Development and Validation of Method for Zika Virus Detection Method development and validation for Zika virus disease detection

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Sumalee Chanama
Pattara Wongjaroen
Sirirat Naemkhunthot
Laddawan Meephaendee
Arisara Posanacharoen
Pongsiri Tanthong
Husneeyah Vateh
Sarinee Chumnanraksa
Wararat Jamfa
Areerat Sa-ngasang


          Zika virus (ZIKV), member of the Flaviviridae family, is spread mostly by the bite of an infected Aedes aegypti or Ae. albopictus and can be passed from mother to fetus, sexual transmission and blood transfusion. The most common symptoms are fever, rash, joint pain and red eyes. ZIKV infection during pregnancy can cause microcephaly. The most appropriate ZIKV assay is the detection of ZIKV RNA in serum and urine by real-time reverse transcription-polymerase chain reaction (real time RT-PCR). The objectives of this study were the development and validation of ZIKV real-time RT-PCR, and also the production of ZIKV RNA control. ZIKV real-time RT-PCR test was modified from US-CDC’s protocol, using 3 sets of primer/probe specific to prM, E and NS2b genes. ZIKV RNA control containing 350 base pairs of ZIKV nucleotide including prM and E gene was produced and synthesized by In vitro transcription (IVT). The method validation of ZIKV RNA control and diluted ZIKV various concentrations was defined as 100% of specificity and Limit of Detection (LOD) was 0.15 PFU/ml. Furthermore, the proficiency testing was performed and showed 100% concordance. These results showed that the ZIKV real-time RT-PCR test was successful developed to detect Zika viral RNA both African and Asian lineages.


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