Currently, clinical diagnosis of ZIKV infection relies on serological assays for the detection of antibodies (including rapid lateral-flow immunochromatographic assays, Ig M capture enzyme-linked immunosorbent assay [MAC-ELISA], and plaque reduction neutralization test [PRNT]) and molecular-based assays for the detection of viral nucleic acids (conventional or quantitative, real-time reverse transcription polymerase chain reaction [RT-q PCR]) , and an NS2B-specific RT-q PCR assay recently developed by the Pan American Health Organization (PAHO) in response to the ZIKV outbreak in South America which intends to replace the CDC-validated ZIKV pr M RT-q PCR assay of lower sensitivity .
However, the use of RT-q PCR assays as diagnostic tests requires centralized laboratory facilities, trained personnel, expensive equipment, and extended turnaround times associated with sample transportation over large distances.
However, in adult individuals where clinical manifestations do occur they are usually mild, self-limiting, and non-specific associated with an acute febrile illness characterized by low-grade (~38 °C) and short-term (2–7 days) fever, fatigue, rash, arthralgia, myalgia, headache, and conjunctivitis.
These clinical signs are indistinguishable from those induced by many other flaviviral or alphaviral infections.
For these reasons, point-of-need (PON) molecular detection tools for easy, rapid, reliable, inexpensive, and on-site ZIKV testing can not only significantly improve the quality of the health care system in vulnerable areas, but also ensure rapid testing in blood banks and provide enhanced field surveillance of ZIKV transmission with an overall impact of major significance on public health.
To date, only three potential PON, molecular-based assays to detect ZIKV RNA have been developed, although not extensively evaluated on target diagnostic specimens .The recent emergence of Zika virus (ZIKV) in Brazil and its precipitous expansion throughout the Americas has highlighted the urgent need for a rapid and reliable on-site diagnostic assay suitable for viral detection.Such point-of-need (PON), low-cost diagnostics are essential for ZIKV control in vulnerable areas with limited resources.Briefly, confluent monolayers of Vero cells were inoculated with a 1/10 dilution of ZIKV PRVABC59, FLR, and MR 766 strains in a minimal volume of maintenance media without fetal bovine serum.After 1 h adsorption at 37 °C, monolayers were overlaid with complete EMEM and incubated at 37 °C and 5% CO g for 15 min at 4 °C, aliquoted, and stored at −80 °C.Hence, laboratory diagnosis of ZIKV is mandatory to confirm the clinical diagnosis .Therefore, the availability of rapid, reliable, and relatively low cost diagnostic tools is of utmost importance for ZIKV control and management.Vero cells (ATCC® CCL-81™) were maintained in Eagle’s minimum essential medium (EMEM, Mediatech, Inc., Manassas, VA) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Flowery Branch, GA), 2 m M L-glutamine (Gibco®, Carlsbad, CA), and penicillin and streptomycin (100 U/ml and 100 μg/ml, respectively; Gibco®) at 37 °C in 5% CO) were kindly provided by Dr. C6/36 were maintained in 1X Dulbecco’s modified minimum essential medium (DMEM, Gibco®) supplemented with 7.5% sodium bicarbonate (Gibco®), 10% fetal bovine serum, 1X non-essential amino acids (Gibco®), and 1 m M sodium pyruvate (Gibco®) at 30 °C in 5% CO atmosphere.AP-61 were maintained in 1X Leibovitz’s L-15 medium (Gibco®) supplemented with 2 m M L-glutamine, 10% fetal bovine serum, and 7.5% tryptone phosphate broth (Sigma-Aldrich, St. Tissue culture fluid (TCF) derived from Vero cells infected with ZIKV PRVABC59 (ATCC® VR-1843™), FLR (ATCC® VR-1844™), and MR766 (ATCC® VR-1838™) strains were used for analytical sensitivity and specificity evaluation of ZIKV-specific RT-q PCR and RT-ii PCR assays.Integration of the hydrolysis probe technology and an optical detection module allows automatic detection and interpretation of ii PCR results in the form of “positive” or “negative” readouts in a relatively low-cost device [ POCKIT™ system workflow for point-of-need detection of Zika virus RNA.This system includes a compact automatic nucleic acid extraction device (taco™ mini) and a portable PCR device (POCKIT™).