Universal primers for HBV genome DNA amplification across subtypes: a case study for designing more effective viral primers

Qingrun Zhang,Changqing Zeng,Shan'Gang Jia,Elliott Richards,Guanghua Wu

doi:10.1186/1743-422x-4-92

Abstract

BackgroundThe highly heterogenic characteristic of viruses is the major obstacle to efficient DNA amplification. Taking advantage of the large number of virus DNA sequences in public databases to select conserved sites for primer design is an optimal way to tackle the difficulties in virus genome amplification.ResultsHere we use hepatitis B virus as an example to introduce a simple and efficient way for virus primer design. Based on the alignment of HBV sequences in public databases and a program BxB in Perl script, our method selected several optimal sites for HBV primer design. Polymerase chain reaction showed that compared with the success rate of the most popular primers for whole genome amplification of HBV, one set of primers for full length genome amplification and four sets of walking primers showed significant improvement. These newly designed primers are suitable for most subtypes of HBV.ConclusionResearchers can extend the method described here to design universal or subtype specific primers for various types of viruses. The BxB program based on multiple sequence alignment not only can be used as a separate tool but also can be integrated in any open source primer design software to select conserved regions for primer design.

Highlights

The highly heterogenic characteristic of viruses is the major obstacle to efficient DNA amplification
This means that hepatitis B virus (HBV) circulates as a complex mixture of genetically distinct but closely related variants that are in equilibrium at a certain time point of infection in a given circumstance
Identification of candidate regions for primer design by BxB We analyzed 1123 sequences, 1020 from public databases (Additional file 1) and103 sequences identified in our laboratory, with the BxB program. 10 regions were selected according to BxB analysis (Table 1)

Summary

Introduction

The highly heterogenic characteristic of viruses is the major obstacle to efficient DNA amplification. Taking advantage of the large number of virus DNA sequences in public databases to select conserved sites for primer design is an optimal way to tackle the difficulties in virus genome amplification. It has been widely reported that it is possible to have two HBV genotypes or recombinant types in one infected individual [3,4,5]. HBV, like other viruses such as HIV, HCV and poliovirus, has a high mutation rate of 2 × 10-5/site/year [6,7]and quasispecies distribution in infected individuals (page number not for citation purposes). A mixture of HBV quasispecies is a mixture of HBV haplotypes, which is a more important concept to researchers, such as in drug resistant mutant studies-different haplotypes of HBV may represent different types of drug resistance [9,10,11,12]

Methods

Results

Discussion

Conclusion