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.
Highlights
We developed a positive external control based on a MS2 bacteriophage vector to be used in assays to detect the nucleic acids from Zika, chikungunya, and dengue virus serotypes 1–4. e pET47b(+)-MS2-ZDC vector was constructed after the insertion of a synthetic sequence into a unique BamHI site of pET47b(+)-MS2, which falls within the replicase gene of the MS2 genome
Our study was focused on creating a positive control for a RT-qPCR-based diagnostic assay that detects Zika, dengue
We proposed an external positive control based on the MS2 bacteriophage modified
Summary
Emerging and reemerging viruses transmitted by arthropod vectors, such as yellow fever virus, Zika, dengue, chikungunya, Rift Valley fever virus, Japanese encephalitis virus, West Nile virus, Saint Louis encephalitis virus, Murray Valley encephalitis virus, Usutu virus, Spondweni virus, and O’nyong-nyong virus have transmission cycles in urban environments. ese viruses cause overlapping clinical symptoms, and many patients develop serious physiological manifestations that can include death in severe cases [1, 2].e anthropogenic environmental factors, such as disorganized urbanization, population displacement, and precariousness of basic sanitation, have favored the transmission and spread of these viruses. e last decade was chaotic in Brazil with a large number of infected people and many complications caused by these viruses, especially microcephaly (Zika), hemorrhagic syndromes (dengue), and BioMed Research International severe arthralgias (chikungunya). is high prevalence brought loss for public health and country economy [3,4,5].Since the clinical symptoms of arbovirus infections overlap, laboratory diagnostics are necessary to distinguish between them. Ese viruses cause overlapping clinical symptoms, and many patients develop serious physiological manifestations that can include death in severe cases [1, 2]. E last decade was chaotic in Brazil with a large number of infected people and many complications caused by these viruses, especially microcephaly (Zika), hemorrhagic syndromes (dengue), and BioMed Research International severe arthralgias (chikungunya). Viral infections can be diagnosed through different methods including viral culture, serology, and molecular methods, and these same techniques are used to test for arbovirus infections [6, 7]. Real-time reverse-transcription polymerase chain reaction (RT-qPCR) has been used to detect dengue (a virus that is part of family Flaviviridae, genus Flavivirus), Zika (a virus that is part of family Flaviviridae, genus Flavivirus), and chikungunya (a virus that is part of family Togaviridae, genus Alphavirus) worldwide in different samples such as serum, urine, cerebrospinal fluid, and saliva. RT-qPCR is easier to conduct than the other methods used to detect pathogens, and its quality depends on the samples, the human operator, the nucleic acid test (NAT) kit, and PCR equipment [9,10,11]
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