carried out to verify the stable repeatability of the detection method provided in Example 2.

As shown in Table 3, the standard deviations of each detection experiment of Takaribe virus and Taimiami virus were all controlled within 0.5, and the coefficient of variation was below 1%, indicating that the detection method of Example 2 has good stability and repeatability.

Table 3 Stability evaluation of real-time fluorescent quantitative RT-PCR detection methods for Takaribe virus and Taimiami virus

The present invention has been described in detail with general descriptions and specific embodiments above, but it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

sequence listing

<110> Institute of Viral Disease Prevention and Control, Chinese Center for Disease Control and Prevention

<120> Primer-probe combination, kit and method for detecting Takaribe virus and Taimi virus

<130> KHP211110058.9

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

agggtgagca gtgtcttatc 20

<210> 2

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

tgccttgaga gtccttccta c 21

<210> 3

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ataacctcca atatccaacac cc 22

<210> 4

<211> 19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tgtcgtttcc cacagcatc 19

<210> 5

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

attcaacctg aagactggcc gactgactg 29

<210> 6

<211> 32

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ttgtgagagc caggaggatt ctcagtttct tc 32

<210> 7

<211> 200

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atttacagag gacgtgaaga accctgtttt gagggtgagc agtgtcttat caaaattcaa 60

cctgaagact ggccgactga ctgtaaggct gaccacacaa aacacattccg ctttttatca 120

aggagccaaa aaagcatagc tgtaggaagg actctcaagg catttttctc ctggtcgctt 180

actgaccctt tagggaacga 200

<210> 8

<211> 200

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ttacatggag tcagcagtgt cacgagaatc acatgagtgc catgcagctt ataacctcca 60

atatccacac ccaggttgtg agagccagga ggattctcag tttcttcacc tggtcgttgt 120

cagatgctgt gggaaacgac atgccaggag ggtattgttt agaaaaatgg atgctgattg 180

ccagccaact aaaatgtttt 200

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

taatacgact cactataggg 20

<210> 10

<211> 20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

acatccactt tgcctttctc 20

Claims (12)

1. the primer probe combination that is used for the real-time fluorescent quantitative RT-PCR detection of Takaribe virus and Tai Miami virus is characterized in that, contains two pairs of specific primers and two specific probes, wherein, all The sequence of the specific primer is shown in SEQ ID NO.1-4, and the sequence of the specific probe is shown in SEQ ID NO.5 and 6. 2. primer-probe combination according to claim 1, is characterized in that, described specific probe label produces the fluorophore of different fluorescent colors respectively, and described fluorophore comprises fluorescent reporter group and fluorescence quencher group group; The fluorescent reporter group is any one selected from FAM, HEX, Texas Red, CY5, TET, JOE, CY3, TAMRA, ROX, LCRED640, LC RED705; the fluorescent quenching group is selected from BHQ1, Any of BHQ2, BHQ3, Dabcyl. 3. according to the described primer probe combination of claim 1 or 2, it is characterized in that, the 5' end labeling FAM fluorescent reporter group of the specificity probe shown in sequence as SEQ ID NO.5, 3' end labeling BHQ 1 fluorescent quenching group; the 5' end of the specific probe with the sequence shown in SEQ ID NO.6 is labeled with a HEX fluorescent reporter group, and the 3' end is labeled with a BHQ 1 fluorescent quenching group. 4. Use of the primer-probe combination according to any one of claims 1 to 3 in the preparation of a test kit for detecting Takaribe virus and Tamiamivirus. 5. A kit for the detection of Takaribe virus and Taimi virus, characterized in that it comprises the primer-probe combination according to any one of claims 1-3. 6. The kit according to claim 5, wherein the reaction procedure of the kit for real-time fluorescent quantitative RT-PCR detection is: reverse transcription at 50°C for 30min; pre-denaturation at 95°C for 10min, denaturation at 95°C for 15s , 40 cycles of annealing at 60°C for 60s. 7. according to claim 5 or 6 described test kits, it is characterized in that, in the reaction system when described test kit carries out real-time fluorescent quantitative RT-PCR detection, the molar ratio of upstream primer, downstream primer and probe is 2. :2:1. 8. The kit according to claim 7, wherein the 25 μL reaction system of the kit when performing real-time fluorescent quantitative RT-PCR detection is: 12.5 μL of 2× buffer, 0.5 μL of 10 μM upstream primer, 10 μM 0.5 μL downstream primer, 0.25 μL 10 μM probe, 1 μL enzyme, 5 μL template, make up to 25 μL with RNA hydrolase-free purified water. 9. A method for non-diagnostic purposes utilizing real-time fluorescent quantitative RT-PCR to detect Hitakaribe virus and Taimiamivirus, characterized in that, the RNA of the sample to be tested is a detection template, utilizing such as SEQ ID NO The specific primers shown in .1-4 and the specific probes shown in SEQ ID NO.5-6 carry out real-time fluorescent quantitative RT-PCR, and judge whether the sample to be tested contains Takaribe virus and Taimiami virus. 10. The method according to claim 9, wherein the reaction program of the real-time fluorescent quantitative RT-PCR is reverse transcription at 50°C for 30 minutes; pre-denaturation at 95°C for 10 minutes, denaturation at 95°C for 15 seconds, and annealing at 60°C for 60 seconds for 40 seconds. cycles. 11. The method according to claim 9 or 10, characterized in that, in the reaction system of the real-time fluorescent quantitative RT-PCR, the molar ratio of the upstream primer, the downstream primer and the probe is 2:2:1. 12. The method according to claim 11, wherein the 25 μL reaction system of the real-time fluorescence quantitative RT-PCR is: 12.5 μL of 2× buffer, 0.5 μL of 10 μM upstream primer, 0.5 μL of 10 μM downstream primer, 10 μM probe Needle 0.25 μL, enzyme 1 μL, template 5 μL, RNase Free Water to make up to 25 μL.

Images