Development and evaluation of the detection method of triplex real-time fluorescent quantitative RT-PCR assays for dengue, yellow fever and chikungunya viruses
10.3760/cma.j.cn112866-20220420-00094
- VernacularTitle:登革病毒、黄热病毒和基孔肯雅病毒三重实时荧光定量RT-PCR检测方法的建立及评价
- Author:
Naipeng KAN
1
;
Yuwei WENG
;
Tingting YU
;
Jinzhang WANG
Author Information
1. 福建省疾病预防控制中心，福州 350012
- Keywords:
Reverse transcription-polymerase chain reaction;
Dengue virus;
Yellow fever virus;
Chikungunya virus
- From:
Chinese Journal of Experimental and Clinical Virology
2022;36(6):707-711
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To develop a triplex real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay for dengue virus (DENV), yellow fever virus (YFV) and chikungunya virus (CHIKV), so as to achieve the rapid detection of these three viruses.Methods:The complete genome sequences of DENV(Ⅰ, Ⅱ, Ⅲ, Ⅳ), YFV and CHIKV were retrieved from Global Shared Database for comparative analysis, estimate its conservative region, specific primers and probes were designed, then a triplex real-time RT-PCR assay was developed. The specificity was evaluated by other viral nucleic acids. The sensitivity was evaluated by in vitro transcribed RNAs of DENV, YFV and CHIKV. The repeatability of the method was evaluated by independent repeated experiments with different concentrations of viral nucleic acids. DENV detection method was validated with dengue patient serum. YFV and CHIKV detection methods were validated with simulated positive samples. The sera from healthy people were used for negative validation. Results:This method has no cross-reaction with other viral nucleic acids. The limit of detection (LOD) of DENV (Ⅰ、Ⅱ、Ⅲ、Ⅳ), YFV and CHIKV in vitro transcribed RNAs were less than 21.55 copies/PCR, 21.25 copies/PCR, 21.85 copies/PCR, 22.75 copies/PCR, 22 copies/PCR, 45.65 copies/PCR. The standard deviation of Ct values of each concentration was less than 0.5 and the coefficient of variation was less than 3%. The positive rate of clinical and simulated positive samples was 100%, and the negative rate of healthy serum was 100%. Conclusions:A triplex real-time fluorescent quantitative RT-PCR assay for DENV, YFV and CHIKV detection was established, and proved to be specific, sensitive and repetitive.
