5种外来病病毒多重RT-PCR检测方法的建立

通过GenBank中对小反刍兽疫病毒N蛋白基因、亨德拉病毒及尼帕病毒H蛋白基因、西尼罗河热病毒PrM蛋白基因和非洲猪瘟病毒P72基因这5种外来病病毒的主要基因的分析比较,选择相应基因的保守序列进行人工合成,并对每段基因设计2对引物,扩增片段大小均在350-550bp之间。通过对条件的反复优化,建立了1种能够同时检测到这5种病原的并联PCR/RT-PCR方法。结果显示,该方法最低可检测到10-5 mg/L的病毒的DNA/cDNA,而且特异性强、重复性好。在45份猪样品及56份蝙蝠样品的检测中,只有阳性对照出现了目的条带,而其他均未出现目的条带。这说明本研究建立的5种主要外来病病毒并联PCR/RT-PCR的检测方法具有灵敏度高、特异性好的特点,可用于ASFV、PPRV、HeV、NiV和WNV的预防、检测及扑灭。

The multiple RT-PCR assay for five viruses

This work synthesized the conserved sequences of peste des petits ruminants virus N pro- tein gene, Hendra virus H protein gene, Nipah virus H protein gene,West Nile fever virus PrM pretein gene and African swine fever virus P72 gene by analysising and comparing the five kinds of viruses in GenBank. We design two sets of primers for each virus,and the amplified fragment size is between 350-550 bp. We established a muhiple PCR/RT-PCR assay for above viruses simulta- neously by repeatedly optimizing reaction condition. The sensitivity was consistently observed to be 10-s mg/L per PCR/RT-PCR mixture in the detection limits of the test. A retrospective study was also performed by analysis of 101 frozen samples,including 45 swine samples and 56 bat sam- ples. PCR gave positive results for positive control, but PCR did not give any positive results for other samples. This work provides a novel, highly sensitive multiple PCR/RT-PCR method for rapid and specific detection of ASFV,PPRV, HeV, NiV and WNV, that can be used as a routine di- agnostic test in surveillance,control and eradication programs.