Abstract
Infection with Zika virus (ZIKV) is of growing concern since infection is associated with the development of congenital neurological disease. Quantitative reverse transcription PCR (qRT-PCR) has been the standard for ZIKV detection; however, Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) may allow for faster and cheaper testing. Studies have suggested that ZIKV detection in urine is more sensitive and has a longer window of detection compared to serum and saliva. The objective of this study was to develop a urine diagnostic test that could be completed in under 30 minutes. Urine samples spiked with ZIKV or dengue virus were tested using RT-LAMP as well as by conventional quantitative qRT-PCR. These techniques were then validated using crude lysates made from ZIKV infected mosquitoes in addition to urine and serum samples from ZIKV infected patients. RT-LAMP specifically detected ZIKV in urine and serum for ZIKV infected patients and crude mosquito lysates. This test was performed in under 30 minutes and did not require RNA extraction from urine nor mosquitos. This approach could be used for monitoring of exposed individuals, especially pregnant women, couples wanting to conceive, or individuals with suspicious symptoms as well as surveillance of mosquito populations.
Highlights
Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a one-step nucleic acid amplification method based on PCR technology that has been used to diagnose infectious diseases6
The Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) reaction required all 6 primers to work under the optimized conditions; removing the forward and backward inner primers or the loop primers did not result in a positive result (Fig. 1B)
In order to determine the lower detection limit of the RT-LAMP reaction for Zika virus (ZIKV), a dilution series ranging from 1 × 103 to 1 × 108 plaque-forming units (PFU) ZIKV was amplified (Fig. 2)
Summary
Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a one-step nucleic acid amplification method based on PCR technology that has been used to diagnose infectious diseases. RT-LAMP include: (1) high specificity; (2) high sensitivity; (3) short turn-around time; (4) robustness in various pH and temperature ranges; (5) low cost and stability of reagents at room temperature; (6) it has been an applied technology for bacterial infection in urine. This study describes a RT-LAMP methodology that can detect ZIKV in patient samples and crude Aedes aegypti lysates without RNA isolation. The test could be used at the point-of-care by untrained personnel for the monitoring of exposed individuals as well as surveillance of disease vectors
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