L Xue1,2, W Cai1,2, Q Wu1,2, J Zhang1,2, W Guo2. 1. Guangdong Institute of Microbiology, State Key Laboratory of Applied Microbiology Southern China, Guangzhou, China. 2. Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of Applied Microbiology, Guangzhou, China.
Abstract
AIMS: This study aims to develop a quick and sensitive method for obtaining GII.17 norovirus genome sequences based on a novel amplification strategy. METHODS AND RESULTS: Based on multiple alignments of GII.17 norovirus genome sequences available in GenBank, a set of primer pairs were first rationally designed, which could amplify six overlapping fragments encompassing the whole genome. Two sequencing primers II.17-Seq1R and II.17-Seq6F were also designed to complement sequences at both ends. The sensitivity of new primers was then evaluated by end-point dilution RT-PCR that was comparable to detection primers G2SKF/G2SKR. In practice, genome sequences of nine Guangzhou GII.17 strains were successfully obtained by the new method in one working day. All genomes comprised 7495 nucleotides with three complete ORFs, and their phylogenetic relationships were verified with other GII norovirus reference strains. CONCLUSIONS: Based on the new amplification strategy, a quick and sensitive method for direct sequencing of GII.17 norovirus genomes was successfully established. SIGNIFICANCE AND IMPACT OF THE STUDY: The newly developed method can be used as an important tool to collect genetic information of GII.17 noroviruses, and new obtained viral genomes in Guangzhou also provide reference data for norovirus research in future.
AIMS: This study aims to develop a quick and sensitive method for obtaining GII.17 norovirus genome sequences based on a novel amplification strategy. METHODS AND RESULTS: Based on multiple alignments of GII.17 norovirus genome sequences available in GenBank, a set of primer pairs were first rationally designed, which could amplify six overlapping fragments encompassing the whole genome. Two sequencing primers II.17-Seq1R and II.17-Seq6F were also designed to complement sequences at both ends. The sensitivity of new primers was then evaluated by end-point dilution RT-PCR that was comparable to detection primers G2SKF/G2SKR. In practice, genome sequences of nine Guangzhou GII.17 strains were successfully obtained by the new method in one working day. All genomes comprised 7495 nucleotides with three complete ORFs, and their phylogenetic relationships were verified with other GII norovirus reference strains. CONCLUSIONS: Based on the new amplification strategy, a quick and sensitive method for direct sequencing of GII.17 norovirus genomes was successfully established. SIGNIFICANCE AND IMPACT OF THE STUDY: The newly developed method can be used as an important tool to collect genetic information of GII.17 noroviruses, and new obtained viral genomes in Guangzhou also provide reference data for norovirus research in future.