山羊和绵羊皮下脓肿3种主要病原菌三重TaqMan荧光定量PCR检测方法的建立

Development of a triplex TaqMan real-time PCR assay for detection of three major bacterial pathogens causing subcutaneous abscess in goats and sheep

为建立山羊和绵羊皮下脓肿3种主要病原菌即金黄色葡萄球菌(S.aureus)、伪结核棒状杆菌(C.pseudotuberculosis)和化脓隐秘杆菌(A.pyogenes)的三重TaqMan荧光定量PCR检测方法,本研究分别根据S.aureus的耐热核酸酶nuc基因以及C.pseudotuberculosis和A.pyogenes的16S rRNA基因的特异性保守核苷酸序列设计了3套引物和探针,经反应体系和反应条件的优化及特异性、敏感性和重复性的评价,并对山羊和绵羊临床样品进行检测,建立了3种病原菌的三重TaqMan荧光定量PCR检测方法。利用建立的方法检测大肠杆菌、肠炎沙门菌、多杀性巴氏杆菌等山羊和绵羊常见的20种细菌的DNA样品,结果显示仅S.aureu、C.pseudotuberculosis和A.pyogenes DNA样品检测为阳性,其他细菌DNA样品均为阴性,表明该方法具有较强的特异性。利用建立的方法分别检测不同稀释度的3个重组质粒标准品,结果显示最低检出浓度≤88 copies/μL,表明该方法具有较高的敏感性。重复性试验结果显示变异系数≤2.14%,表明该方法具有较好的重复性。利用建立的三重TaqMan荧光定量PCR方法和普通PCR方法检测174份山羊和绵羊皮下脓肿DNA样品,结果显示2种方法的总符合率为97.13%。结果表明,本研究建立的三重TaqMan荧光定量PCR方法可用于山羊和绵羊皮下脓肿3种主要病原菌的高通量快速诊断。

In order to establish a triplex TaqMan real-time PCR assay for detection of the three major bacterial pathogens(S.aureus,C.pseudotuberculosis and A.pyogenes)causing subcutaneous abscess in goats and sheep,three sets of primers and probes,based on the specific conserved nucleotide sequences of the thermonuclease(nuc)gene of S.aureu and the 16S rRNA genes of C.pseudotuberculosis and A.pyogenes,were designed and the triplex TaqMan real-time PCR assay was established by optimizing of reaction system and reaction conditions,evaluating of specificity and sensitivity and detecting of clinical samples of goats and sheep.DNA samples of 20 common bacterial species in goats and sheep,such as E.coli,Salmonella enteritidis and Pasteurella multocida,were detected by the established assay and the results showed that only S.aureu,C.pseudotuberculosis and A.pyogenes DNA samples were positive and the other bacterial species DNA samples were negative,which indicated that this assay has strong specificity.Three recombinant plasmid standards at different dilutions were detected by the established assay and the results showed that the minimum detected concentration was less than or equal to 88 copies/μL,which indicated that the assay has strong sensitivity.The results of repeatability test showed that the coefficient variation was less than or equal to 2.14%,which indicated that this assay has good repeatability.One hundred and seventy-four goats and sheep subcutaneous abscess DNA samples were detected by the triplex TaqMan real-time PCR assay and conventional PCR assay,the results showed that the total coincidence rate of the two assays was 97.13%.These results showed that the triplex TaqMan real-time PCR assay established in this study can be used for the high-throughput and rapid diagnosis of the three major bacterial pathogens causing subcutaneous abscess in goats and sheep.