检测猪繁殖与呼吸综合征病毒高致病性和经典毒株可视化RT-LAMP方法的建立

Development of a visual RT-LAMP method for detection of highly pathogenic and classical strains of porcine reproductive and respiratory syndrome virus

猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)是目前影响养猪业的重要病毒之一,国内PRRSV是以美洲型为主,根据致病性的强弱又可分为高致病性(HP)和经典性PRRSV毒株,高致病性PRRSV是引起国内2006年'猪高热病'的主要病原。PRRSV属于条件性致病病毒,对PRRSV感染猪和发病猪的准确、快速检测是采取有效措施的前提和依据。环介导等温扩增技术(LAMP)是一种敏感、快速、操作相对简便的核酸扩增技术,整个反应在单一温度下进行,反应结果可以通过肉眼观察,在动物病原检测方面应用较多。本试验通过GenBank获得了HP-PRRSV和经典PRRSV分离株序列,选择2种毒株共同的保守基因区段,设计了用于检测PRRSV的RT-LAMP引物。通过对反应体系、反应温度、特异性、敏感性等优化,建立了检测PRRSV的RT-LAMP方法。建立的方法总体系为25μL,外引物终浓度为0.20μmol/L、内引物终浓度为1.60μmol/L,63.0℃恒温作用1 h,给反应产物中加入1μL SYBR GreenⅠ,阳性结果呈绿色,紫外光下可见荧光,阴性结果呈橙色,紫外光下无荧光。方法对PRRSV RNA的最低检测质量浓度为9.44×10-5 mg/L。用建立的方法对临床送检的19份猪样品(血清和组织)进行检测,与国标(GB/T 27517-2011)的HP-PRRSV和PRRSV双重RT-PCR检测方法进行对比,双重RT-PCR方法检测出6份阳性,RT-LAMP方法检测出8份阳性。本试验建立了检测PRRSV经典毒株和高致病毒株的RT-LAMP方法,为PRRSV的检测提供了可选择的方法。

Porcine reproductive and respiratory syndrome virus(PRRSV)is one of the most important virus affecting swine industry.The main PRRSV strain is the American type strain in China.The American strain also further is divided into highly pathogenic(HP)and classical PRRSV strains according to the pathogenicity.The highly pathogenic PRRSV is the main cause of the'Pig fever'in China in 2006.PRRSV belongs to the conditional pathogenic virus.The accurate and rapid detection of PRRSV-infected pigs and diseased pigs is a prerequisite and basis for taking some measures.Loop-mediated isothermal amplification(LAMP)is a sensitive,rapid,and relatively simple method of nucleic acid amplification.The whole reaction is carried out at a single temperature.The results can be observed by the naked eye and used in animal pathogen detection.In this study,RT-LAMP primers for HP-PRRSV and classical PRRSV were designed by analyzing the sequence of HP-PRRSV and classical PRRSV isolates obtained in GenBank and selecting the conserved gene segments of the two strains.The RT-LAMP method for detecting PRRSV was established by optimizing the reaction system,reaction temperature,specificity and sensitivity.In the reaction system,the total amount was 25μL,the final concentration of the external primers was 0.20μmol/L,the final concentration of the internal primer was 1.60μmol/L,and the reaction conditions were 63.0℃constant temperature for 1 h.Add 1μL of SYBR GreenⅠto the reaction product.Positive results present green and fluorescence is visible under the ultraviolet lamp;negative results present orange,no fluorescence under ultraviolet light.The minimum detectable concentration of PRRSV RNA was 9.44×10-5 mg/L.The established RT-LAMP method is used for detection of 19 samples(serum and tissues),and compared with the HP-PRRSV and PRRSV dual RT-PCR methods of the national standard(GB/T 27517-2011).As a result,six positive samples were detected by the PCR method,and eight positive samples were detected by the RT-LAMP method.This study established a RT-LAMP method that can simultaneously detect PRRSV classic strains and highly virulent strain,providing an alternative method for the detection of PRRSV in production.