CN100339488C - Detection method of hepatitis B virus genome drug resistance mutation - Google Patents

Abstract

The invention relates to a method for detecting mutant hepatitis B virus, in particular to a method for quickly and accurately distinguishing wild-type and lamivudine-resistant mutant hepatitis B virus in clinical blood samples by using DNA reverse dot hybridization technology. The invention further relates to kits for this clinical assay.

Description

Detection method of drug resistance mutation in hepatitis B virus genome

field of invention

The invention relates to a method for detecting mutant hepatitis B virus, in particular to a method for quickly and accurately distinguishing wild-type and lamivudine-resistant mutant hepatitis B virus in clinical blood samples by using DNA reverse dot hybridization technology. The invention further relates to kits for this clinical assay.

Background of the invention

Hepatitis B virus (HBV) is the main pathogen causing viral hepatitis. Hepatitis B caused by HBV is a serious infectious disease. At present, there are at least 350 million chronic HBV-infected persons in the world, and China, as a high prevalence area of hepatitis B, has about 9% of the total population (about 120 million) as chronic HBV-infected persons. Persistent chronic HBV infection can be clinically manifested as various types of diseases, including asymptomatic carrier status, acute or chronic hepatitis, liver cirrhosis, etc., and severe cases may develop into primary liver cancer (HCC). At present, 1.2 million people die from diseases caused by HBV infection every year in the world (Maynard, Vaccine, 1990, R (Suppl): S18-S20; Clarke and Bloor, J Clin Virol, 2002, 25: S41-S45.).

The HBV genome contains 4 transcription units, each of which constitutes an independent open reading frame (ORF), respectively P, X, C, S, and the total length of these 4 open reading frames is 4.7Kb. The P open reading frame is 2532bp long and encodes the P protein (i.e. HBV DNA polymerase) of 844 amino acids, which is the main functional unit of virus replication and the main target of antiviral drug development. The P protein includes 4 functional regions (Lanford et al, J.Virol., 1999, 73: 1885-1893; Locarnini SA., Hepatology, 1998, 27: 294-297), starting from the amino terminal: (1) terminal Protein region (TP), which binds to negative-strand DNA during reverse transcription; (2) spacer region (SD), which has tolerance mutations, and deletion does not affect the activity of polymerase; (3) reverse transcriptase Region (RT), which contains the sequence necessary to maintain reverse transcriptase activity, and this region contains 5 functional conserved sequences A-E: A (AA421-436), B (AA506-528), C (AA546-550) , D (AA576-589) and E (AA592-600), wherein A, C, and D are the binding domains of reverse transcriptase and nucleoside triphosphates, and B and E are RNA templates and primer positioning domains; (4) RNase H district.

The main action sites of nucleoside analogue drugs currently used clinically are the B and C conserved sequences located in the reverse transcriptase region of HBV DNA polymerase. The catalytically active region of HBV polymerase is the YMDD motif located in the C conserved sequence (Hilleman, AIDS Res. Hum. Retroviruses, 1994, 10(11): 1409-1419). Although the catalytic structure of HBV polymerase seldom changes under natural conditions, it is very easy to change after long-term drug treatment, leading to drug resistance in drug users.

Lamivudine is currently the safest and most effective nucleoside analogue drug for inhibiting hepatitis B virus replication. Its principle is to inhibit the reverse transcription of HBV pre-genomic RNA into negative-strand DNA and Terminate DNA chain extension and effectively inhibit HBV DNA replication (Lai et al., N Engl J Med, 1998, 339: 61-68) (Cammack et al., Biochem.Pharmacol., 1992, 43 (10): 2059- 2064), reduce HBV DNA levels. However, short-term lamivudine treatment cannot completely eliminate HBV DNA, and lamivudine has no direct effect on covalently closed circular DNA (cccDNA) of HBV. Most patients need long-term antiviral treatment to deplete the cccDNA pool in the body To achieve the purpose of eliminating HBV (Moraleda et al. J Viral, 1997, 71: 9392-9399), and after long-term treatment with lamivudine, the virus may develop drug resistance under the pressure of drugs and human immune selection. Studies have shown that lamivudine resistance mutations in most hepatitis B viruses are mainly concentrated in the tyrosine-methionine-aspartic acid-aspartic acid (YMDD) motif of viral DNA polymerase, showing A base substitution occurs for the nucleotide encoding methionine (ATG→ATT/GTG), so that the encoded amino acid undergoes a corresponding change (M→I/V). Mutations lead to decreased efficacy of lamivudine, increased serum viral titers and elevated alanine aminotransferase (ALT) activity. Therefore, regular monitoring of viral genome changes during lamivudine treatment will be of great clinical significance.

The YMDD motif is required for HBV DNA polymerase activity and is a highly conserved region of HBV reverse transcriptase. Sequencing results found that lamivudine resistance mutations usually occur in the YMDD motif and its adjacent regions (Marchelle IA. et al, Hepatology, 1998, 27: 1670-1677), mainly manifested in amino acid 552 of DNA polymerase The M mutation in the gene sequence is I/V, and the ATG mutation in the corresponding gene sequence is ATT/GTG. There is now sufficient evidence that the M552V mutation is the major form of lamivudine resistance mutation. At present, the detection methods of drug-resistant mutations mainly include the following: mass spectrometry (Pyo Hong et al., J Hepatol., 2004, 40 (5): 837-844), fluorescence polarization (Bai et al., World J. Gastroenterol , 2003, 9(10): 2344-2347), PCR-peptide nucleic acid (Ohishi and Chayama, Proc.Natl.Acad.Sci.USA, 1989, 86: 7059-7062), restriction fragment length polymorphism method RFLP ( Sun Jian, Hou Jinlin, Xiao Lei et al., Chinese Journal of Experimental and Clinical Virology, 2003, 17(1): 18-20), Linear Probe Analysis (LiPA) (Stuyver et al., J.Clin.Microbiol., 2000, 38: 702-707), fluorescent quantitative PCR melting curve analysis (Whalley, et al, J.Clin.Microbiol., 2001, 39: 1456-1459) and gene chip technology (JeongH, et al, J.Korean Med. Sci., 2004, 19:541-546). However, most of these methods are expensive and cumbersome to operate, which is not conducive to the implementation of the majority of primary hospitals. Therefore, the inventors tried to combine the known nucleic acid dot blot hybridization technique and filter hybridization method to establish a method capable of detecting and analyzing the lamivudine-resistant mutant genotype of hepatitis B virus in a short time.

purpose of invention

An object of the present invention is to provide a method for detecting wild-type and mutant hepatitis B virus, in particular to provide a method for using DNA reverse dot hybridization (RDB) technology to identify and distinguish wild-type and Lamif in clinical blood samples A method for determining resistance to mutant hepatitis B virus, the method comprising the following steps: (1) providing a serum sample to be tested and extracting DNA therefrom; (2) using synthetic specific oligonucleotide primers, utilizing polymerase chain reaction technology Amplify target DNA fragments containing tyrosine-methionine-aspartic acid-aspartic acid (YMDD) motifs and mutant forms thereof; (3) make labeled target DNA with specific oligonucleotides Acid probe hybridization; (4) After the hybridization membrane is developed, analyze and judge the existence and relative amount of the hepatitis B virus lamivudine resistance mutation according to the color development result, which is characterized in that in the polymerase chain reaction amplification system The forward and reverse primers used were:

(1) 5'-TGCTGCTATGCCTCATCT-3' (SEQ ID NO: 1)

(2) Biotin-5'-CAGAGACAAAAGAAAATT-3' (SEQ ID NO: 2),

The 5' end of the reverse primer is labeled with biotin, and the 5' amino-labeled oligonucleotide probes used for hybridization are:

(3) NH ₂ -5'-TTTCAGTTATATGGATGATGT-3' (SEQ ID NO: 3);

(4) NH ₂ -5'-TTTCAGTTATATTGATGATGT-3' (SEQ ID NO: 4);

(5) NH ₂ -5'-TTTCAGTTATGTGGATGATGT-3' (SEQ ID NO: 5),

And 5' end biotin-labeled and 3' end amino-labeled chromogenic control oligonucleotide probe 6:

(6) Biotin-5'-GCCGTAACCGTCACAATCCGT-3'-NH ₂ (SEQ ID NO: 6).

According to a preferred embodiment of the present invention, wherein the hybridization process is carried out in a nucleic acid molecular hybridization device equipped with a porous filter plate and a vacuum suction unit.

According to a preferred embodiment of the present invention, wherein said marker molecule is biotin.

According to a preferred embodiment of the present invention, wherein said hepatitis B virus is from any known variant of hepatitis B virus in the serum of the subject to be tested.

According to a preferred embodiment of the present invention, said mutation is a mutation of wild-type hepatitis B virus to lamivudine-resistant hepatitis B virus.

According to a preferred embodiment of the present invention, wherein said wild-type hepatitis B virus has a tyrosine-methionine-aspartic acid-aspartic acid (YMDD) motif.

According to a preferred embodiment of the present invention, wherein said lamivudine-resistant mutant hepatitis B virus has tyrosine-isoleucine-aspartic acid-aspartic acid (YIDD) or tyrosine Acid-valine-aspartic acid-aspartic acid (YVDD) motif.

Another object of the present invention is to provide a test kit for detecting lamivudine resistant mutant hepatitis B virus, which kit includes (1) nucleic acid extraction reagent; (2) polymerase chain reaction reagent; (3) Hybridization membrane and reverse dot hybridization reaction reagent, it is characterized in that forward and reverse primer (primer 1 and 2) used in the polymerase chain reaction amplification system are respectively:

(1) 5'-TGCTGCTATGCCTCATCT-3' (SEQ ID NO: 1);

(2) Biotin-5'-CAGAGACAAAAGAAAATT-3' (SEQ ID NO: 2),

Where the 5' end of the reverse primer is labeled with biotin, the 5' amino-labeled oligonucleotide probe used for hybridization is:

(3) NH ₂ -5'-TTTCAGTTATATGGATGATGT-3' (SEQ ID NO: 3);

(4) NH ₂ -5'-TTTCAGTTATATTGATGATGT-3' (SEQ ID NO: 4);

(5) NH ₂ -5'-TTTCAGTTATGTGGATGATGT-3' (SEQ ID NO: 5),

As well as 5′ biotin-labeled and 3′ amino-labeled chromogenic control oligonucleotide probes for monitoring the hybridization process are:

(6) Biotin-5'-GCCGTAACCGTCACAATCCGT-3'-NH ₂ (SEQ ID NO: 6).

According to a preferred embodiment of the present invention, wherein said nucleic acid extraction reagent is a DNA extraction solution.

According to a preferred embodiment of the present invention, wherein said polymerase chain reaction reagent is made of polymerase chain reaction (PCR) buffer, upstream primer, downstream primer, deoxyuridine triphosphate (dUTP) including deoxyuridine A polymerase chain reaction system consisting of ribonucleoside triphosphates (dNTPs), thermostable Taq enzyme and uracil-DNA-glycosylase (UDG enzyme), and positive standards (wild-type and mutant HBV DNA plasmids).

According to a preferred embodiment of the present invention, said reverse dot hybridization reaction reagents include hybridization solutions I and II, solutions I, II, III and IV and standard controls.

According to yet another preferred embodiment of the present invention, wherein the forward and reverse primers used for the target polynucleotide amplification reaction are respectively primer 1: (1) 5'-TGCTGCTATGCCTCATCT-3' (SEQ ID NO: 1) and Biotin-labeled primer 2: (2) Biotin-5'-CAGAGACAAAAGAAAATT-3' (SEQ ID NO: 2).

According to another preferred embodiment of the present invention, the oligonucleotide probes labeled with the 5' terminal amino group for nucleic acid hybridization are respectively:

(3) NH ₂ -5'-TTTCAGTTATATGGATGATGT-3' (SEQ ID NO: 3);

(4) NH ₂ -5'-TTTCAGTTATATTGATGATGT-3' (SEQ ID NO: 4);

(5) NH ₂ -5'-TTTCAGTTATGTGGATGATGT-3' (SEQ ID NO: 5),

And the chromogenic control oligonucleotide probes used to monitor the hybridization process are:

(6) Biotin-5'-GCCGTAACCGTCACAATCCGT-3'-NH ₂ (SEQ ID NO: 6).

Contents of the invention

The present invention provides a method for detecting lamivudine-resistant mutant hepatitis B virus, and in particular provides a method for quickly and accurately distinguishing wild-type and lamivudine-resistant in clinical blood samples using DNA reverse dot hybridization technology A method for mutating hepatitis B virus. The present invention further provides a kit for clinical lamivudine resistance mutation detection.

In order to overcome the defects and deficiencies in the prior art, the present invention provides a method suitable for detecting hepatitis B virus lamivudine drug-resistant mutations, the method comprising: (1) providing a serum sample to be tested and extracting DNA therefrom; (2) Using synthetic oligonucleotide primers, carry out conventional polymerase chain reaction to DNA containing tyrosine-methionine-aspartic acid-aspartic acid (YMDD) respectively to the serum sample and control sample DNA ) motif and its mutant target DNA fragments; (3) hybridize the labeled target DNA with the specific oligonucleotide probe; (4) after the biotin-labeled fragment is combined with the chromogenic reaction group, hybridize the membrane After developing the color, analyze and judge the existence of the hepatitis B virus lamivudine drug-resistant mutation and its relative amount according to the result of the color development. and reverse primers are:

(1) 5'-TGCTGCTATGCCTCATCT-3' (SEQ ID NO: 1);

(2) Biotin-5'-CAGAGACAAAAGAAAATT-3' (SEQ ID NO: 2).

According to a preferred embodiment of the present invention, the 5' terminal amino-labeled oligonucleotide probes used for nucleic acid hybridization are respectively:

(3) NH ₂ -5'-TTTCAGTTATATGGATGATGT-3' (SEQ ID NO: 3);

(4) NH ₂ -5'-TTTCAGTTATATTGATGATGT-3' (SEQ ID NO: 4);

(5) NH ₂ -5'-TTTCAGTTATGTGGATGATGT-3' (SEQ ID NO: 5),

And the chromogenic control oligonucleotide probes used to monitor the hybridization process are:

(6) Biotin-5'-GCCGTAACCGTCACAATCCGT-3'-NH ₂ (SEQ ID NO: 6).

According to another preferred embodiment of the present invention, wherein said serum sample to be tested is from the serum of a subject infected with hepatitis B virus.

According to another preferred embodiment of the present invention, the polymerase chain reaction conditions for amplifying the nucleic acid to be detected in the specimen are pretreatment at 50°C for 3 minutes, pre-denaturation at 95°C for 5 minutes, and then 93°C for 30 seconds, 55°C 30 seconds, 40 seconds amplification at 72°C, a total of 35 cycles; the last 72°C incubation for 7 minutes.

According to yet another preferred embodiment of the present invention, wherein the nucleic acid hybridization process is carried out in a nucleic acid molecular hybridization device equipped with a porous filter plate and a vacuum suction unit.

According to yet another preferred embodiment of the present invention, wherein said marker molecule is biotin or amino.

According to yet another preferred embodiment of the present invention, the genotypes of the target DNA comprising the YMDD motif include wild-type (YMDD) and lamivudine-resistant mutants (YIDD or YVDD).

As we all know, the commonly used blot hybridization method is to immobilize the target DNA on a nylon membrane or nitrocellulose membrane, use a labeled probe to contact and hybridize with the target DNA to be detected, and determine the result after developing or developing color. With this method, each hybridization reaction can only detect whether a DNA to be tested contains the complementary sequence of a certain probe, that is, only one genotype can be judged at a time. If a gene locus has multiple alleles (such as HLA-A, HLA-B, HLA-DR loci) or to detect multiple target sequences (such as thalassemia mutation genes, ABO blood type, etc.), use this point The one-time detection of the hybridization method is very complicated and even difficult to complete. The reverse dot hybridization method is to design specific probes for each allele first, and add the probe points to the hybridization matrix, and then the DNA sample to be tested (usually using a 5'-end labeled biological PCR amplification of the target DNA fragment with primers such as digoxigenin or digoxigenin) to hybridize with it, so that the sample to be tested will bind to the probe with a complementary sequence, and after washing to remove unbound DNA, it can be detected specific binding target sequence. Therefore, using this method, it is possible to simultaneously detect normality at a certain site and multiple related mutant forms, or normality or mutation at different sites.

In order to complete the method of the present invention, the serum sample to be tested is first routinely separated and prepared, and stored at -20°C for future use. For detection, take about 40 μl of serum sample, add an equal amount of DNA extraction solution, mix well, and then bathe in boiling water for about 10 minutes. In order to fully lyse the virus particles contained in the specimen, if necessary, the mixture of serum specimen and DNA extraction solution can be transferred to 4°C and allowed to stand for 8-12 hours. After centrifugation, the supernatant was used for polymerase chain reaction (PCR). The 50 μl reaction system used included: 1× qualitative PCR buffer, 0.2mM dNTPs, 2U Taq enzyme, 0.2 μM primers 1 (SEQ ID NO: 1) and 2 (SEQ ID NO: 2). In the presence of UDG enzyme (1U), carry out conventional PCR reaction on the processed specimen or the standard for subsequent parallel detection. The reaction conditions used are: pretreatment at 50°C for 3 minutes, pre-denaturation at 94°C for 5 minutes, followed by amplification at 93°C for 30 seconds, 55°C for 30 seconds, and 72°C for 40 seconds, a total of 35 cycles; the final incubation at 72°C 7 minutes. When hybridizing, first denature the PCR amplification product at 95°C for 5 minutes, then place it in ice and cool it for more than 2 minutes, and then in the presence of preheated hybridization solution I at about 44°C, place it in a vacuum chamber equipped with a porous filter plate and a negative pressure suction pump. In the hybridization tank of the nucleic acid molecular rapid hybridization instrument, the PCR amplification product was hybridized with the 5′-terminal amino-labeled probe for about 7 minutes. The probes used were:

(3) NH ₂ -5'-TTTCAGTTATATGGATGATGT-3' (SEQ ID NO: 3)

(4) NH ₂ -5'-TTTCAGTTATATTGATGATGT-3' (SEQ ID NO: 4)

(5) NH ₂ -5'-TTTCAGTTATGTGGATGATGT-3' (SEQ ID NO: 5),

After washing (three times) the hybridization membrane with 44°C preheated hybridization solution II, the PCR amplification product and the color control probe (6) Biotin-5'-GCCGTAACCGTCACAATCCGT-3'-NH ₂ (SEQ ID NO : 6) React with the binding solution (25°C) for about 2 minutes. After the hybridization membrane was washed (three times) with hybridization solution I, a freshly prepared chromogenic solution was added to develop color for about 6 minutes. After vacuum-drying the developing solution, observe the spot color on the hybridization membrane to determine the existence and relative amount of the hepatitis B virus lamivudine drug-resistant mutation site in the specimen. Within the color development time, a clearly visible blue circular spot on the hybridization membrane is a positive test result, and colorless or very light color development is a negative result.

In the method of the present invention, since the primers designed for various types of HBV nucleic acid sequences are used to amplify the target nucleic acid sequence, and the chromogenic control probe and the oligonucleotide for detecting wild-type YMDD and mutant YIDD/YVDD motifs are designed Nucleic acid hybridization analysis with nucleotide probes, so this method can accurately and effectively identify and distinguish HBV wild-type YMDD and its lamivudine-resistant mutants YIDD and YVDD. Moreover, the method of the present invention can not only be used to detect single HBV infection, but also qualitatively and quantitatively detect mixed infection or mutation of HBV, thereby providing molecular virological evidence for clinical individualized treatment of hepatitis B, and providing effective clinical medicine. meaningful reference. On the other hand, since the method of the present invention uses a nucleic acid molecular hybridization device with a unique principle of diversion hybridization, the diffusion rate of nucleic acid molecules, the concentration of local reactions and their hybridization efficiency are greatly improved. Generally speaking, using the method of the present invention, it only takes 2-3 hours at the fastest from sample processing to result interpretation (traditional hybridization method needs at least 2 working days). Therefore, compared with the traditional hybridization method, the invention of the method obviously has the advantages of simple operation, rapidity and convenience.

Another object of the present invention is to provide a test kit for detecting lamivudine resistant mutant hepatitis B virus, which kit includes (1) nucleic acid extraction reagent; (2) polymerase chain reaction reagent; (3) The hybridization membrane and reverse dot hybridization reaction reagent are characterized in that the forward and reverse primers used in the polymerase chain reaction amplification system are respectively:

(1) 5'-TGCTGCTATGCCTCATCT-3' (SEQ ID NO: 1)

(2) Biotin-5'-CAGAGACAAAAGAAAATT-3' (SEQ ID NO: 2),

The 5' end of the reverse primer was labeled with biotin.

The 5′ amino-labeled oligonucleotide probes used for hybridization are:

(3) NH ₂ -5'-TTTCAGTTATATGGATGATGT-3' (SEQ ID NO: 3);

(4) NH ₂ -5'-TTTCAGTTATATTGATGATGT-3' (SEQ ID NO: 4);

(5) NH ₂ -5'-TTTCAGTTATGTGGATGATGT-3' (SEQ ID NO: 5);

And 5'-end biotin-labeled and 3'-end amino-labeled chromogenic control oligonucleotide probe 6:

(6) Biotin-5'-GCCGTAACCGTCACAATCCGT-3'-NH ₂ (SEQ ID NO: 6).

According to a preferred embodiment of the present invention, wherein said nucleic acid extraction reagent is a DNA extraction solution.

According to a preferred embodiment of the present invention, wherein said polymerase chain reaction reagent comprises PCR buffer, upstream primer (primer 1), downstream primer (primer 2), deoxyuridine nucleoside triphosphate (dUTP) in The PCR reaction system composed of deoxyribonucleoside triphosphates (dNTPs), thermostable Taq enzyme and uracil-DNA-glycosylase (UDG enzyme), and positive standards wild-type (YMDD) and mutant (YIDD and YVDD) HBV positive plasmid.

According to a preferred embodiment of the present invention, said reverse dot hybridization reaction reagents include hybridization solutions I and II, solutions I, II, III and IV and standard controls.

According to yet another preferred embodiment of the present invention, wherein the forward and reverse primers used in the target DNA amplification reaction are respectively primer 1: (1) 5'-TGCTGCTATGCCTCATCT-3' (SEQ ID NO: 1) and biotin Labeled primer 2: (2) Biotin-5'-CAGAGACAAAAGAAAATT-3' (SEQ ID NO: 2).

According to another preferred embodiment of the present invention, the oligonucleotide probes labeled with the 5' terminal amino group for nucleic acid hybridization are:

(3) NH ₂ -5'-TTTCAGTTATATGGATGATGT-3' (SEQ ID NO: 3)

(4) NH ₂ -5'-TTTCAGTTATATTGATGATGT-3' (SEQ ID NO: 4)

(5) NH ₂ -5'-TTTCAGTTATGTGGATGATGT-3' (SEQ ID NO: 5),

And the chromogenic control oligonucleotide probes used to monitor the hybridization process are:

(6) Biotin-5'-GCCGTAACCGTCACAATCCGT-3'-NH ₂ (SEQ ID NO: 6).

The judging principle of the result is that the chromogenic control probe 6 (SEQ ID NO: 6) used to monitor the hybridization process should be chromogenic in all detections. If probes 3 (SEQ ID NO: 3), 4 (SEQ ID NO: 4), and 5 (SEQ ID NO: 5) show a single color, it means that there are YMDD wild type, YIDD mutant type, and YVDD mutant type in the sample, respectively. Existence of single HBV DNA; If probe 3 (SEQ ID NO: 3) and 4 (SEQ ID NO: 4) develop color simultaneously, then show that there is YMDD wild type and YIDD mutant mixed infection in the sample; As probe 3 (SEQ ID NO: 4) ID NO: 3) and 5 (SEQ ID NO: 5) develop color simultaneously, then show that there is YMDD wild type and YVDD mutant type mixed infection in the sample; Such as probe 4 (SEQ ID NO: 4) and 5 (SEQ ID NO: 5) Simultaneous color development indicates that there is mixed infection of YVDD and YIDD mutant HBV in the sample; such as probe 3 (SEQ ID NO: 3), 4 (SEQ ID NO: 4), 5 (SEQ ID NO: 5) Simultaneous color development of the three probes indicates that mixed infection of YMDD wild type, YIDD and YVDD mutant HBV exists in the sample at the same time.

The kit of the invention is mainly used for detecting and distinguishing wild-type (YMDD) and lamivudine-resistant mutants (YIDD and YVDD) HBV DNA, so as to judge the clinical typing of HBV lamivudine-resistant virus. The entire detection process includes the main steps of extracting HBV DNA in serum samples, PCR amplification of target DNA fragments containing YMDD/YIDD/YVDD motifs, and hybridization of PCR amplification products. In order to complete these steps, the kit of the present invention can be further divided into two parts, a PCR reaction kit and a reverse dot hybridization kit. Wherein, the former includes DNA extract, Taq enzyme, positive standard (YMDD wild type and YIDD/YVDD mutant HBV DNA plasmid) and uracil-DNA-glycosylase (UDG enzyme). The latter includes hybridization solutions I and II, solutions I, II, III and IV, and hybridization membranes. Reagents for hybridization are mainly conventional 20×SSC, sodium dodecylsulfonate (SDS), streptavidin-horseradish peroxidase conjugate (POD), and tetramethylbenzidine (TMB). The kit consists of hybridization solution I (2×SSC-0.1% SDS), hybridization solution II (0.5×SSC-0.1% SDS), solution I (250u/mlPOD), solution II (0.1mol/L sodium citrate), Solution III (2 mg/ml TMB), Solution IV (3% H ₂ O ₂ ), negative and positive standard controls, and membrane strips used as carriers for the hybridization reaction.

The kit of the present invention not only provides specific probes corresponding to various types of HBV, but also provides chromogenic control probes and positive standards for wild-type and mutant HBV, so that the kit has a complete quality control system . At the same time, because it is equipped with a nucleic acid molecular hybridization device with a unique flow-through hybridization system, it is extremely beneficial to the diffusion of nucleic acid molecules and the increase of local reaction concentration to improve the efficiency of nucleic acid hybridization. Generally speaking, it only takes about 3 hours from sample processing to result judgment. Especially due to the use of carefully designed nucleic acid probes, the detection of lamivudine-resistant HBV has better sensitivity and specificity. Therefore, test kit of the present invention provides new tool for the rapid detection of clinical specimen and large sample census (one can detect 15 people's parts or more samples), and provides reliable laboratory for the clinical use of lamivudine in accordance with.

Description of drawings

Fig. 1 shows the electrophoresis pattern of target DNA amplified fragments by 2% agarose gel. Wherein the 1st, 2nd and 3 swimming lanes are the specific target DNA fragments (233bp) amplified from the serum HBV DNA; the 4th, 5th and 6 swimming lanes are the specific target DNA fragments (233bp) amplified from the plasmid DNA ); M lane is the standard molecular weight mark.

Figure 2 shows the detection results of six different HBV genotype serum samples by reverse dot blot hybridization. Among them: A: YMDD infection, B: YIDD infection, C: YVDD infection, D: YMDD, YIDD mixed infection, E: YMDD, YVDD mixed infection, F: YIDD, YVDD mixed infection.

The specific embodiment of the invention:

Embodiment 1: the extraction of serum DNA

Aseptically collect about 1ml of venous blood from the hepatitis B patient to be tested, let it stand at room temperature and 4°C for 2 hours and 1 hour respectively, then centrifuge (8,000rpm, 5 minutes) to separate and collect 200μl of serum sample. Then take 40 μl of serum sample, add an equal amount of DNA extraction solution to it, mix thoroughly and place in a boiling water bath for 10 minutes. In order to ensure that the virus particles contained in the serum can be fully lysed, the mixture was further left to stand at 4° C. for 10 hours. It was then centrifuged (10,000 rpm, 5 minutes) and 2 μl of the supernatant was collected and used for further PCR reactions.

Embodiment 2: PCR amplification of the fragment comprising YMDD motif

Take several tubes of single-person PCR reaction solution, add 1 μl UDG enzyme to each tube, and then directly add 2 μl template (or negative and positive standards), mix well and then centrifuge briefly (3 seconds). Then put each reaction tube into the PCR instrument, pretreat at 50°C for 3 minutes, and amplify according to the following conditions: 94°C for 5 minutes, then press 93°C for 30 seconds, 55°C for 30 seconds, and 72°C for 40 seconds, a total of 35 cycles, A final 72°C extension was performed for 7 minutes. The amplified product was detected by 2% agarose gel electrophoresis (see Figure 1). The PCR amplification system used included: 1× qualitative PCR buffer, 0.2mM dNTPs, 2U Taq enzyme, 0.2μM primers 1 (SEQ ID NO: 1) and 2 (SEQ ID NO: 2).

Example 3: Reverse dot blot detection of samples of three known genotypes

Before hybridization, preheat hybridization solution I (2×SSC-0.1% SDS) and hybridization solution II to 44°C for use. Take three 1.5ml centrifuge tubes according to the number of samples to be tested, add 0.5ml hybridization solution I to each tube and preheat to 44°C. After the PCR amplification product was denatured at 95° C. for 10 minutes, it was placed in an ice-water mixture for 4 minutes. Then take 1000 μl hybridization solution I+2 μl solution I (1000:2) mixed solution as the binding solution and store it at 4°C for later use, and take the mixture of solution II: solution III: solution IV (1900:200:1) (1.9ml solution II+ 0.2ml of solution III + 1 μl of solution IV) is used as a chromogenic solution and stored away from light for later use.

Before hybridization, fill the reaction chamber with distilled water, place the metal perforated plate, and turn on the negative pressure pump to discharge the water on the perforated plate. Set the temperature of the hybridization instrument at 44°C, first place the plastic gasket on the metal porous plate and place the drug resistance detection membrane strip on the plastic gasket, and cover the silica gel compartment and the snap-on cover. Then add the denatured PCR amplification product into 0.5ml hybridization solution I preheated at 44°C, mix well, add it to the hybridization tank and incubate for 7 minutes, and turn on the pump for diversion hybridization. Then add hybridization solution II preheated at 44°C to wash the membrane strip (1 ml per well, repeat washing 3 times). Set the temperature of the hybridization instrument to 25°C (or room temperature) and add binding solution (1ml per well). After standing for 2 minutes, pump out the binding solution, and then add room temperature hybridization solution I to wash the membrane strip (1ml per well, repeat washing 3 times). Then add solution II and let it stand for a while, then pump dry, and finally add freshly prepared chromogenic solution (1ml per well), develop color for about 6 minutes and turn on the pump to drain the chromogenic solution to observe the results (see Figure 2).

From the results shown in Figure 2, it can be seen that the color development of each probe is clearly visible, and there is no non-specific hybridization.

Example 4: Reverse Dot Hybridization Genotyping Detection of Different Genotype Samples and Judgment of Results

Using the method and kit of the present invention, 49 serum samples of highly suspected drug-resistant hepatitis B patients collected from Guangzhou Eighth People's Hospital were detected for HBV drug-resistant mutations. Results: YMDD (4 cases), YIDD (2 cases), YVDD (10 cases), YMDD+YIDD (1 case), YMDD+YVDD (26 cases), YIDD+YVDD (2 cases), YMDD+YIDD+ YVDD (4 cases). In order to further confirm the accuracy of the method of the present invention, the PCR products of all samples were sequenced. The result shows that the detection result using the kit of the present invention is basically consistent with the sequencing result, and the specificity reaches 97.1%.

Embodiment 5: the clinical application of the inventive method

The invention of the method can provide scientific and individualized molecular biology experiment data for formulation of clinical lamivudine medication regimen, and provide reliable molecular virological evidence for the treatment of hepatitis B patients. If there is a lamivudine-resistant mutant virus in the patient's serum, the dosage of lamivudine should be immediately reduced or even stopped, and other drugs should be used for treatment, so as to avoid wasting time and money, and avoid delaying the disease and treatment. unwanted consequences. The result interpretation of the present invention can have seven kinds of situations:

Probes 6 (SEQ ID NO: 6) and 3 (SEQ ID NO: 3) were determined to be YMDD wild-type infection by color development;

Probes 6 (SEQ ID NO: 6) and 4 (SEQ ID NO: 4) were determined to be YIDD mutant infection by color development;

Probes 6 (SEQ ID NO: 6) and 5 (SEQ ID NO: 5) were determined to be YVDD mutant infection by color development;

The color development of probes 6 (SEQ ID NO: 6), 3 (SEQ ID NO: 3) and 4 (SEQ ID NO: 4) can be judged as mixed infection of YMDD wild type and YIDD mutant type;

The color development of probes 6 (SEQ ID NO: 6), 3 (SEQ ID NO: 3) and 5 (SEQ ID NO: 5) can be judged as mixed infection of YMDD wild type and YVDD mutant type;

The color development of probes 6 (SEQ ID NO: 6), 4 (SEQ ID NO: 4) and 5 (SEQ ID NO: 5) can be judged as mixed infection of YIDD and YVDD mutants;

Probes 6 (SEQ ID NO: 6), 3 (SEQ ID NO: 3), 4 (SEQ ID NO: 4) and 5 (SEQ ID NO: 5) can be judged as YMDD wild type, YIDD and YVDD mutant Mixed infection (see Table 1 and Figure 2).

Table 1 Seven different HBV infection conditions and their judgment sample number Chromogenic Probe Set Virus infection 1 6 (SEQ ID NO: 6) and 3 (SEQ ID NO: 3) YMDD infection 2 6 (SEQ ID NO: 6) and 4 (SEQ ID NO: 4) YIDD infection 3 6 (SEQ ID NO: 6) and 5 (SEQ ID NO: 5) YVDD infection 4 6 (SEQ ID NO: 6), 3 (SEQ ID NO: 3), 4 (SEQ ID NO: 4) YMDD+YIDD mixed infection 5 6 (SEQ ID NO: 6), 3 (SEQ ID NO: 3), 5 (SEQ ID NO: 5) YMDD+YVDD mixed infection 6 6 (SEQ ID NO: 6), 4 (SEQ ID NO: 4), 5 (SEQ ID NO: 5) YIDD+YVDD mixed infection 7 6 (SEQ ID NO: 6), 3 (SEQ ID NO: 3), 4 (SEQ ID NO: 4), 5 (SEQ ID NO: 5) YMDD+YIDD+YVDD mixed infection

Sequence Listing

<110> Sun Yat-sen University Anda Gene Co., Ltd.

<120> Detection method of hepatitis B virus genome drug resistance mutation

<140>

<141>

<160>6

<210>1

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> is designed according to the specific nucleotide sequence to be used as a primer for PCR amplification.

<400>1

TGCTGCTATG CCTCATCT

<210>2

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> is designed according to the specific nucleotide sequence to be used as a primer for PCR amplification.

<400>2

CAGAGACAAA AGAAAAATT

<210>3

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> is designed according to a specific nucleotide sequence to be used as a hybridization probe.

<400>3

TTTCAGTTAT ATGGATGATG T

<210>4

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> is designed according to a specific nucleotide sequence to be used as a hybridization probe.

<400>4

TTTCAGTTAT ATTGATGATG T

<210>5

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> is designed according to a specific nucleotide sequence to be used as a hybridization probe.

<400>5

TTTCAGTTAT GTGGATGATG T

<210>6

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> is designed according to a specific nucleotide sequence to be used as a hybridization probe.

<400>6

GCCGTAACCG TCACAATCCG T

Claims (2)

1. A kit for detecting lamivudine-resistant mutant hepatitis B virus, which includes (1) polymerase chain reaction reagents, (2) hybridization membrane and reverse dot hybridization reagents; wherein (1) ) consists of DNA extraction solution, heat-resistant Taq enzyme, positive standard and uracil-DNA-glycosylase; (2) consists of hybridization solution I and II, solutions I, II, III and IV and hybridization membrane; its The forward and reverse primers characterized for use in the target polynucleotide amplification reaction are, respectively: (1) 5'-TGCTGCTATGCCTCATCT-3' (SEQ ID NO: 1) (2) Biotin-5'-CAGAGACAAAAGAAAATT-3' (SEQ ID NO: 2). 2, according to the test kit of claim 1, it is characterized in that the oligonucleotide probe that also increases the 5' terminal amino label of molecular hybridization is: (3) NH2-5'-TTTCAGTTATATGGATGATGT-3' (SEQ ID NO: 3); (4) NH2-5'-TTTCAGTTATATTGATGATGT-3' (SEQ ID NO: 4); (5) NH2-5'-TTTCAGTTATGTGGATGATGT-3' (SEQ ID NO: 5); And the 5′ biotin-labeled and 3′ amino-labeled oligonucleotide probes used for color control are: (6) Biotin-5'-GCCGTAACCGTCACAATC CGT-3'-NH2 (SEQ ID NO: 6).

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