溶藻弧菌实时荧光定量PCR快速检测方法的建立
Development of a dual real-time PCR for the rapid detection of Vibrio alginolyticus
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摘要: 为建立溶藻弧菌（VA）的快速检测方法,本研究以VA Collagenase基因为靶基因设计合成引物及Taq Man探针,建立了实时荧光定量PCR快速检测VA的方法。结果显示,对15株实验菌株进行荧光定量PCR检测,只有VA菌株检测为阳性,表明该检测方法特异性强;该方法的灵敏度为18 cfu/m L;稳定性和重复性实验结果表明,同一样品重复检测4次Ct值的变异系数均小于2%;利用该检测方法对采集的165份样品进行检测,共计检出2份VA阳性样品,与SN/T2564-2010行标法检测结果一致,显示了良好的实用性。该检测方法灵敏度高、特异性强,具有良好的实用性。Abstract: To establish a rapid assay for Vibrio alginolyticus(VA) detection,a dual real-time PCR method was developed targeting Collagenase gene of VA. The results showed that the test for 15 bacteria strains of the dual real- time PCR method, only VA test was positive, indicating that the method had high specificity. In addition,the sensitivity of a dual real-time PCR was 18 cfu/m L. Stability and reproducibility of the test results showed that the coefficient of variation of the same sample repeat the test four times Ct values were less than2%. Furthermore,a total 2 positive samples for VA were detected from 165 clinical samples by the real-time method,which was in accordance with the testing result by SN/T 2564-2010 standard detection protocol.Therefore,the real-time method provides a novel rapid and sensitive detection method for VA infection.
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