Establishment of TaqMan RT-PCR for detection of TIBOV virus
10.3760/cma.j.cn112866-20201013-00262
- VernacularTitle:西藏环状病毒TaqMan反转录聚合酶链式反应方法的建立
- Author:
Panpan FENG
1
;
Qikai YIN
;
Jierong ZHAO
;
Shihong FU
;
Fan LI
;
Ying HE
;
Songtao XU
;
Guodong LIANG
;
Kai NIE
;
Huanyu WANG
Author Information
1. 中国疾病预防控制中心病毒病预防控制所，北京 102206
- Keywords:
Tibet orbivirus;
TaqMan RT-PCR;
Molecular detection
- From:
Chinese Journal of Experimental and Clinical Virology
2021;35(2):209-212
- CountryChina
- Language:Chinese
-
Abstract:
Objective:A highly sensitive and specific real-time quantitative TaqMan reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for rapid and accurate detection of Tibet orbivirus (TIBOV).Methods:The TIBOV genomic sequences from GenBank were analyzed by Clustal X 2.1 and the specific primers and probe were designed in the conserved segment of VP4 gene. RNA standard was obtained from in vitro transcription and a TaqMan RT-PCR assay for TIBOV was established. The sensitivity, specificity and repeatability of this method were evaluated. Results:The assay showed a good amplification curve within the range of 1.0×10 2~8 copies/reaction template, the detection limit was 1.0×10 2 copies/reaction, the coefficients of variation of Ct values in repeat detections were all less than 1.5%. No cross-reaction was found in this assay. Variable mosquito samples were screened by this assay and the result showed TIBOV negative. The prepared TIBOV simulated positive samples were 100% detected. Conclusions:The assay developed in this study is specific and sensitive for detection of TIBOV and can be used for laboratory detection and routine surveillance.
