Establishment And Clinical Application Of A New Genotyping Method For Hepatitis B Virus Using Real Time Fluorimetry Polymerase Chain Reaction

Hepatits B poses a great threat to life and health. In China Hepatits B virus (HBV) prevails at a high infection rate and causes considerable morbidity. The division of HBV genotypes which resulted from virus mutation and long-term filtration and accumulation by the host, provides a significant means for further research into the clinical characteristics of HBV. The present research suggests that besides the factors existing in the host, the heterogeneity of virus gene may also contribute to the differences between HBV genotypes in some clinical phenomenon like virus mutation, consequent diseases and treatment response. Thus It's necessary that larger scale of research into epidemiology should be made to further probe the relation between genotypes and clinical outcome. The method to determine HBV genotypes can be applied to study epidemiology, so as to make clinical medicine and therapy program more efficient and to restrain the prevalence of HBV. To establish and test the veracity of a convenient, sensitive, quick and low-costing method to determine HBV genotypes, namely the real-time Polymerase Chain Reaction (PCR) method, this study is begun. Meanwhile, in this study, this method is applied to determine HBV genotypes in different areas of China, other HBV genotypes determining methods are compared with it and the clinical significance of HBV is also analyzed. The establishment of the real-time fluorimetry PCR method to determinegenotypes B~D of hepatitis B virusHBV has a high gene heterogeneity. Eight different genotypes named from A to H have been distinguished according to the differences in complete gene sequence of HBV strains. Genotypes A,B,C and D are all found in China and they have obvious geographical distribution. Genotypes B and C are predominant types, genotype A is rarely discovered, and genotype D is mainly found in the minority people. Many recent researches indicate that HBV genotypes play an important role in the clinical therapy and disease prognosis of HBV carriers. However, due to lack of a convenient, reliable, quick and low-costing clinical method to determine HBV genotypes, cosmic research on epidemiology is hard to carry out, and as a result, the relation between HBV genotypes and diseases caused by HBV is unlikely to be illuminated. To solve this problem, considering the distribution of genotypes in China, primers and probes for genotypes B, C and D have been designed and applied to detect the genotypes of 132 CHB patients, using the PCR method.This study has detected genotypes division on the basis of HBV genotype-specific nucleotide sequence. The PCR primers and Taqman probes of each genotype have been designed according to the analysis of 143 complete HBV genomes from GeneBank by DNASIS analyzing software. With the help of ABI 7000 software, the results are analyzed as follows: The expansion of PCR occurs only when a certain type of primer and probe is combined with the same type of template, so that the particularity of the detection can be guaranteed. Meanwhile, short segments expanded by this method and higher annealing temperature also guarantee the particularity of the expansion. The reaction system can distinguish HBV genotypes in a convenient, sensitive and quick way, after the expansion done by the fluorimetry PCR device. According to the results, in the 132 samples, 26 (19.7%) are genotype B, 92 (69.7%) are genotype C, 10 (7.6%) are genotype D and 4 (3%)are unidentified.The standard basis for HBV genotypes division is the homology of complete gene sequence up to and above 92% or that of gene S up to 96% and above. Gene S can be used to distinguish genotypes. After three strains from each of the genotypes B, C and D are selected, special primer BS1/S1R is used to expand gene S in each strain. Then each gene S of 18 PCR-produced clones of the strains (two samples of each strain are cloned) is sequenced. With the help of DNASIS analyzing software, it is discovered that the results of sequencing by this method are completely in accordance to those from GenBank. 71 samples and 4 undistributed were randomly selected and their S gene was sequenced throught using special primer. The sequenceswere analyzed by DNASIS and the evolutional tree was founded. There is a different sample in which were detected by real time fluorimetry PCR and 4 samples were not distributed. The exact probability of real time fluorimetry PCR method is 93.3% (70/75) .From the facts above, it is certain that the real-time PRC method can identify HBV genotypes in a convenient, sensitive, quick and reliable way, and therefore, it is suitable for cosmic clinical and epidemiological research.clinical application of a new genotyping method for hepatitis B virus using real time fluorimetry polymerase chain reactionDifferent genotypes result in different diseases. This, besides other factors, may be originated from the facts that virus gene controls the expression of antigen and that different strains have different frequency of variation and immunity-clearing resistance.In this study, the established method to determine HBV genotypes with real time fluorimetry PCR is applied to detect the genotypes of 128 CHB patients in Lanzhou region, and at the same time, clinical data such as HBV DNA and HBV-M are collected to study HBV genotypes and clinical performances of diseases. Conclusions based on this study are as follows. First, in the CHB patients in Lanzhou Region, genotype C is the main genotype, next are genotypes B and D. Second, there are no significant differences in the proportions of genotypes between male and female. Third, carriers of genotype C have a higher HBV DNA level and a much higher positive rate for HbeAg than those of genotype B. Since HbeAg is the target antigen of CTL cell, it is likely to be attacked, causing damage to liver. Fourth, carriers of genotype B has a higher positive rate for anti-HBe than those of genotype C.Comparison with three methods detected HBV genotypesTechnology of HBV genotypes division has been developing since the idea of genotype was brought forward. At present, there are four major methods to determine genotypes, namely the nucleotide sequence mensuration, the PCR-RFLP method ,the PCR using type-specific primers method and the ELISA with monoclonal antibodies method. Although the nucleotide sequence mensuration is reliable and can produce accurate results, with its complicated technology, demanding experiment conditions and high cost, it is still hard to be put into general use and thus not suitable for cosmic clinical and epidemiological research. Other methods have their advantages and disadvantages with a development in process from complicated to simple and fast. Base on PCR method has greatly predigested the prosess to determine genotypes. However, the information of the PCR method is relatively inadequate for it is gainedonly from gene S rather than complete gene sequence; what's more, there is pollution matter unavoided in the process of this method. In a word, there are still some problems to be solved when these methods are used in large-scale epidemiology research.In this study, genotypes in the samples of blood serum from 75 CHB patients are detected, using the HBV gene S sequence mensuration, the PCR-RFLP method and the real-time PCR method respectively. And the results from these three methods are compared and analyzed. In the 75 samples, based on the HBV gene S sequence mensuration, genotype B are 14, genotype C 55 and genotype D 6, based on the PCR-RFLP method, genotype B are 14, genotype C 52, genotype D 8 and one is unidentified while based on the real-time PCR method, genotype B are 12, genotype C 52 genotype D 7 and 4 are unidentified. Compared the PCR-RFLP method with the real-time PCR method, it can be inferred that there are no significant differences in identifying rate, sensitivity, particularity and stability between these two methods. Although the HBV gene S sequence mensuration is accurate and reliable, it's impractical to be put into general clinical use due to its complicated technology, long experiment process and demanding experiment conditions. While the PCR-RFLF and the PCR methods greatly predigest the process, improve the particularity and stability, shorten the time and cut the cost of genotypes detecting, and therefore they can be applied to detect large numbers of samples in clinic, although it's true that these two methods more or less have some limitation.It is very important for detailed molecular imformation of virus type and It is the base of individual treatment. So we must realize detailedly HBV genotypes. Throught detecting HBV genotypes we can study amply epidemiology> etiology > therapeutics. With perfecting HBV genotyping method, we will discover new genotype and know power of different genotype HBV . Then we can also learn the realation of genotype and curative effect.