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目的为提高RT-PCR的检出水平,进一步改进手足口病的检测方法提供参考依据。方法同时采用RT-PCR和real-time RT-PCR方法检测2011年2—5月河南省新乡市各级医院临床诊断为手足口病患者的100份粪便标本及50份非肠道病人粪便标本中肠道病毒通用型(PE)、柯萨奇病毒A组16型(CVA16)和肠道病毒71型(EV71)核酸;改进引物,RT-PCR检测CVA16;在NCBI中对RT-PCR使用的各组引物进行BLAST比对,并使用Primer Premier 6.0及Oligo 6.62软件分析各引物。结果 Real-time RT-PCR法检出CVA16、EV71和PE阳性率分别为36%、33%和71%,常规RT-PCR法分别检出20%、27%和64%,2者的PE、EV71检出率无统计学差异,而CVA16的检出率差异有统计学意义(χ2=6.349,P<0.05);改进引物后,RT-PCR法CVA16阳性检出率提高到35%(χ2=5.643,P<0.05),与临床诊断的符合率增加10%,接近real-time RT-PCR检测水平;2种方法的特异性、漏诊率及误诊率无明显差异。结论常规RT-PCR法对CVA16的检出率较低,采用改进后的引物,其检出率达到real-time RT-PCR水平。
Objective To provide a reference for improving the detection level of RT-PCR and further improving the detection of HFMD. Methods 100 stool samples and 50 non-intestinal samples of stool specimens from patients with clinically diagnosed HFMD at all levels of hospitals in Xinxiang City of Henan Province from February to May in 2011 were simultaneously detected by RT-PCR and real-time RT-PCR. (PE), coxsackievirus A group 16 (CVA16), and enterovirus 71 (EV71) nucleic acids; improved primers for detecting CVA16 by RT-PCR; The group primers were BLAST aligned and each primer was analyzed using Primer Premier 6.0 and Oligo 6.62 software. Results The positive rates of CVA16, EV71 and PE detected by Real-time RT-PCR were 36%, 33% and 71%, respectively. The positive rates of PE, There was no significant difference in the detection rate of EV71, but the detection rate of CVA16 was significantly different (χ2 = 6.349, P <0.05). After the primers were improved, the positive rate of CVA16 in RT-PCR was increased to 35% (χ2 = 5.643, P <0.05), and the coincidence rate with clinical diagnosis increased by 10%, which was close to the real-time RT-PCR detection. The specificity, misdiagnosis rate and misdiagnosis rate of the two methods had no significant difference. Conclusion The detection rate of CVA16 by conventional RT-PCR method is low. The detection rate of real-time RT-PCR was achieved by using the improved primers.