Rapid colorimetric detection of Zika virus from serum and urine specimens by reverse transcription loop-mediated isothermal amplification (RT-LAMP)

Abstract

Zika virus (ZIKV) has emerged as a major global public health concern in the last two years due to its link as a causative agent of human birth defects. Its rapid expansion into the Western Hemisphere as well as the ability to be transmitted from mother to fetus, through sexual transmission and possibly through blood transfusions has increased the need for a rapid and expansive public health response to this unprecedented epidemic. A non-invasive and rapid ZIKV diagnostic screening assay that can be performed in a clinical setting throughout pregnancy is vital for prenatal care of women living in areas of the world where exposure to the virus is possible. To meet this need we have developed a sensitive and specific reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay to detect ZIKV RNA in urine and serum with a simple visual detection. RT-LAMP results were shown to have a limit of detection 10-fold higher than qRT-PCR. As little as 1.2 RNA copies/μl was detected by RT-LAMP from a panel of 178 diagnostic specimens. The assay was shown to be highly specific for ZIKV RNA when tested with diagnostic specimens positive for dengue virus (DENV) and chikungunya virus (CHIKV). The assay described here illustrates the potential for a fast, reliable, sensitive and specific assay for the detection of ZIKV from urine or serum that can be performed in a clinical or field setting with minimal equipment and technological expertise.

Fig 1. Visual detection of specificity of ZIKV RT-LAMP assay with the arbovirus urine panel.

Photos represent 1 of 3 replicates. Reactions were incubated at 65°C for 40 minutes before results were recorded. DENV1, dengue virus 1, DENV2, dengue virus 2; DENV3, dengue virus 3; DENV4, dengue virus 4; WNV, West Nile virus; SLEV, St. Louis encephalitis virus; CHIK, chikungunya virus; ZIKV, zika virus; NTC, non-template control.

Fig 2. Evaluation of ZIKV RT-LAMP assay with diagnostic specimens.

Negative (N) ZIKV RT-LAMP results and positive (P) ZIKV RT-LAMP results were separated and plotted based on Ct values (x-axis) and RNA concentrations (y-axis) from the corresponding RT-PCR. Urine samples are represented by black diamonds. Serum samples are represented by red diamonds. The solid horizontal line represents the 1 RNA copy/μl cutoff value. The dashed vertical line represents the Ct value 38.0. All Ct values ≥ 38.0 are considered negative in the RT-PCR.

Fig 3. Predictive values for the ZIKV RT-LAMP assay based on pre-test probability percentages of ZIKV infection.

Positive (solid black line) and negative (solid red line) predictive values (%), and 95% Cis (positive: dashed black line, negative: dashed red line), plotted against pre-test probability (%) for the RT-LAMP assay using all diagnostic samples combined.