猪细小病毒7型荧光定量PCR检测方法的建立与应用

Establishment and application of real-time quantitative PCR detection method for porcine parvovirus type 7

猪细小病毒7型(PPV7)是近年来新发现的一种新型PPV。目前,缺乏更为高效的针对PPV7的检测方法,为了建立能够快速检测PPV7的方法,本研究根据PPV7 NS1基因的保守区序列,设计1对特异性引物及TaqMan探针。经过各反应条件的优化,初步建立了一种便捷、灵敏、能够快速准确检测PPV7的荧光定量PCR(qPCR)方法,并对其特异性、敏感性、稳定性以及与SYBR GreenⅠqPCR检测方法的符合率进行检测与对比。结果显示,该方法除对PPV7的基因组DNA有特异性扩增,对猪伪狂犬病毒、猪传染性胃肠炎病毒、猪流行性腹泻病毒、猪轮状病毒、猪瘟病毒、猪繁殖与呼吸综合征病毒、日本乙型脑炎病毒及PPV1核酸均无交叉反应,特异性较强。该方法对重组质粒标准品检测限为1.76拷贝/μL,而常规PCR方法检测限为1.76×10^(2)拷贝/μL,表明本研究建立的方法敏感性较高。该方法对不同浓度质粒标准品的组内和组间重复性试验变异系数均小于1.7%,重复性较好。利用本研究建立的qPCR方法和SYBR GreenⅠqPCR方法对采自福建地区的150份样品进行检测,结果显示,两种检测方法的阳性率分别为24.67%(37/150)和6.67%(10/150),二者的总符合率为80.67%(121/150)。表明本研究建立的qPCR方法可以用于临床样品的检测。本研究建立的qPCR检测方法特异性强、敏感性高、重复性稳定性好,为PPV7的流行病学调查及检测提供了新的技术支撑。

Porcine parvovirus type 7(PPV7)is a newly discovered type of porcine parvovirus in recent years.At present,there is a lack of efficient diagnostic methods for PPV7.In order to establish a rapid and accurate detection method for PPV7,this study designed a pair of specific primers and a TaqMan probe based on the conserved region of PPV7 NS1 gene sequence.After optimization,a convenient,sensitive and rapid fluorescent quantitative PCR detection method was established for the diagnosis of PPV7,and its specificity,sensitivity,stability,and agreement with the SYBR Green I fluorescent quantitative PCR(qPCR)detection method were validated.Results showed that this method specifically amplified the genomic DNA of PPV7 and no cross-reactivity with other viruses,including pseudorabies virus,porcine epidemic diarrhea virus,porcine rotavirus,classical swine fever virus,porcine reproductive and respiratory syndrome virus,Japanese encephalitis virus,and PPV1,which indicates this method has good specificity.The detection limits were 1.76 copies/μL for recombinant plasmid standards and 1.76×10^(2) copies/μL for conventional PCR methods,indicating that the method established in this study is highly sensitive.The coefficients of variation(CV)of the intra-and inter-group repetitive tests were both less than 1.7%,which shows good repeatability.The diagnostic results of 150 samples collected in Fujian Province were compared with the SYBR Green I qPCR method and the fluorescent quantitative PCR method established in this study.The results showed that the positive rate of the fluorescent quantitative PCR detection method was 24.67%(37/150),the positive rate of SYBR Green I qPCR detection method was 6.67%(10/150),total compliance rate of the qPCR established in this study and SYBR Green I qPCR was 80.67(121/150),which suggests this method is extremely sensitive compared with SYBR Green I qPCR method.The fluorescent quantitative PCR assay established in this study is highly sensitive,specific and reproducible and stable,providing new technical support for the epidemiological investigation and detection of PPV7.