Objective To establish a loop-mediated isothermal amplification( LAMP) method for detecting diarrhea pathogens( Shigella and Salmonella) in rhesus monkeys and evaluate the application of the LAMP method for detecting ...Objective To establish a loop-mediated isothermal amplification( LAMP) method for detecting diarrhea pathogens( Shigella and Salmonella) in rhesus monkeys and evaluate the application of the LAMP method for detecting bacterial diseases in nonhuman primate laboratory animals. Materials and Methods A total of 205 fecal samples of rhesus monkeys were detected in this LAMP assay. The specificity and sensitivity of LAMP for Shigella and Salmonella were analyzed,and real-time polymerase chain reaction( REAL-TIME PCR) assay was employed as control. Results The LAMP method established here needed only 45 min to complete the reaction at 63℃. Its detection limit was 10 pg / μL and with a high specificity. The positive rate of Shigella and Salmonella was 1. 5% and 6. 3%,respectively. Conclusions Here we have established a fast and simple Shigella and Salmonella LAMP detection method that has strong specificity and high sensitivity and is suitable for rapid detection of bacterial disease in macaques. The development of this rapid detection kit is underway,and it will be helpful to the diarrhea detection.展开更多
环介导等温扩增技术(LAMP)是一种广泛应用的恒温下快捷检测技术,但其极易发生气溶胶污染导致假阳性结果,将LAMP与簇状规则间隔短回文重复序列(CRISPR)系统结合可以有效避免假阳性结果。为建立针对绵羊泰勒虫18S r RNA基因的LAMP-CRISPR/...环介导等温扩增技术(LAMP)是一种广泛应用的恒温下快捷检测技术,但其极易发生气溶胶污染导致假阳性结果,将LAMP与簇状规则间隔短回文重复序列(CRISPR)系统结合可以有效避免假阳性结果。为建立针对绵羊泰勒虫18S r RNA基因的LAMP-CRISPR/Cas12a检测方法,本研究根据绵羊泰勒虫18S rRNA基因设计LAMP的特异性引物,构建靶基因的质粒标准品作为模板,优化反应条件后建立LAMP方法。进一步将LAMP产物加入CRISPR/Cas12a检测体系作为模板,优化CRISPR/Cas12a检测体系,获得最佳反应体系。结果显示,优化后LAMP方法的最佳反应温度为65℃,最佳反应时间30 min,内外引物浓度比≥6:1时最佳,LAMP-CRISPR/Cas12a的最佳反应体系为Cas12a蛋白与CRISPR RNA(crRNA)浓度比为1:2,探针浓度为2.5μmol/L。利用建立的LAMP-CRISPR/Cas12a方法检测绵羊泰勒虫、东方泰勒虫、中华泰勒虫、马泰勒虫、驽巴贝斯虫、绵羊无形体、弓形虫的DNA,结果显示仅绵羊泰勒虫为阳性结果,其他病原均为阴性;将构建的质粒标准品10倍倍比稀释(10^(0)~10^(7)倍)后作为模板,利用该方法检测,结果显示该方法的检测限为4.04拷贝/μL;于同一时间和不同时间利用该方法检测重组质粒标准品pMD19-T-T.ovis(4.04×10^(3)拷贝/μL~4.04×10^(1)拷贝/μL),分析其重复性,结果显示该方法批内变异系数小于5%,批间变异系数小于10%,具有良好的重复性。利用建立的LAMP-CRISPR/Cas12a方法与套式PCR检测临床绵羊血液样品,结果显示二者的阳性符合率达100%(14/14)、阴性符合率96.43%(54/56),总符合率97.14%(68/70)。本实验基于绵羊泰勒虫18S rRNA基因首次建立了特异性、敏感性及重复性均良好的LAMP-CRISPR/Cas12a检测方法,为绵羊泰勒虫病的监测和防控奠定了技术基础。展开更多
文摘Objective To establish a loop-mediated isothermal amplification( LAMP) method for detecting diarrhea pathogens( Shigella and Salmonella) in rhesus monkeys and evaluate the application of the LAMP method for detecting bacterial diseases in nonhuman primate laboratory animals. Materials and Methods A total of 205 fecal samples of rhesus monkeys were detected in this LAMP assay. The specificity and sensitivity of LAMP for Shigella and Salmonella were analyzed,and real-time polymerase chain reaction( REAL-TIME PCR) assay was employed as control. Results The LAMP method established here needed only 45 min to complete the reaction at 63℃. Its detection limit was 10 pg / μL and with a high specificity. The positive rate of Shigella and Salmonella was 1. 5% and 6. 3%,respectively. Conclusions Here we have established a fast and simple Shigella and Salmonella LAMP detection method that has strong specificity and high sensitivity and is suitable for rapid detection of bacterial disease in macaques. The development of this rapid detection kit is underway,and it will be helpful to the diarrhea detection.
