直接PCR法在口腔牙龈卟啉单胞菌检测中的应用

Direct PCR Assay for Detection of Porphyromonas gingivalis in Human Oral Cavities

目的为了便于食管癌大规模流行病学筛查工作的开展,探讨一种能快速敏感地检测口腔牙龈卟啉单胞菌(Porphyromonas gingivalis,P.gingivalis)DNA的免核酸抽提直接PCR方法。方法收集我院门诊100例体检人员的200份口腔拭子(每人双份),以TE buffer(10 mM Tris,0.1 mM EDTA,pH 8.0)、商品化裂解液以及核酸抽提试剂盒等作为基因组提取试剂,使用P.gingivalis特异引物探针进行直接定量PCR检测。结果与裂解液-直接qPCR法相比,TE-直接qPCR法的灵敏度为94.12%,特异性为100%;与试剂盒-qPCR法相比,TE-直接qPCR法的灵敏度与特异性均为100%;TE-直接qPCR法与裂解液-直接qPCR法以及与试剂盒-qPCR法检测P.gingivalis均具有高度一致性(分别为Kappa=0.954,Kappa=1);TE-直接qPCR法与试剂盒-qPCR法检测阳性P.gingivalis的Ct均值无显著差异(P=0.907)。结论TE-直接qPCR法检测口腔P.gingivalis灵敏度高、特异性强,是快速高效检测口腔P.gingivalis的理想方法,可用于食管癌大规模分子流行病学研究。

Objective To establish an rapid and sensitive direct polymerase chain reaction(PCR)procedure for P.gingivalis without DNA extraction in oral swab for esophageal carcinoma(EC)epidemiological study.Methods Two hundred gingival swabs from 100 physical examinations(2 swabs each person)were analyzed using direct quantitative PCR(qPCR),with specificprimer and probe for P.gingivalis.The template DNAs of samples were extracted from gingival swab by Tris-EDTA(TE)buffer,compared with commerciallysis buffer and MicroElute Genomic DNA Kit prior to PCR ampli fication.Results Compared with lysis-based direct qPCR,the sensitivity and specificity of TE-based direct qPCR were 94.12%and 100%;while compared with kit-based qPCR,they were 100%.TE-based direct qPCR had almost perfect agreement compared with lysis-direct PCR(Kappa=0.954)and with kit-qPCR(Kappa=1).The difference of mean cycle threshold(Ct)values between TE-direct qPCR and kit-qPCR was not significant(P=0.907).Conclusion TE-direct qPCR assay is a simple,effective method for detecting oral P.gingivalis with high sensitivity and specificity,and it should be useful for esophageal squamous cell carcinoma(ESCC)in large-scale molecular epidemiological investigation in the future.