External Control Viral-Like Particle Construction for Detection of Emergent Arboviruses by Real-Time Reverse-Transcription PCR

Abstract

Arboviruses have been emerging and reemerging worldwide, predominantly in tropical and subtropical areas. As many arbovirus infections, including dengue (DENV), Zika (ZIKV), and chikungunya (CHIKV), have similar signs and symptoms, clinical diagnosis of arbovirus infections is challenging. Therefore, reliable laboratory tests are necessary to improve the clinical management of patients with suspected arbovirus infections. Real-time reverse-transcription PCR (RT-qPCR) is among the more effective methods to distinguish these viruses. The aim of this study was to construct a unique positive external control derived from a unique plasmid using genetic engineering for specific use in RT-qPCR assays to detect Zika, dengue (1–4), and chikungunya. An external control derived from the MS2 bacteriophage was constructed using sequences from arbovirus and human genomes. Laboratories were asked to test the control in the ZDC Biomol kit, a RT-qPCR kit which is able to detect Zika, dengue serotypes 1–4, chikungunya, and an internal human control. RNA extracted from the external control was able to be amplified and detected in RT-qPCR assays for each virus detected by using the ZDC Biomol kit. The external control, samples from viral culture, and infected patient samples display similar amplification using this assay. The pET47b(+)MS2-ZDC vector is a viable expression system for the production of external control viral-like particles (MS2-ZDC). The RNA from the recombinant particles can be easily extracted and can function as a tool to validate all steps of process from the extraction to the amplification of all targets in specific reaction. Thus, the MS2-ZDC particles are laboratory-safe in order to avoid risk for operators, and the phages are effective as positive control for use in the ZDC Biomol kit amplifying all kit targets making them effective for commercial profile.

Evidence of co-circulation of multiple arboviruses transmitted by Aedes species based on laboratory syndromic surveillance at a health unit in a slum of the Federal District, Brazil

“…Each of these four subgroups on the plate is composed of 23 wells to test the samples (38 µl/4 subgroups = 9.5 µl of sample eluate for each subgroup), and one positive control from the kit itself. For the real-time PCR (qPCR) probes, the ZDC kit uses three triplex reactions and two duplex reactions in the same plate: DENV-1 (HEX), DENV-4 (FAM), and internal control (Quasar 670); DENV-3 (HEX), DENV-2 (FAM), and internal control (Quasar 670); CHIKV (FAM) and internal control (HEX); and ZIKV (FAM) and internal control (HEX) [ 29 ].…”

Development, verification, and validation of an RT-qPCR-based protocol for Yellow Fever diagnosis

“…This vector was used to express a non-propagating MS2 particle. The use of MS2-like particles as positive control was evaluated in a RT-qPCR assay described in the literature and the proposed multiplex prototype (Borghetti et al, 2019;Domingo et al, 2012;Zambenedetti et al, 2017).…”