荧光定量RT-PCR快速检测手足口病病原研究

Rapid detection of pathogen from hand foot mouth disease by real time quantitative RT-PCR

目的建立荧光定量RT-PCR技术用于检测手足口病的肠道病毒。方法利用TaqMan技术,设计一对共引物及探针,建立、优化反应体系后,构建质粒标准品,利用10倍稀释法建立相对定量标准曲线,根据TCID50倍比稀释,建立灵敏度曲线。特异性检验后利用临床标本与传统的RT-PCR法进行比较。结果定量标准曲线的灵敏度为105/μl(R2=0.999),PCR扩增效率为94%。灵敏度曲线显示检测到的最低浓度值为5.0TCID50/ml(R2=0.99),PCR扩增效率为97.6%。荧光RT-PCR检出率为80.9%,显著高于RT-PCR(检出率为62.92%)。结论研究建立的荧光定量RT-PCR方法检测肠道病毒敏感性高、特异性好、效率高,可以作为肠道病毒的快速检测方法。

Objective To develop real time quantitative RT - PCR for rapid detection of enterovirus from hand - foot - mouth disease. Methods TaqMan technique was used and a set of universal primers and probe were designed, Real - time RT - PCR for specific sensitive detection of Enterovirus was established. The positive recombinant plasmid was constructed and was used as standard, after ten -fold dilutation, it was used to make quantitative standard curve, TCID50 was diluted ten -fold and then used to make sensitivity standard curve. After specificity was determined clinical specimens were detected to compare real time RT - PCR with traditional RT - PCR. Results The sensitivity of the assay was 10^5/μl and the regression coefficient was 0. 999 of the quantitative standard curve. The sensitivity was TCID50 and the regression coefficient of the sensitivity standard curve was 0.99. Positive rate of real time RT - PCR is 80.9%, and that of RT - PCR is 62. 92%. There is signifantly statistical difference. Conclusion A real time quantitative RT - PCR for detection of enterovirus have successfully developed and it has high sensitivity, good specificity and high efficiency, thus it can be used cot rapid detection of enterovirus.