National Veterinary Institute, Technical University of Denmark, Frederiksberg C, Denmark.
J Virol Methods. 2013 Nov;193(2):697-705. doi: 10.1016/j.jviromet.2013.07.019. Epub 2013 Jul 24.
PRRSV is a positive-sense RNA virus with a high degree of genetic variability among isolates. For diagnostic sensitivity and vaccine design it is essential to monitor PRRSV genetic diversity. However, to date only a few full genome sequences of PRRSV isolates have been made publicly available. In the present study, fast and robust methods for long range RT-PCR amplification and subsequent next generation sequencing (NGS) were developed and validated on nine Type 1 and nine Type 2 PRRSV viruses. The methods generated robust and reliable sequences both on primary material and cell culture adapted viruses and the protocols performed well on all three NGS platforms tested (Roche 454 FLX, Illumina HiSeq2000, and Ion Torrent PGM™ Sequencer). These methods will greatly facilitate the generation of more full genome PRRSV sequences globally.
PRRSV 是一种正链 RNA 病毒，其分离株之间具有高度的遗传变异性。为了提高诊断的灵敏度和疫苗设计的效果，监测 PRRSV 的遗传多样性至关重要。然而，迄今为止，仅有少数 PRRSV 分离株的全基因组序列可供公开使用。在本研究中，针对 9 株 1 型和 9 株 2 型 PRRSV 病毒，开发并验证了用于长距离 RT-PCR 扩增和后续下一代测序（NGS）的快速而稳健的方法。这些方法在原始材料和细胞培养适应的病毒上均产生了稳健可靠的序列，并且在三种测试的 NGS 平台（Roche 454 FLX、Illumina HiSeq2000 和 Ion Torrent PGM™ Sequencer）上均表现良好。这些方法将极大地促进全球范围内更多 PRRSV 全基因组序列的产生。
