Development of Universal and Lineage-Specific Primer Sets for Rapid Detection of the Zika Virus (ZIKV) in Blood and Urine Samples Using One-Step Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP)

摘要

Zika is a mosquito-borne disease that has been posing a significant threat to public health in recent years. The Zika virus (ZIKV), the causative agent of this disease, is classified into 2 distinct genetic lineages, namely Asian and African. While molecular nucleic acid analysis methods have been shown to be useful for the diagnosis of ZIKV infection, the development of assays based on one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) offers several advantages, such as shorter incubation times, ease of handling, and rapid detection. In this study, a universal LAMP primer set was developed to target conserved sequences of known ZIKV lineages. Additionally, the Af7462 and As1788 primer sets were designed based on LAMP-based single-nucleotide polymorphism (SNPs) typing for the specific detection of the African and Asian lineages. The developed RT-LAMP assays could specifically detect the African and Asian lineages of ZIKV, with a detection limit ranging from 0.17 FFU/mL to 2.3×102 FFU/mL. As ZIKV viremia ranges between 102 to 106 PFU/mL or 103 to 106 copies/mL, the data indicate that the viremia range of clinical samples is within the detection range of our assay. Due to the high specificity and sensitivity, as well as the ease of use of our assay, it could potentially be used for early clinical diagnosis applications.