猪圆环病毒2型和3型双重PCR鉴别检测方法的建立及应用

Establishment and application of a double PCR differential detection method for porcine circovirus types 2 and 3

为建立一种鉴别检测猪圆环病毒2型(PCV-3)和3型(PCV-3)的方法,本研究根据GenBank中PCV-2、PCV-3基因序列分别设计合成特异性检测引物,经过双重PCR反应条件的优化,建立了鉴别检测PCV-2和PCV-3的双重PCR方法。该方法对PCV-2、PCV-3、PCV-2/PCV-3混合物可分别扩增出300 bp、518 bp、300 bp和518 bp的特异性条带,检测PRV、PPV、PRRSV、CSFV、TGEV、PEDV均为阴性,对PCV-2/PCV-3混合物的检测灵敏度达2.1 ng DNA,与PCV-2和PCV-3单项PCR方法的符合率均为100%。该方法在检测136份临床病料样品的应用中,PCV-2阳性率达23.53%(32/136),PCV-3阳性率达18.38%(25/136),PCV-2/PCV-3混合感染率达7.35%(10/136),说明青海省部分地区猪群中存在PCV-2、PCV-3的流行,且存在PCV-2和PCV-3的混合感染。表明该双重PCR具有良好的特异性、敏感性、重复性、准确性和临床适用性,可以用于临床病料中PCV-2和PCV-3的快速低含量鉴别检测,将为PCV-2和PCV-3感染的临床鉴别诊断和流行病学监测提供了一种简便适用的方法,为我国猪圆环病毒病的综合防控提供技术支持。

This study was to establish a method for differential detection of porcine circovirus type 2(PCV-3) and type 3(PCV-3).specific primers were designed and synthesized according to Gen Bank PCV-2 and PCV-3 gene sequences.Double PCR reaction conditions were optimized,and a dual PCR method to detect PCV-2 and PCV-3 was established.With this method,PCV-2,PCV-3,PCV-2/PCV-3 mixture could be amplified to bands of 300 bp,518 bp,300 bp and 518 bp.Detections of PRV,PPV,PRRSV,CSFV,TGEV,PEDV were negative.The detection sensitivity for PCV-2/PCV-3 mixture was 2.1 ng DNA,and was 100% in coincidence with the single PCT diction of PCV-2 or PCV-3.In using the method for detection of 136 clinical samples,the positive rate of PCV-2 was 23.53%(32/136),that of PCV-3 was 18.38%(25/136),and that of PCV-2/PCV-3 mixed infection was 7.35%(10/136).PCV-2 and PCV-3 currently spread in swine in some areas of Qinghai Province,and there is even mixed infection PCV-2 and PCV-3 infection.This study showed that the double PCR was of high specificity,sensitivity,repeatability,accuracy and clinical applicability,and could be used for rapid detection of low-content PCV-2 and PCV-3 in clinical samples.This method would be of strong specificity,high sensitivity,fast detection,easy operation,and high accurate in clinical diagnosis and epidemiological surveillance of PCV-2 and PCV-3,and would provide technical support for comprehensive prevention and control of porcine circovirus disease in China.