鸡新城疫病毒和禽肺炎病毒二重RT-PCR检测方法的建立及应用

Establishment and application of duplex RT-PCR detection for Newcastle disease virus and avian pneumovirus

  • 摘要: 目的建立一次性可同时扩增鉴别鸡新城疫病毒(NDV)和禽肺炎病毒(APV)的二重RT-PCR,为有效防控鸡新城疫和禽肺炎病毒病提供技术支持.方法根据GenBank已发表NDV的F基因序列(GenBank登录号JX840454)和APV的F基因序列(GeneBank登录号AF187154)分别设计2对特异性引物,应用这2对引物建立可同时检测鉴别NDV和APV的二重RT-PCR,对扩增条件进行优化,并通过特异性试验、敏感性试验和临床样品检测等验证其实用性.结果优化后的二重RT-PCR可特异性扩增出参试NDV毒株(LaSota株、F48E9株、I株)及APV/MN-10毒株的目的条带,其大小分别为247和424 bp,而其他对照参试毒株在247和424 bp处均无特异条带出现.二重RT-PCR针对NDV和APV的最低检出限均为10 pg的RNA.应用建立的二重RT-PCR对132份从广西采集的病鸡样品进行检测,结果得到NDV阳性样品26份、APV阳性样品2份,与血清学方法的鉴定结果一致.随机选取的2份NDV阳性PCR产物与NDV(GenBank登录号JX840454)的F基因序列一致,同源性达100%;2份APV阳性PCR产物与APV(GenBank登录号AF187154)的F基因序列也完全一致,同源性达100%.结论针对NDV和APV建立的二重RT-PCR具有特异性好、灵敏度高、简便快速的特点,可用于临床快速鉴别诊断,为有效防控鸡新城疫和禽肺炎病毒病提供了技术支持.

     

    Abstract: ObjectiveA method has been established for simultaneous detection of Newcastle disease virus(NDV) and avian pneumovirus(APV) by duplex reverse transcriptase polymerase chain reaction(RT-PCR), which could provide tech-nical support for control of NDV and APV. MethodTwo pair of specific primers based on F gene sequence of NDV(Gen-Bank accession number JX840454) and F gene sequence of APV(GenBank accession number AF187154) were designed. A duplex RT-PCR was set to detect NDV and APV with the designed primers. Amplification condition was optimized. Prac-ticability of the detection was verified by specificity test, sensitivity test and clinical sample validation. ResultSpecified amplification results by optimized duplex RT-PCR showed that objective bands of tested NDV strains (Strain Lasota, Strain F48E9 and Strain Ⅰ) and APV/MN-10 strain were 247 and 424 bp in length but there were no special bands were detected at 247 and 424 bp in other control strains. The minimum detection limit was 10 pg RNA for NDV and APV. In clinical samples detection using duplex RT-PCR, out of 132 sick chickens collected in Guangxi, there were 26 NDV positive samples and 2 APV positive samples. The result was in accordance with that by serological method. Two NDV positive samples were randomly selected. PCR products of the two NDV positive samples and 2 APV positive samples were se-quenced. Homology analysis showed that PCR products of two NDV samples were 100% homologous with F gene sequence of NDV(GenBand accession number JX840454), and PCR products of APV samples were 100% homologous with F gene se-quence of APV(GenBand accession number AF187154). ConclusionDuplex RT-PCR for NDV and APV is a detection method with high specificity, high sensibility and rapid detection. It can be applied to rapid clinical identification and serve as a technical support for control of Newcastle disease viruse and avian pneumovirus.

     

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