Development and evaluation of a duplex RT-qPCR assay for the detection and identification of Mayaro and chikungunya viruses.

IF 6.1 2区 医学 Q1 MICROBIOLOGY
Konrad M Wesselmann, Cécile Baronti, Antoine Nougairède, Laurence Thirion, Xavier de Lamballerie, Remi Charrel, Laura Pezzi
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Abstract

Mayaro virus (MAYV) is a mosquito-borne alphavirus that is widespread in the Amazon basin, where it co-circulates with the closely related chikungunya virus (CHIKV). Due to the limited surveillance and technical limitations of diagnostic assays (scarcity of commercial assays, serology cross-reactivity), the true burden of MAYV is uncertain. We designed a new RT-qPCR assay targeting the nsp1 gene for MAYV detection, which can be used in monoplex or duplex format. In the duplex format, the new MAYV assay is combined with a CHIKV assay and a second MAYV assay, both previously published. The lower limit of detection with a 95% positivity rate was determined to be <10 RNA copies/μL in monoplex and duplex formats for both MAYV and CHIKV. Monoplex and duplex assays proved to be linear within the tested range of approximately 108 to 102 RNA copies/μL and showed 100% specificity against a wide panel of arboviruses as well as several other pathogens in clinical samples. The testing of CHIKV-positive sera and MAYV-spiked plasma samples confirmed the suitability of the assays in a clinical setting. These assays offer a reliable tool for detection and differentiation of MAYV and CHIKV in endemic settings.IMPORTANCEMolecular diagnostic capabilities for detecting alphaviruses other than chikungunya virus (CHIKV) remain limited. Mayaro virus (MAYV), an emerging mosquito-borne alphavirus, co-circulates with CHIKV in the Americas, making clinical differentiation between the two viruses challenging. To address this, we developed a novel RT-qPCR assay specifically for the detection of MAYV, which can also be used in a duplex format to simultaneously detect and distinguish CHIKV. The assay was thoroughly evaluated in both monoplex and duplex formats and demonstrated high sensitivity and specificity. This new tool is particularly valuable for the detection of MAYV, especially in resource-limited settings, where its duplex format offers efficient and accurate differentiation of acute infections.

马雅罗病毒和基孔肯雅病毒双重RT-qPCR检测鉴定方法的建立和评价。
马雅罗病毒(MAYV)是一种蚊媒甲病毒,在亚马逊盆地广泛传播,与密切相关的基孔肯雅病毒(CHIKV)共同传播。由于监测有限和诊断检测的技术限制(缺乏商业检测,血清学交叉反应性),MAYV的真正负担尚不确定。我们设计了一种新的针对nsp1基因的检测MAYV的RT-qPCR方法,该方法可以单链或双链检测。在复式格式中,新的MAYV检测与先前发表的CHIKV检测和第二种MAYV检测相结合。检测下限为8 ~ 102个RNA拷贝/μL,阳性率为95%,对多种虫媒病毒和临床样品中的几种其他病原体具有100%的特异性。对chikv阳性血清和mayv加标血浆样本的检测证实了该检测方法在临床环境中的适用性。这些检测方法为在流行环境中检测和区分甲型h1n1流感病毒和甲型h1n1流感病毒提供了可靠的工具。除基孔肯雅病毒(CHIKV)外,检测甲病毒的分子诊断能力仍然有限。马雅罗病毒(MAYV)是一种新出现的蚊媒甲病毒,与CHIKV病毒在美洲共同传播,这使得区分这两种病毒具有挑战性。为了解决这个问题,我们开发了一种新的RT-qPCR检测方法,专门用于检测MAYV,该方法也可以以双工格式同时检测和区分CHIKV。该分析在单形和双形格式中进行了全面评估,并显示出高灵敏度和特异性。这种新工具对于检测MAYV特别有价值,特别是在资源有限的环境中,其双工格式提供了有效和准确的急性感染区分。
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来源期刊
Journal of Clinical Microbiology
Journal of Clinical Microbiology 医学-微生物学
CiteScore
17.10
自引率
4.30%
发文量
347
审稿时长
3 months
期刊介绍: The Journal of Clinical Microbiology® disseminates the latest research concerning the laboratory diagnosis of human and animal infections, along with the laboratory's role in epidemiology and the management of infectious diseases.
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