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.

Loop-Mediated Isothermal Amplification (LAMP) for the Diagnosis of Zika Virus: A Review

“…In contrast, the two-step LAMP protocol needs the addition of a reverse transcriptase enzyme together with the DNA polymerase ( Figure 2). The use of the two-step LAMP protocol to detect ZIKV has been described by several groups [27,28,57,59]. However, the two-step protocol does carry some disadvantages that make it less practical than a one-step LAMP.…”

“…High analytical specificity for LAMP-based ZIKV assays has been reported in both mosquito and human samples. In these studies, no detectable cross-reaction was seen against dengue virus (DENV 1-4), chikungunya virus (CHIKV), yellow fever virus (YFV), West Nile virus (WNV), Saint Louis encephalitis (SLEV), Western equine encephalitis virus (WEEV), Venezuelan equine encephalitis virus (VEEV), Rift Valley fever virus (RVFV), Bussuquara virus (BSQV), Langat virus (LGTV), Powassan virus (POWV), Ilheus virus (ILHV), Plasmodium falciparum, influenza virus and other pathogens [37,39,41,42,59].…”

Rapid diagnosis of Zika virus through saliva and urine by Loop-mediated isothermal amplification (LAMP)

“…The assay uses primers designed to target the capsid gene and generate real-time amplification that can be detected in less than 30 min. These characteristics make LAMP assays ideal to use in portable devices, which can be configured to diagnose diseases at point of care facilities [14,15]. Therefore, the detection of ZIKV RNA by LAMP provides the basis of a test that can readily facilitate the diagnosis of new cases.…”

Valve‐Enabled Sample Preparation and RNA Amplification in a Coffee Mug for Zika Virus Detection

“…[4] While reverse transcription PCR (RT-PCR) is often used as aN AT,R T-LAMP has also been extensively used for virus detection owing to its robustness,h igh sensitivity and specificity,s hort incubation time,a nd simplified thermal management. [7][8][9][10][11][12][13][14] Although RT-LAMP allows virus samples to be used for amplification without sample preparation, the small sample volume and dilution steps involved decrease detection sensitivity. [7][8][9][10][11][12][13][14] Although RT-LAMP allows virus samples to be used for amplification without sample preparation, the small sample volume and dilution steps involved decrease detection sensitivity.…”