Rapid and Specific Genotyping System for Hepatitis B Virus Corresponding to Six Major Genotypes by PCR Using Type-Specific Primers

Abstract

A simple and precise genotyping system based on PCR using type-specific primers was developed for the determination of genotypes A through F of hepatitis B virus (HBV). This assay system is considered to be a useful tool for the molecular diagnosis of HBV infection and for large-scale surveys.Hepatitis B virus (HBV) is a well-known agent of acute and chronic hepatitis, with an estimated 350 million chronic carriers around the world. HBV has a circular and partially doublestranded DNA genome of 3.2 kb containing four overlapping open reading frames. HBV strains isolated worldwide have been classified into six genomic groups deduced from genome comparisons and designated genotypes A to F (3, 6, 7). The HBV genotypes have a characteristic geographic distribution. HBV genotyping by phylogenetic analysis based on nucleotide sequences produces the most reliable and certain genotyping results. However, this is not an appropriate method for largescale genotyping. On the other hand, several groups have reported the genotyping of HBV by the restriction fragment length polymorphism method (1, 2, 4, 8). However, their methods were not so sensitive and specific compared with our PCR genotyping method. In this paper, we report a simpler, more rapid, and more specific genotyping system for HBV involving PCR using type-specific primers.A total of 55 HBV DNA-positive serum samples obtained from individuals in six different countries, including Japan, Vietnam, the United States, Egypt, Ghana, and Bolivia, were used for the evaluation of our genotyping system. We selected the HBV DNA-positive samples by nested PCR. The sequences of PCR primers used in this study are shown in Table 1. The first-round PCR primers (outer primer pairs) and second-round PCR primers (inner primer pairs) were designed on the basis of the conserved nature of nucleotide sequences in regions of the pre-S1 through S genes, irrespective of the six HBV genotypes. P1 (sense) and S1-2 (antisense) were universal outer primers (1,063 bases). B2 was used as the inner primer (sense) with a combination called mix A for genotypes A, B, and C. Mix A consisted of antisense primers BA1R (type A specific), BB1R (type B specific), and BC1R (type C specific). B2R was used as the inner primer (antisense) with a combination called mix B for genotypes D, E, and F. Mix B consisted of sense primers BD1 (type D specific), BE1 (type E specific), and BF1 (type F specific). These primer combinations for second-round PCR were designed on the basis of the differences in the sizes of the genotype-specific bands. The type-specific primers were designed on the basis of the conserved nature of those sequences within a genotype and on the basis of their poor homology with the sequences derived from other HBV genotypes. The strategy for HBV genotyping is illustrated in Fig.

Detection of hepatitis B virus genotypes using oligonucleotide chip among hepatitis B virus carriers in Eastern China

“…Since Okamoto et al [18] established the first method to classify HBV, several methods for HBV genotyping have been reported except sequence analysis, such as multiplex PCR [19] , restriction fragment length polymorphism (RFLP) [20] , serological assay [21] and reverse hybridization [22] . These other genotyping methods have a number of advantages over sequence analysis as they are inexpensive and easy to perform.…”

A comparison of molecular methods for hepatitis B virus (HBV) DNA detection from oral fluid samples

“…To increase the HBV DNA detection from oral fluid samples, several PCR parameters were evaluated using spiked HBV oral fluid samples that had the DNA extracted with the QIAamp DNA Mini kit and that were detected using semi-nested PCR (Naito et al, 2001). The QIAamp DNA Mini kit has been used in other studies for DNA extraction from saliva samples and exhibited good efficiency (Viltrop et al, 2010;Durdiaková et al, 2012), so this assay was used in the initial experiments for the optimization of the PCR conditions.…”

“…Oligonucleotides specific for the core and surface regions of the HBV genome were employed for the following PCR experiments (Table 1): a semi-nested PCR amplifying HBV surface protein genes (Naito et al, 2001) with slight modifications performed in the present study to generate a product of 1100 bp; a nested PCR for the surface regions of the HBV genome (developed in the present study) with a product of 542 bp; and a one-round PCR specific for the core region of HBV (Olioso et al, 2007), which generated a product of 441 bp.…”

“…In the first round, this assay uses the sense primer PS1 and a mixture of the antisense primers S2 and S22, which permits the amplification of all HBV genotypes. The second round of amplification was performed on the entire first-round PCR product, using the primers PS1 and SR (Naito et al, 2001). …”

“…For the standardization of PCR for use on oral fluid samples, viral DNA obtained from oral fluid samples extracted with the QIAamp DNA Mini kit was first subjected to semi-nested PCR using oligonucleotides previously described by Naito et al (2001). The best conditions for the first round of amplification were obtained when using 1.5 U Platinum Taq DNA polymerase, 5 ml DNA and 40 cycles of amplifications with a primer annealing temperature of 47 u C. For the second round of amplification, using Platinum Taq DNA polymerase at 1.5 U with 30 cycles of amplification and 1 min of extension of the DNA gave the best result.…”