叠氮溴化丙锭结合荧光定量PCR测定布鲁氏菌活菌数方法的建立

Development of a PMA-qPCR assay for detection of viable Brucella

为简便、快速测定宿主体内或细胞内布鲁氏菌的活菌数,本研究以布鲁氏菌单拷贝bcsp31基因为检测靶标,设计特异性引物,经优化反应条件及叠氮溴化丙锭(PMA)最佳使用浓度与曝光时间,建立了一种测定布鲁氏菌活菌数的叠氮溴化丙锭--荧光定量PCR方法(PMA-qPCR),并评估了该方法的特异性、敏感性、重复性。结果显示,建立的qPCR标准曲线Ct值与质粒标准品拷贝数对数值之间线性关系良好;特异性试验结果显示,该方法对布鲁氏菌S2株扩增结果为阳性,对大肠杆菌、沙门氏菌、小肠结肠炎耶氏菌、副溶血弧菌扩增结果均为阴性;其对布鲁氏菌S2的检测限为10~3cfu/mL,比常规PCR方法敏感性高100倍;重复性试验结果显示,批间及批内重复试验的变异系数均为0.63%~2.04%,重复性好。利用该方法对不同比例布鲁氏菌S2活菌/死菌混合样品定量测定,结果显示随活菌比例的增大,测得的活菌数与常规qPCR测定值越接近,表明经优化处理的PMA有效抑制了qPCR对死菌DNA的扩增,但对活菌的qPCR扩增无影响。将该方法与平板计数法共同测定TSB纯培养的布鲁氏菌S2活菌数,结果二者的测定值无统计学差异。随后利用这两种方法同时测定两种不同浓度的S2侵染巨噬细胞后的胞内活菌数,结果显示两种方法测定值虽然均差异显著(p<0.05),但测得的活菌数均在一个数量级且变化趋势一致,并均与侵染的活菌数呈正相关。上述结果表明,本研究建立的PMA-qPCR检测方法可以对布鲁氏菌活菌实现快速定量检测,为布鲁氏菌活菌数测定相关研究提供可行技术。

In order to easily and rapidly determine the number of viable Brucella in vivo or in cells,in this study,the single-copy gene bcsp31 of Brucella was used as the detection target,and specific primers were designed.After optimizing the reaction conditions and the optimal concentration and exposure time of PMA,a propidium monoazide-fluorescent quantitative PCR method(PMA-q PCR)was developed to determine the viable number of Brucella,and the specificity,sensitivity,and repeatability of the method were evaluated.The results showed that there was a good linear correlation between the Ct value of the established PMA-q PCR standard curve and the logarithm of the copy number of the standard plasmid.Specificity test results showed that the method was positive for B.suis S2 amplification,but negative for E.coli,S.typhimurium,Y.enterocolitica,V.parahaemolyticus and RNase-free water.The sensitivity of the method was 10~3 cfu/m L,which is 100 times more sensitive than conventional PCR;The results of repeatability tests showed that the coefficient of variation between inter-and intra-repeatability tests was 0.63%-2.04%,and the repeatability was good.Viable or dead B.suis S2 of the known concentrations were mixed in different proportions for the PMA-q PCR detection,and sample without PMA treatment served as controls.The results showed that with the increase of the proportion of viable Brucella,the number of viable Brucella measured was closer to that measured by conventional q PCR,which indicated that PMA could effectively inhibit the amplification of dead Brucella DNA by q PCR.The concentrations of B.suis S2 strain cultured in TSB were measured by the PMA-q PCR and plate counting methods,yielding no significant differences.Subsequently,the number of viable Brucella determined by the two methods were significantly different within S2-infected cells(p<0.05),which might be attributed to the viable but non-culturable(VBNC)cells of some plate-cultured B.suis S2 that failed to grow.The above results show that the PMA-q PCR detection method established in this study can achieve rapid quantitative detection of viable Brucella,and provides a feasible technique for related research and application of viable Brucella determination.