Simultaneous detection of Zika, chikungunya, dengue, yellow fever, West Nile, and Japanese encephalitis viruses by a two-tube multiplex real-time RT-PCR assay

Zhixiang Xu; Yun Peng; Minghui Yang; Xiaohe Li; Jun Wang; Rongrong Zou; Jinhu Liang; Shisong Fang; Yingxia Liu; Yang Yang

doi:10.1002/jmv.27658

Simultaneous detection of Zika, chikungunya, dengue, yellow fever, West Nile, and Japanese encephalitis viruses by a two-tube multiplex real-time RT-PCR assay

J Med Virol. 2022 Jun;94(6):2528-2536. doi: 10.1002/jmv.27658. Epub 2022 Feb 22.

Authors

Zhixiang Xu^{1

2

3}, Yun Peng^{1

2}, Minghui Yang¹, Xiaohe Li^{1

2}, Jun Wang^{1

2}, Rongrong Zou^{1

2}, Jinhu Liang^{1

2}, Shisong Fang³, Yingxia Liu^{1

2}, Yang Yang^{1

2}

Affiliations

¹ Shenzhen Key Laboratory of Pathogen and Immunity, National Clinical Research Center for Infectious Disease, State Key Discipline of Infectious Disease, Shenzhen Third People's Hospital, Second Hospital Affiliated to Southern University of Science and Technology, Shenzhen, China.
² Savid Medical School, University of Chinese Academy of Sciences, Beijing, China.
³ Shenzhen Center for Disease Control and Prevention, Shenzhen, China.

PMID: 35146775
DOI: 10.1002/jmv.27658

Abstract

Due to the concurrent prevalence and increasing risk of coinfection of the clinically important Arboviruses, timely and accurate differential diagnosis is important for clinical management and the epidemiological investigation. A two-tube multiplex real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for the simultaneous detection of Zika virus (ZIKV), chikungunya virus (CHIKV), dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) was developed and optimized with high specificity and sensitivity. The detection limit for all the six viruses could reach as low as five genome equivalent copies and 2.8 × 10^-³ tissue culture infectious doses (TCID₅₀ ) for ZIKV, YFV, CHIKV and 2.8 × 10^-2 TCID₅₀ for JEV per reaction, with high accuracy and precision (R² > 0.99). The coefficient of variation of intra-assay and inter-assay for our quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was low, and the obtained positive rates ad C_t values of this assay were comparable with singleplex commercial kits. Moreover, the multiplex qRT-PCR assay was able to detect possible co-infections without competitive inhibition of target viral genomes. In conclusion, our rapid, sensitive, cost-effective multiplex qRT-PCR will be of great use for differential diagnosis in a clinical setting and epidemiological investigation during surveillance.

Keywords: arbovirus; coinfection; detection; differential diagnosis; multiplex qRT-PCR.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Chikungunya Fever* / diagnosis
Chikungunya virus* / genetics
Dengue Virus* / genetics
Dengue* / diagnosis
Encephalitis Virus, Japanese* / genetics
Encephalitis Viruses, Japanese* / genetics
Humans
Reverse Transcriptase Polymerase Chain Reaction
West Nile Fever* / diagnosis
Yellow Fever* / diagnosis
Yellow fever virus / genetics
Zika Virus Infection*
Zika Virus* / genetics