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禽肾炎病毒与鸡细小病毒双重PCR检测方法的建立

26458 2019-12-16 10:18:29 张艳芳,谢芝勋,邓显文,等 中国动物检疫
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为了同时检测禽肾炎病毒(avian nephritis virus,ANV)和鸡细小病毒(chicken parvovirus,ChPV),根据GenBank中ANV的ORF基因和ChPV的 NS1基因的保守序列,设计筛选了扩增片段大小分别为511 bp和242 bp的特异性引物,通过反应条件优化、特异性和敏感性试验,建立了一种双重PCR检测方法。该方法最佳引物比例为ANV上下引物各0.4 μL(20 mol/L),ChPV上下引物各0.6 μL(20 mol/L);最佳退火温度为53.8℃;灵敏度可达到55 fg(ANV)和58 fg(ChPV);对常见鸡病病原体进行检测,结果全为阴性。本研究建立的PCR方法具有特异、敏感、快速、稳定的优点,可用于同时鉴别诊断ANV和ChPV的混合感染。

Establishment of a Duplex PCR Assay for Detection of Avian Nephritis Virus and Chicken Parvovirus

A duplex PCR assay for simultaneous detection of both avian nephritis virus(ANV)and chicken parvovirus(ChPV)was established. Specifically,one pair of specific primers were designed according to the conserved sequences of ORF gene of ANV and NS1 gene of ChPV published in GenBank,after optimization of reaction conditions,specificity and sensitivity tests,the duplex PCR assay was established and evaluated. The results showed that the best ratio of primers was 0.4 μL(20 mol/L)respectively for upper and lower primers of ANV,and 0.6 μL(20 mol/L)respectively for those of ChPV;the best annealing temperature was 53.8 ℃;the detection limits could be up to 55 fg(ANV)and 58 fg(ChPV);then the assay was used to detect common pathogens of chicken diseases,and all detection results were negative. A conclusion was given that,the established assay was specific,sensitive,rapid and stable,and the assay could be used to identify and diagnose mixed infection of ANV and ChPV.

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