鸡传染性喉气管炎病毒可视化LAMP快速诊断方法的建立和应用

试验旨在建立简易、快速、高效的鸡传染性喉气管炎病毒(infectious laryngotracheitis virus,ILTV)检测和诊断方法。根据GenBank上公布的ILTV TK基因序列,设计检测ILTV的特异性环介导的等温扩增(loop media-ted isothermal amplification,LAMP)技术反应引物,通过对LAMP反应体系和反应条件的优化,以及特异性、敏感性和临床样品的检测,建立了ILTV LAMP检测方法。结果显示,以内引物ILT9-FIP和ILT9-BIP、外引物ILT9-F3和ILT9-B3、环引物ILT9-LB和ILT9-LF为LAMP反应引物,反应温度为66℃时,所建立的LAMP检测方法反应效率最高;所建立的LAMP检测方法能够特异性地检测ILTV(匈牙利株和王岗株),不与新城疫病毒(NDV,B株)、鸡传染性支气管炎病毒(IBV,H52株和H120株)、大肠杆菌、鸡副嗜血杆菌、巴氏杆菌等发生交叉反应,且能够检测到的病毒最低浓度达到0.06pg/μL,其灵敏度是普通PCR方法的100倍;采用建立LAMP方法对50个临床样本进行检测,阳性率为14%,且与PCR检测结果的符合率达96%。本研究建立了特异性强、灵敏度高、操作简单的LAMP检测方法,适用于临床上ILTV的快速检测和诊断。

Establishment and Application of Visual LAMP Detection Method for Infectious Laryngotracheitis Virus

This study was aimed to establish a simple,rapid and efficient method for detection and diagnosis of infectious laryngotracheitis virus (ILTV).The specific perimers were designed for the loop mediated isothermal amplification (LAMP) reaction based on the conservative region of ILTV TK gene in GenBank.The LAMP detection method for ILTV was established through the optimization of the LAMP reaction system and reaction conditions,as well as the specificity,sensitivity and detection of clinical samples.The results showed that the established LAMP reaction exhibited the highest amplification efficiency under the parameters of internal primers (ILT9 FIP and ILT9 BIP),outer primers (ILT9-F3 and ILT9-B3),ring primers (ILT9-LB and ILT9-LF) and reaction temperature (66℃).The established LAMP detection method could specifically detect ILTV (Hungarian and Wanggang strains),there was no cross-react with Newcastle disease virus (NDV,B strain),avian infectious bronchitis virus (IBV,H52 and H120 strains),Escherichia coli,Haemophilus parasuis, Pasteurella,etc.Meanwhile,the sensitivity of the established LAMP detection method was 0.06 pg/L,and its sensitivity was 100 times of the conventional PCR method.50 clinical samples were tested by establishing LAMP method,the positive rate was 14%,and the coincidence rate was 96% with PCR method.This study established a LAMP detection method with high specificity,high sensitivity and simple operation,which was suitable for rapid detection and diagnosis of ILTV in clinical application.