摘要
为建立霍乱弧菌(VC)的快速检测方法,以VC的hlyA基因为靶基因设计合成引物及Taq Man探针建立实时荧光定量PCR快速检测法,同时对该方法进行了特异性、灵敏性、稳定性和重复性试验,并对145份临床样品进行了检测。结果表明:特异性试验中15株试验菌株荧光定量PCR检测只有VC菌株为阳性,表明该检测方法特异性强;灵敏性试验表明该方法的灵敏度为25 cfu/m L;稳定性和重复性试验表明同一样品重复检测4次,Ct值的变异系数均小于2%;对145份临床样品进行检测,共计检出2份VC阳性样品,与行标法(SN/T 2425—2010)检测结果一致。说明该检测方法灵敏度高、特异性强、重复性好,具有良好的实用性。
To establish a rapid detection assay for Vibrio cholerae（ VC）,a real- time fluorescent quantitative PCR assay was established using hly A gene of VC as a targeting gene to design and synthesize the primers with a Taq Man probe. At the same time,the specificity,sensitivity,stability and repeatability of the assay was also performed,and 145 clinical samples were detected. The results showed that in the specificity test,only VC strains were detected as positive among 15 test strains by real- time fluorescent quantitative PCR assay,indicating that the assay had strong specificity. In addition,the sensitivity of the assay was 2. 5 cfu/m L. Stability and reproducibility tests showed that the coefficients of variation of the Ct values were less than 2% when the detection for same sample was repeated for 4 times. Furthermore,a total of 2 VC positive samples were detected among 145 clinical samples,and the result was in accordance with the testing result by SN/T 2425—2010 standard detection protocol. The results indicate that the detection assay has the advantages of high sensitivity,strong specificity,and good reproducibility with good practicability.
基金
国家质检总局科技项目(2013IK031
2013IK051
2015IK089)
海南省社会发展科技专项(2015SF29)
海南省应用技术研究与开发专项(ZDXM20130025)
重庆市科技计划项目(cstc2014yykfA80017)
广东检验检疫局科技计划项目(2013GDK04
2015GDK53)
