南方农业学报2017，Vol.48Issue(3)：546-551,6.DOI:10.3969/j:issn.2095-1191.2017.03.027
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
Abstract
[Objective]A 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. [Method]Two 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. [Result]Specified 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). [Conclusion]Duplex 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.关键词
鸡新城疫病毒(NDV)/禽肺炎病毒(APV)/二重RT-PCR/检测Key words
Newcastle disease virus(NDV)/avian pneumovirus(APV)/duplex RT-PCR/detection分类
农业科技引用本文复制引用
谢志勤,王盛,刘加波,庞耀珊,梁亮,冯务玲,谢芝勋,邓显文,谢丽基,范晴,罗思思,黄莉,黄娇玲,张艳芳..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[J].南方农业学报,2017,48(3):546-551,6.基金项目
国家"万人计划"领军人才专项项目(2016-37) （2016-37）
广西科技重点研发计划项目(桂科AB16380054) （桂科AB16380054）
南宁市科学研究与技术开发计划项目(20152308) （20152308）
