Establishment and application of dual real-time fluorescent RT-PCR method for detection of Enterovirus
10.3969/j.issn.1671-8348.2016.33.026
- VernacularTitle:双重实时荧光RT-PCR法检测肠道病毒方法的建立及应用
- Author:
Lianhong XU
;
Yulin YUE
;
Yongfang WANG
;
Ying CHU
;
Lixin JIANG
- Keywords:
hand foot and mouth disease;
enterovirus;
enterovirus71;
dual real-time fluorescence RT-PCR method
- From:
Chongqing Medicine
2016;45(33):4688-4690,4741
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop a dual real-time fluorescent RT-PCR method for rapid detection of enterovirus(EV)and en terovirus type 71(EV71).Methods Specific primers and probes were designed and the dual real-time fluorescent RT-PCR reaction system was established.The quantitative standard curve was drawn;its sensitivity and precision were evaluated.Feces and throat swab specimens of 109 clinical patients with hand foot and mouth disease were collected and tested by using this method.Then the obtained results were compared with those detected by commercial EV71 PCR kit.Results The relative coefficient(2)of EV and EV71 standard curve established by the dual real-time fluorescent RT-PCR method were both 0.998.Its sensitivity reached 0.5 TCID50/mL for detecting EV and 0.05 TCID50/mL for detecting EV71.The within-run precision for detecting EV and EV71 was ＜3％ and total precision≤4％.The results showed good specificity for the detection of enterovirus and non-enterovirus.In 109 detected clinical samples,84 cases of EV positive samples were detected,in which 56 cases were EV71 positive with the total positive rate of 51.4 ％,which was consistent with the result of simple fluorescent RT-PCR commercialization kit(P=1.000).Conclusion The established dual real-time fluorescent RT-PCR method has high sensitivity and good stability,which has an important significance for early high throughput rapid diagnosis of hand foot and mouth disease.
