Detection and discrimination of multiple strains of Zika virus by reverse transcription-loop-mediated isothermal amplification

Hiroka Aonuma,Shigeru Tajima,Fumihiro Kato,Masayuki Saijo,Tokio Hoshina,Itoe Iizuka-Shiota,Seiji Hori,Hirotaka Kanuka

doi:10.1186/s41182-020-00274-z

Abstract

BackgroundMonitoring both invasion of Zika virus disease into free countries and circulation in endemic countries is essential to avoid a global pandemic. However, the difficulty lies in detecting Zika virus due to the large variety of mutations in its genomic sequence. To develop a rapid and simple method with high accuracy, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) was adopted for the detection of Zika virus strains derived from several countries.ResultsCommon primers for RT-LAMP were designed based on the genomic sequences of two standard Zika strains: African lineage, MR-766, and Asian lineage, PRVABC59. RT-LAMP reactions using a screened primer set, targeting the NS3 region, detected both Zika virus strains. The minimum detectable quantity was 3 × 10−2 ng of virus RNA. Measurable lag of reaction times among strains was observed. The RT-LAMP method amplified the target virus sequence from the urine and serum of a patient with a travel history in the Caribbean Islands and also provided a prediction about which lineage of Zika virus strain was present.ConclusionsThe RT-LAMP method using a well-optimized primer set demonstrated high specificity and sensitivity for the detection of Zika virus strains with a variety in genomic RNA sequences. In combination with the simplicity of LAMP reaction in isothermal conditions, the optimized primer set established in this study may facilitate rapid and accurate diagnosis of Zika fever patients with virus strain information.

Highlights

Monitoring both invasion of Zika virus disease into free countries and circulation in endemic countries is essential to avoid a global pandemic
Design and screening of optimized primer set for Zika virus (ZIKV) reverse transcription-loop-mediated isothermal amplification (RT-Loop-mediated isothermal amplification (LAMP)) To identify the infection of both ZIKV MR-766-NIID (MR) and PRVABC59 (PR) strains by Reverse transcription (RT)-LAMP, a common primer set was designed targeting a region covering the whole ZIKV genome (Fig. 1a)
The primer set included several mismatches with the sequence of the PR strain (Fig. 1b). These primer sets were screened for their sensitivity in the RT-LAMP reaction for detecting genomic RNA of the ZIKV MR strain, and a primer set targeting the NS3 region of ZIKV showed the highest sensitivity (Table 1)

Summary

Introduction

Monitoring both invasion of Zika virus disease into free countries and circulation in endemic countries is essential to avoid a global pandemic. To develop a rapid and simple method with high accuracy, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) was adopted for the detection of Zika virus strains derived from several countries. LAMP amplifies a target DNA with high specificity using multiple primers, and it has been used to type single nucleotide polymorphisms in a DNA sequence [6,7,8]. It has been widely used for the detection of mosquitoborne pathogens such as parasites and viruses from both human and vector mosquitoes [9,10,11,12,13]. Reverse transcription (RT)-LAMP has been employed to detect genomic RNA of ZIKV [14,15,16,17], because recent outbreaks have occurred in resource-limited settings, increasing the need for simple assays suitable for reliable, cost-effective, high-throughput ZIKV diagnosis

Methods

Results

Discussion

Conclusion