Technical Method
Development of a triplex real-time RT-PCR assay to detect Zika, Chikungunya and Mayaro viruses
Lijin Lai, Aqian Li, Quanfu Zhang, Lina Sun, Chuan Li, Wei Wu, Qin Wang, Mifang Liang, Dexin Li, Yan Wei, Jiandong Li
Published 2019-12-30
Cite as Chinese J Exp Clin Virol, 2019, 33(6): 632-636. DOI: 10.3760/cma.j.issn.1003-9279.2019.06.015
Abstract
ObjectiveTo establish a method for the simultaneous identification of Zika, Chikungunya and Mayaro viruses.
MethodsThe complete genome sequences of Zika, Chikungunya and Mayaro virus were retrieved from Global Shared Database for comparative analysis, estimate its conservative region and determine the target gene location, specific primers and probes were designed, then a triplex real-time RT-PCR assay was developed. The specificity, sensitivity and repeatability of the assay were assessed by viral nucleic acid of Zika virus, Chikungunya virus a, in vitro transcriptional RNA of Mayaro virus, normal human serum and related virus simulation sample.
ResultsThe result showed that the established method could detect Zika virus, Chikungunya virus, as well as simulated Mayaro virus samples, the limit of detection (LOD) of Zika and Chikungunya virus was 16.22 Copy/PCR and 12.02 Copy/PCR, respectively, the LOD for simulated Mayaro virus RNA was 2.82 Copy/PCR, no significant difference was detected between the triplex and monoplex assays. No cross reaction was found in the detection of dengue virus, Hantavirus, severe fever with thrombocytopenia syndrome (SFTS) virus, yellow fever virus and influenza virus, and 100 healthy adults blood samples, the specificity of the method was 100%. The repeatability result showed that the standard deviation of all three detections were blow 0.5 and the coefficient of variation was less than 2% by selecting viral nucleic acids or transcribed RNA with high, medium and low concentration gradients.
ConclusionsA triplex real-time RT-PCR assay for detection of Zika, Chikungunya and Mayaro virus has been established with an acceptable specificity, sensitivity and repeatability.
Key words:
Zika virus; Chikungunya virus; Mayaro virus; Triplex Real-time RT-PCR
Contributor Information
Lijin Lai
Key Laboratory of Environmental Pollution Monitoring and Disease Control, Ministry of Education, Department of Occupational and Environmental Health, School of Public Health, Guizhou Medical University, Guiyang 550025, China
Aqian Li
National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
Quanfu Zhang
National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
Lina Sun
National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
Chuan Li
National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
Wei Wu
National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
Qin Wang
National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
Mifang Liang
National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
Dexin Li
National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
Yan Wei
Key Laboratory of Environmental Pollution Monitoring and Disease Control, Ministry of Education, Department of Occupational and Environmental Health, School of Public Health, Guizhou Medical University, Guiyang 550025, China
Jiandong Li
National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
