实时荧光PCR技术应用于手足口病病原学检查的体会

Analysis of application of real- time fluorogenic quantitative PCR in etiological examination of hand foot and mouth disease

目的分析实时荧光定量PCR(FQ-PCR)技术在手足口病病原学检查中的应用价值。方法选取2013年1月—2014年1月经临床确诊为手足口病的患儿125例,采集其临床标本共167份并均应用FQ-PCR技术行病原学检查,其中123份咽拭子标本同时采用逆转录聚合酶链反应(RT-PCR)技术检查。结果 167份临床标本的肠道病毒检出率为74.85%(125/167),不同标本类型的EV71、Cox A16与混合感染阳性检出率组间比较,无统计学意义(P>0.05);重症病例的EV71阳性率58.82%与总阳性率76.47%高于普通病例的16.67%与56.48%,Cox A16阳性率11.76%低于普通病例的39.81%,有统计学意义(P<0.01);咽拭子标本应用FQ-PCR检查的EV71、Cox A16阳性检出率69.11%、46.34%均比RT-PCR检查的52.03%、31.71%高,有统计学意义(P<0.05)。结论手足口病的主要病原体是EV71、Cox A16,FQ-PCR技术的病原菌检出率与标本采集方式无相关性,但可相应提高咽拭子标本病原的确诊率,改善临床治疗与预后效果。

Objective To analyze the application value of real - time fluorogenic quantitative PCR （ FQ - PCR） in the etiological examination of hand foot and mouth disease. Methods Totally 125 cases clinically diagnosed as hand foot and mouth disease in our hospital from January 2013 to January 2014 were selected for the study, from whom 167 clinical specimens were collected and undergone etiological examination through FQ -PCR and 123 throat swab specimens were examined through reverse transcription polymerase chain reaction （ RT - PCR）. Results The detection rate of intestinal virus in the 167 clinical specimens was 74. 85% （ 125/167 ）. The positive rates of infection by EVT1, CoxA16 and mixed infection of different specimens between the two groups were no statistical significance （ P 〉 0. 05 ）. The positive rate of EV71 in severe cases was 58.82% and the total positive rate of all the cases was 76.47% , higher than the 16.67% and 56.48% among normal cases. The positive rate of CoxA16 was 11.76% , lower than that of the 39.81% among normal cases, the difference being statistically significant （P 〈 0. 01 ）. The positive rates of EV71 and CoxA16 in throat swab specimens examined by FQ - PCR were 69.11% and 46. 34% respectively, higher than the 52. 03% and 31.71% examined through RT - PCR, statistically significant （ P 〈 0. 05 ）. Condusions The pathogen of hand foot and mouth disease is EV71 andCoxA16. The detection rate of pathogenic bacteria through FQ -PCR is of no correlation with sampling methods, but FQ - PCR can increase the pathogenic diagnosis rate of throat swab specimens and improve the effect of clinical treatment and prognosis.