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Communication

RT-dPCR in Mosquito Samples for ZIKV Detection: Effects of RNA Extraction and Reverse Transcription in Target Concentration

Laboratório de Saúde Única, Feevale Techpark, Universidade Feevale, Campo Bom 93700-000, Brazil
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Author to whom correspondence should be addressed.
Viruses 2020, 12(8), 827; https://doi.org/10.3390/v12080827
Received: 3 July 2020 / Revised: 25 July 2020 / Accepted: 27 July 2020 / Published: 30 July 2020
Zika virus (ZIKV) is an important arbovirus, responsible for recent outbreaks of Guillain Barré Syndrome and Congenital Zika Syndrome (CZS). After thousands of CZS cases, ZIKV is under constant surveillance in Brazil. Reliable and robust detection techniques are required to minimize the influence of host inhibitors from clinical samples and mosquito pool samples. Reverse transcription Digital Polymerase Chain Reaction (RT-dPCR) is a technique that allows the accurate quantification of DNA targets with high sensitivity, and it is usually less affected by inhibitors than RT-qPCR. This study aimed to assess the influence of mosquito tissue, RNA extraction and cDNA synthesis in ZIKV PCR detection. Samples containing 0, 3 and 10 mosquitoes were spiked with ZIKV MR766 and serially diluted prior to RNA extraction and RT-dPCR for ZIKV. Two reverse transcription protocols were tested. Assay sensitivity allowed the detection of 1.197 copies/µL. A higher correlation between dilution factor and target quantification was observed in 10 mosquito pool samples. The lower quantification in samples diluted without mosquitoes highlights the critical role of the reverse transcription step in RNA detection, since it could be attributed to reverse transcriptase variable performance in samples with low overall RNA concentration. The results in mosquito pools indicate that mosquito tissues do not inhibit ZIKV RT-dPCR, and the RT-dPCR technique has good sensitivity and robustness for ZIKV detection in mosquito pool samples regardless of mosquito tissue concentration. View Full-Text
Keywords: RT-dPCR; ZIKV; quantification; mosquito RT-dPCR; ZIKV; quantification; mosquito
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MDPI and ACS Style

Almeida, P.R.d.; Eisen, A.K.A.; Demoliner, M.; Spilki, F.R. RT-dPCR in Mosquito Samples for ZIKV Detection: Effects of RNA Extraction and Reverse Transcription in Target Concentration. Viruses 2020, 12, 827. https://doi.org/10.3390/v12080827

AMA Style

Almeida PRd, Eisen AKA, Demoliner M, Spilki FR. RT-dPCR in Mosquito Samples for ZIKV Detection: Effects of RNA Extraction and Reverse Transcription in Target Concentration. Viruses. 2020; 12(8):827. https://doi.org/10.3390/v12080827

Chicago/Turabian Style

Almeida, Paula R.d., Ana K.A. Eisen, Meriane Demoliner, and Fernando R. Spilki. 2020. "RT-dPCR in Mosquito Samples for ZIKV Detection: Effects of RNA Extraction and Reverse Transcription in Target Concentration" Viruses 12, no. 8: 827. https://doi.org/10.3390/v12080827

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