Development of a multiplex luminex assay for nucleic acid detection of Phlebovirus

Abstract

Objective To establish a multiplex Luminex assay for nucleic acid detection of Phlebovirus. Methods Specific primers and probes of Rift Vally fever virus(RVFV), Severe fever with thrombocytopenia syndrome bunyavirus(SFTSV) and Heartland virus(HLV) were designed. Target fragments of the three viruses were amplified using target enriched multiplex(Tem) PCR, and then a multiplex Luminex assay of nucleic acid detection was established.The specificity of the developed Luminex assay was evaluated withTem-PCR products of RVFV, SFTSV, HLV and the amplification products of 7 hemorrhagic fever viruses (HFVs) including Hantand virus(HTNV), and its sensitivity and reproducibility was evaluated with in vitro transcribed viral RNA standards of gradient dilution. Results Using the developed multiplex Luminex assay of nucleic acid detection, the target sequences of RVFV, SFTSV and HLV could be detected specifically, and there was no cross-reaction with 7 HFVsincluding HTNV. In vitro transcribed RNA of 103 copies/μl, 102 copies/ μl and102copies/μl could be detected for RVFV, SFTSV and HLV detection, respectively.The coefficient of variation of detection values were all less than 15% in each concentration more than 103 copies/μl of RNA standards for both intra-assays and inter-assays. Conclusion A multiplex Luminex assay of nucleic acid detection for phlebovirus was preliminary established to effectively detect RVFV, SFTSV and HLV, laying the foundation for the laboratory diagnosis of related infectious diseases. Key words: Hemorrhagic fevers, viral; Phlebovirus; Suspension array; Polymerase chain reaction