猪圆环病毒1型LAMP检测方法的建立及初步应用

Establishment and Preliminary Application of LAMP Method for Detecting Porcine Circovirus Type 1

为建立猪圆环病毒1型(PCV1)环介导等温扩增(LAMP)检测方法,为现场诊断提供技术支持,本试验根据GenBank公布的PCV1全基因组序列,在其保守区设计了4条特异性LAMP引物,并通过反应条件优化、敏感性试验、特异性试验以及临床样本检测,首次建立了PCV1的LAMP检测方法。结果表明,62℃水浴60min为最佳反应条件;LAMP的检出限为1×10^(1)copies/μL,普通PCR最低检测限为1×10^(3)copies/μL;对猪细小病毒(PPV)、猪伪狂犬病毒(PRV)、猪圆环病毒2型(PCV2)、猪圆环病毒3型(PCV3)进行扩增,结果显示为阴性;应用该方法对103个临床疑似病料进行检测,阳性率为34.0%(35例),普通PCR阳性率为29.1%(30例),两种检测方法符合率为89.5%。本试验为PCV1临床检测提供了一种特异、快速、成本低廉的方法。

In order to establish a detection method of loop-mediated isothermal amplification(LAMP) for porcine circovirus type 1(PCV1) and provide technical support for field diagnosis, according to the PCV1 genome sequence published by GenBank, 4 specific LAMP primers were designed in its conserved region, and through optimization of reaction conditions, sensitivity test, specificity test and clinical sample detection, the LAMP detection method for PCV1 was established for the first time. The results showed that the optimal reaction conditions were water bath at 62℃ for 60 min, the detection limit of LAMP was 1.0×101 copies/μL, and the lowest detection limit of PCR was 1.0×103copies/μL. Nucleic acid amplification results of PPV, PRV, PCV2 and PCV3 were negative. Using this method, 103 clinical suspected samples were detected, and the positive rate was 34.0%(35 cases);the positive rate of ordinary PCR was 29.1%(30 cases), and the coincidence rate of the two detection methods was 89.5%. A specific, rapid and economical clinical testing method was provided for rapid detection of PCV1 in this study.