Hepatitis B virus mutant, mutant amplification kit and use thereof

Abstract

The present invention relates to the field of biotechnology, and discloses a hepatitis B virus mutant, a mutant amplification kit and use thereof. The HBV mutant of the invention takes place mutation of the hepatitis B virus mutant in genome sequence No. 216 and/or 285, the mutation occurred at position 216 is that base T mutates to C, the mutation occurred at position 285 is that base G mutates to A. The two mutations are closely related to the inflammation aggravation of the hepatitis B. The invention further provides a HBV mutant amplification kit to detect the above mutation, the usage of the kit and its application, therefore offering help for the clinical early diagnosis and intervention of severe hepatitis B, and providing references for further studying the functional changes caused by HBV gene mutation.

Claims

1. A kit for identifying hepatitis B (HBV) genetic mutants, wherein the kit comprises: a) two primers, wherein the two primers consist of the sequences of SEQ ID NO:8 and SEQ ID NO:9, b) dNTP, c) PCR buffer, d) DNA polymerization enzyme, and e) ddH.sub.20, wherein the kit provides for the identification of mutations in the HBV genome at positions 216 and/or 285 relative to SEQ ID NO:1, SEQ ID NO:10, or SEQ ID NO:11.

2. The kit according to claim 1, wherein the DNA polymerization enzyme is a high fidelity DNA polymerization enzyme.

3. The kit according to claim 1, wherein the amplification procedure of the kit is: a) pre-denaturation at 94 C. for 3 minutes, b) amplification for 45 cycles of the following: I) 94 C. for 30 seconds II) 57 C. for 15 seconds, III) 72 C. for 2 minutes, and after the amplification cycles are complete, c) extending for 72 C. for 10 minutes.

4. A method for early clinical diagnosis and prevention of severe hepatitis B disease by determining whether a subject's blood comprises a hepatitis B virus (HBV) mutant, wherein the hepatitis B virus mutant comprises a mutation of the HBV genome at position 216 and/or 285 of the HBV according to SEQ ID NO:1, SEQ ID NO:10, or SEQ ID NO:11, wherein said mutation at position 216 is a T to C substitution and wherein, said mutation at position 285 is a G to A substitution, wherein the steps to detect whether the subject's blood comprises said HBV mutant are: 1) preparation of the HBV genomic DNA template by drawing the peripheral venous blood of the test subject and extracting HBV genomic DNA from said blood; 2) amplifying the DNA template acquired in step 1) through PCR amplification in a PCR reaction system to amplify the gene fragments comprising position 216 and 285 from the HBV genome to obtain a PCR product, wherein the PCR reaction system comprises an upstream and downstream primer pair, wherein the upstream and downstream primer pair are SEQ ID NO:8 and 9, respectively; 3) purifying the resulting PCR product from step 2; 4) sequencing the PCR product purified and obtained from step 3); 5) analyzing the results by comparing the sequencing results of step 4) with the sequence of SEQ ID NO:1, SEQ ID NO:10, and/or SEQ ID NO:11 to confirm whether the PCR product comprises a mutation in the HBV genome at position 216 and/or 285; wherein presence of the mutation at position 216 and/or 285 allows the subject to have clinical early diagnosis and/or intervention for severe hepatitis B disease.

5. The method according to claim 4, wherein the PCR reaction system in step 2) further comprises: a) dNTP mix, b) PCR buffer, c) DNA polymerization enzyme, and d) ddH.sub.20.

6. The method according to claim 4, wherein the amplification procedure of step 2) is as follows: a) pre-denaturation at 94 C. for 3 minutes, b) amplification for 45 cycles of the following: I) 94 C. for 30 seconds II) 57 C. for 15 seconds, III) 72 C. for 2 minutes, and after the amplification cycles are complete, c) extending for 72 C. for 10 minutes.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: M1 is that the locus T in HBV 216 of a patient mutates to C, M2 is that the loci T and C coexist in HBV 216 of a patient, WT is that the locus in HBV 216 of a patient does not occur mutation.

(2) FIG. 2: M1 is that the locus G in HBV 285 of a patient mutates to A, M2 is that the loci G and A coexist in HBV 285 of a patient, WT is that the locus in HBV 285 of a patient does not occur mutation.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(3) The present invention is further illustrated by combining with the specific embodiments. It should be understood that the following embodiments are merely used to explain the invention, but are not used to limit the scope of the invention.

First Embodiment

(4) First: Serum Collection from Patients with Severe Hepatitis B and Chronic Hepatitis B

(5) Collect the serum samples of 12 cases with severe hepatitis B and 12 cases with chronic mild hepatitis B from the first affiliated hospital of medical school of Zhejiang university (the patients has been diagnosed explicitly with severe or chronic mild hepatitis B by the indicators of ALT, AST, TBIL, PT, viral titer, etc., and clinical manifestations)

(6) Second: Primer Design

(7) We download 616 complete sequences of HBV genome, and design three pairs of primers (SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7) by using primer 5.0 software according to the complete genome conserved sequence of HBV.

(8) Third: HBV DNA Extraction

(9) An equal volume of nucleic acid extract is added into the 50 ul serum and mixed, followed by water bath at 100 for 10 min, centrifugation for 10 min with 13000 r/min, and then the supernatant is drawn for PCR amplification.

(10) Fourth: PCR Amplification

(11) The amplification for the HBV gene of a patient adopts Prime Star fidelity enzymes of TaKaRa Company, with the brief steps as follows: total volume of the PCR reaction 50 L, wherein template DNA 2 L, upstream primer and downstream primer (10 uM) 1 L, respectively, dNTP (2.5 mM for each four dNTP) 4 L, 5 Prime Star buffer (Mg.sup.2+ Plus) 10 L, Prime Star (5 U/ul) 0.5 L, and dilute with ddH.sub.2O to the total volume of 50 L. Cycle parameters: predenaturation at 94 C. 3 min, then 94 C. 30 s, 57 C. 15 s, 72 C. 2 min for 45 cycles, extending at 72 C. for 10 min. The product is recycled and purified via agarose gel electrophoresis with the concentration of 1%.

(12) Meanwhile, take the expression plasmid HBV1.37 which contained HBV 1.3-fold complete sequence in laboratory as a template, to amplify the above three pieces of HBV gene segment. The product is recycled and purified via agarose gel electrophoresis with the concentration of 1%, for the quality control of the high-throughput sequencing.

(13) Fifth: Sanger Sequencing

(14) The purified PCR product performs sanger sequencing, and three fragments is pieced for a whole HBV DNA complete sequence by DNASTAR software.

(15) Sixth: Solexa High-Throughput Sequencing and Data Analysis Method

(16) Sequencing method: the purified PCR product is sequenced by nano institute, Zhejiang university, the experiment process is briefly described as follows: 1, the genomic DNA is disrupted into small fragments of 200 bp, both ends of each fragment add connectors (each sample add a specific connector). 2, DNA fragments are purified by QIAGEN gel extraction kit. 3, PCR amplifies the DNA fragment with connectors and is purified. 4, detected by Solexa high-throughput sequencing platform.

(17) Data Analysis

(18) (1) Splicing of Sequencing Fragment (Mapping)

(19) Obtain the sequence (reads, tags) data of each sample by the high-throughput sequencing, firstly the sequence data in both directions is composed into a file, and then is spliced into a complete HBV genome by using bowtie software. The Chinese HBV genome standard type B and C sequences reported in Profile of public nucleotide databases HBV complete genome sequence and establishment of Chinese HBV reference sequence (Progress in Natural Science 2008, 18 (2)) acts as splicing reference sequence (SEQ ID NO: 1, SEQ ID NO: 10, SEQ ID NO: 11), while a length of sequence is extracted before and after the gap of the linear genome to compose into a short fragment and the reference sequence is added as well, to ensure the sequence covering on the gap can be correctly spliced. Parameter is set as: seed length=30, allowable mismatch=3. Since according to the principle of high-throughput sequencing, the results of the previous test has the highest accuracy, with the sequencing length increases, the accuracy decreases, thus we choose the first 30 bases for comparison, the sequence with more than three mutations is considered as a sequence with relatively poor sequencing quality, and is removed (i.e., every 10 bases allow a mutation)

(20) (2) Statistical Tests of the Composition Difference of Bases at Each Locus

(21) Perform statistics on the sequence of the splicing file of each sample and its coordinate read in the genome, to obtain the absolute number of the four types of bases measured in all base positions of the complete genome. Then each sample of the severe hepatitis B group and mild hepatitis B group are compared in turn by using combination method. Obtain the base type with significant difference in each locus between two samples (p<0.001) by applying Chi-square test; perform statistics on the above information in all comparison pairs, and take the base type detected with significant difference in more than a half of comparison pairs, while without opposite significant proportion change of base at a certain locus in any comparison pair as the base change with significant difference between the severe hepatitis B group and mild hepatitis B group, and remains (the above steps are completed by writing a script by Python program language (www.python.org).

(22) Through the statistical analysis of the sequencing results of the severe hepatitis B and mild hepatitis B, two new mutation loci are found that: 216T.fwdarw.C and 285G.fwdarw.A. For the 12 cases of severe hepatitis patients, 10 cases exist 216T.fwdarw.C mutation, while 9 cases exist 285G.fwdarw.A mutation.

(23) Though Solexa high-throughput sequencing, it has been detected that, in the severe hepatitis patients with 216T.fwdarw.C mutation, the proportion of 216T.fwdarw.C mutant strain is 8% to 100%; while in the severe hepatitis patients with 285G.fwdarw.A mutation, the proportion of 285G.fwdarw.A mutant strain is 11% to 100%. In the 12 cases of severe hepatitis patients, only 1 case exists duplex mutation of 216T.fwdarw.C and 285G.fwdarw.A, and the proportion of 216T.fwdarw.C mutant strain and 285G.fwdarw.A mutant strain is 17% and 7%, respectively. Further, it is found that in this experiment, when the proportion of 216T.fwdarw.C mutant strain is less than 17%, and the proportion of 285G.fwdarw.A mutant strain is less than 11%, those mutant strains are unable to be detected by conventional sanger sequencing method, such that this type of case is unable to be detected as well.

(24) The results showed that, the solexa high-throughput sequencing technology features much better sensitivity of mutation detection than that of the conventional sanger sequencing, and enables to find the non-dominant strains existed in HBV patients, and is beneficial for us to study the complex quasispecies characteristics in patients. While sanger sequencing method is reliable, accurate, and forms large scale, and is still a best choice for a small number of sequences, thereby commonly being used for the sequencing of PCR products, terminal sequencing of the plasmids and bacterial artificial chromosomes.

(25) In addition, the majority of patients with severe hepatitis have 216T.fwdarw.C and 285G.fwdarw.A mutations, the mutations of the two loci results in a change of amino acid of HBV S region, as shown in the table below:

(26) TABLE-US-00003 Mutation of Mutation nucleotide position Mutation of amino acid 216T.fwdarw.C HBV S region, No change in P region, P region S protein 21L.fwdarw.S 285G.fwdarw.A HBV S region, No change in P region, P region S protein 44G.fwdarw.E

(27) Therefore, 216T.fwdarw.C and 285G.fwdarw.A mutations of hepatitis B virus may be related with the onset of severe hepatitis, further research would offer help for clinical early intervention and diagnostic.

Second Embodiment

(28) First: Serum Collection in Patients with Hepatitis B

(29) Collect serum samples of hepatitis B cases from a plurality of domestic hospitals, wherein there are 102 cases with severe hepatitis B, 127 cases with chronic mild hepatitis B, 95 cases with chronic moderate and severe hepatitis B and 65 cases of carriers (the patients has been diagnosed explicitly with hepatitis B by the indicators of ALT, AST, TBIL, PT, viral titer, etc., and clinical manifestations))

(30) Second: Primer Design

(31) We download 616 complete sequences of HBV genome, and design a pair of primer (SEQ ID NO: 8, SEQ ID NO: 9) by using primer 5.0 software according to the complete genome conserved sequence of HBV, to amplify a length of gene fragment including No. 216 and No. 285 of the HBV genome sequence.

(32) Third: Preparation of Kit

(33) Assemble the above primer (SEQ ID NO: 8, SEQ ID NO: 9) and dNTP, 5 Prime Star buffer, Prime Star, ddH.sub.2O for a HBV mutant amplification kit.

(34) Fourth: HBV DNA Extraction

(35) An equal volume of nucleic acid extract is added into the 50 ul serum and mixed, followed by water bath at 100 for 10 min, centrifugation for 10 min with 13000 r/min, and then the supernatant is drawn for PCR amplification.

(36) Fifth: PCR Amplification

(37) The brief steps of the amplification for the HBV gene of a patient are as follows: total volume of the PCR reaction 50 L, wherein template DNA 2 L, primers (10 uM) 1 L, respectively, dNTP (2.5 mM for each) 4 L, 5 Prime Star buffer (Mg.sup.2+ Plus) 10 L, Prime Star (5 U/ul) 0.5 L, and dilute with ddH.sub.2O to the total volume of 50 L. Cycle parameters: predenaturation at 94 C. 3 min, then 94 C. 30 s, 56 C. 15 s, 72 C. 50 s for 45 cycles, extending at 72 C. for 10 min. The product is recycled and purified via agarose gel electrophoresis with the concentration of 1%.

(38) Sixth: Sanger Sequencing

(39) The purified PCR product performs sequencing according to the conventional sanger sequencing technique in the art.

(40) Seventh: Statistical Method Apply Nonparametric Rank Sum Test, p<0.05 is Considered as a Statistically Significant Difference.

(41) Eighth: Experimental Result

(42) Specific experimental results are shown in Table 1-3. The data from table 1 and table 2 show that: the incidence of 216T.fwdarw.C mutation in patients with severe hepatitis is 45% (46/102), while the incidence of 285G.fwdarw.A mutation is 39% (40/102); loci mutation frequency of 216T.fwdarw.C in patients with chronic moderate and severe hepatitis is 23% (22/95), while the incidence of 285G.fwdarw.A mutation is 29% (28/95); loci mutation frequency of 216T.fwdarw.C in patients with chronic mild hepatitis is 8% (10/127), while the incidence of 285G.fwdarw.A mutation is 5% (6/127); loci mutation frequency of 216T.fwdarw.C in hepatitis B carriers is 5% (3/65), while 285G.fwdarw.A mutation is not found. Compare the hepatitis disease between the mutational sample and non-mutational sample by applying nonparametric rank sum test, showing the result of P<0.001, which indicates that mutational case tends to occur severe inflammation of the liver.

(43) TABLE-US-00004 TABLE 1 Distribution of 216T.fwdarw.C mutation in the four groups of patients with hepatitis B Mutational Wild Total Mutation 216T.fwdarw.C mutation sample sample sample rate Severe hepatitis B 46 56 102 45% chronic moderate and 22 73 95 23% severe hepatitis B chronic mild hepatitis B 10 117 127 8% hepatitis B carrier 3 62 65 5% Note: Compare the hepatitis disease between the mutational sample and non-mutational sample by applying nonparametric rank sum test, showing the result of P <0.001.

(44) TABLE-US-00005 TABLE 2 Distribution of 285G.fwdarw.A mutation in the four groups of patients with hepatitis B Mutational Wild Total Mutation 285G.fwdarw.A mutation sample sample sample rate Severe hepatitis B 40 62 102 39% chronic moderate and 28 67 95 29% severe hepatitis B chronic mild hepatitis B 6 121 127 5% hepatitis B carrier 0 65 65 0% Note: Compare the hepatitis disease between the mutational sample and non-mutational sample by applying nonparametric rank sum test, showing the result of P <0.001.

(45) TABLE-US-00006 TABLE 3 Distribution of 216T.fwdarw.C/285G.fwdarw.A duplex mutation in the four groups of patients with hepatitis B 216T.fwdarw.C/285G.fwdarw.A Mutational Wild Total Mutation duplex mutation sample sample sample rate Severe hepatitis B 32 70 102 31% chronic moderate and 15 80 95 16% severe hepatitis B chronic mild hepatitis B 5 122 127 4% hepatitis B carrier 0 65 65 0% Note: Compare the hepatitis disease between the mutational sample and non- mutational sample by applying nonparametric rank sum test, showing the result of P <0.001.

(46) Table 3 shows that: the incidence of 216T.fwdarw.C/285G.fwdarw.A dual mutation in patients with severe hepatitis is 31% (32/102), the incidence in patients with chronic moderate and severe hepatitis is 16% (15/95), the incidence in patients with chronic mild hepatitis is 4% (5/127), and the incidence in carriers is 0%; nonparametric rank sum test shows P<0.001, the experimental data indicate that 216T.fwdarw.C/285G.fwdarw.A duplex mutational case tends to have severe inflammation of the liver.

(47) The experimental results show that: the 216T.fwdarw.C and 285G.fwdarw.A mutations are closely related to the aggravated liver inflammation, and their further study is beneficial for clarifying the molecular mechanism of hepatitis B aggravation and has positive significance for clinical early warning of severe hepatitis.