Improved reverse transcription-polymerase chain reaction assay for the detection of flaviviruses with semi-nested primers for discrimination between dengue virus serotypes and Zika virus

Abstract

BACKGROUND The genus Flavivirus includes a variety of medically important viruses, including dengue virus (DENV) and Zika virus (ZIKV), which are most prevalent in Brazil. Because the clinical profile of patients affected by different DENV serotypes or ZIKV may be similar, the development of new methods that facilitate a rapid and accurate diagnosis is crucial.OBJECTIVES The current study aimed to develop an improved reverse transcription-polymerase chain reaction (RT-PCR) protocol for universal detection of flaviviruses by using semi-nested primers that discriminate between DENV serotypes and ZIKV.METHODS The bioinformatics workflow adopted for primer design included: (1) alignment of 1,442 flavivirus genome sequences, (2) characterisation of 27 conserved regions, (3) generation of a primer set comprising 77 universal primers, and (4) selection of primer pairs with greatest coverage and specificity. Following primer design, the reaction was validated in vitro. The same approach was applied to the design of primers specific for DENV and ZIKV, using a species-specific sequence database.FINDINGS The new assay amplified an 800-806 nt variable region of the NS5 gene and allowed discrimination of virtually all flavivirus species using reference-sequence comparison. The 800-806 nt fragment was validated as a template for a semi-nested multiplex PCR using five additional primers for the detection of DENV and ZIKV. These primers were designed to generate amplicons of different sizes, allowing differentiation of the four serotypes of DENV, and ZIKV using agarose gel electrophoresis.MAIN CONCLUSIONS The bioinformatics pipeline allowed efficient primer design, making it possible to identify the best targets within the coding region of the NS5 protein. The multiplex system proved effective in differentiation of DENV1-4 and ZIKV on a 2% agarose gel. The possibility of discriminating DENV serotypes and ZIKV in the same reaction provided a faster result consuming less sample. In addition, this simplified approach ensured the reduction of the cost per analysis.

Aedes Aegypti E Arboviroses No Brasil

“…A evolução da produção científica acerca do tema expressa bem a situação frente à epidemia e o impacto gerado na saúde pública mundial. Essa produção, assim como a internacional, apresenta-se em maior número no ano de 2016, ano em que a Organização Mundial de Saúde (OMS) decretou estado de emergência de saúde pública de preocupação internacional (NUNES;PIMENTA, 2016). Como se pode perceber na Figura 1, o aumento da curva justamente quando o tema começa a aparecer como problema de pesquisa e vai crescendo de maneira significativa nos anos seguintes.…”

“…Cita-se também o teste de imunofluorescência que é usado para confirmação do diagnóstico. Nunes et al (2018), em estudo, desenvolveram um protocolo de reação para detecção de flavivírus, que possui a propriedade de discriminar entre os sorotipos de dengue e Zika. Sabe-se que a possibilidade de discriminar sorotipos de DENV e ZIKV na mesma reação fornece resultado mais rápido e usando menos amostra.…”

Detection methods targeting the positive‐ and negative‐sense RNA transcripts from plus‐stranded RNA viruses

“…If this region is conserved among different variants of a virus, the method detects multiple variants without discrimination. If, however, the primers are designed to amplify a variable genomic region, it is possible to only detect a specific variant of the virus [ 32 ]. When using RT‐PCR for diagnosing infection by novel viruses like SARS‐CoV‐2, it can be important to target more than one conserved region to avoid losing sensitivity of the assay, if the virus changes in the target region [ 33 ].…”