Sensitive and rapid detection of Zika virus by loop-mediated isothermal amplification

Abstract

Zika virus (ZIKV) is a mosquito-borne flavivirus, which is a pathogen affecting humans in Africa, Asia, and America. It is necessary to detect ZIKV with a rapid and sensitive molecular method to guide timely treatment. In this study, a loopmediated isothermal amplification (LAMP) assay was described, which is an attractive option as a fast, sensitive, and specific method for ZIKV detection using the NS5 protein coding region and the envelope protein (EP) coding region as target sequences. Two different techniques, a calcein/Mn 2+ complex chromogenic method and real-time turbidity monitoring, were employed. The specificity and sensitivity of the LAMP assay were determined. The assay's detection limit was 0.5 × 10 −9 pmol/µl DNA for NS5 protein coding region and 1.12 × 10 −11 pmol/µl DNA for E coding region, respectively, which is a 100-fold increase in sensitivity compared with real-time reverse transcription-polymerase chain reaction (RT-PCR) and conventional PCR. All 12 non-ZIKA respiratory pathogens tested were negative for LAMP detection, indicating the high specificity of the primers for ZIKV. In conclusion, a visual detection LAMP assay was developed, which could be a useful tool for primary quarantine purposes and clinical screening, especially in situations where resources are poor and in point-of-care tests.

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

“…Additionally, our RT-LAMP assay was also able to detect ZIKV in mosquito samples that had been previously assayed as negative by RT-qPCR with Ct value ranging from 38.6 to 40.3. Taken together, our results suggest that our modification of the RT-LAMP assay could provide sensitivity 10,000 times greater than RT-qPCR for the detection of ZIKV in mosquito samples, thus corroborating previous studies demonstrating that the sensitivity of the LAMP method can be superior to RT-qPCR [42,43,59]. Importantly, there are a number of reasons that might have involved for this variation in sensitivity, including differences in enzymes and research suppliers, primers, detection systems, and type of biological samples.…”

“…Zhao and Feng developed a LAMP assay that detected 0.5 × 10 −9 or 1.12 × 10 −11 pmol/µl DNA for NS5 or E genes, respectively. This represented a 100-fold greater sensitivity compared with conventional PCR and RT-qPCR [43].…”

“…Importantly, the simple, single-temperature incubation allows LAMP reactions to be performed without expensive equipment, directly in the field [36]. Since the 2015 emergence of the ZIKV in Brazil, many LAMP assays have been developed for diagnosis by research groups across the world [27,[37][38][39][40][41][42][43][44][45]. These diagnostic platforms based on LAMP have proven to be specific, sensitive and inexpensive POC tools that can be applied even in resource-limited regions of the world.…”

A Reverse Transcription Loop-mediated Isothermal Amplification for Broad Coverage Detection of Asian and African Zika Virus Lineages

“…Recently, several research groups have developed RT-LAMP assays for the detection of ZIKV by targeting the different regions of the ZIKV genome with excellent sensitivity and speci city [31][32][33][34][35][36][37][38][39][40][41][42][43]. They however, designed the primers for the RT-LAMP assays based only on a single lineage of ZIKV, which is either Asian [33][34][35][36][37][39][40] or African [32] lineage. Many studies designed the RT-LAMP primers based primarily on the sequences of the Asian ZIKV since the recent epidemics in the Paci c islands and Americas [5][6][7]11] were caused by ZIKV from the Asian lineage [53].…”

“…The loop-mediated isothermal ampli cation (LAMP) method that provides simple, sensitive and rapid nucleic acid ampli cation under isothermal conditions promises to be a good alternative to qRT-PCR [30]. The method has been used for the detection of various RNA viruses including ZIKV [31][32][33][34][35][36][37][38][39][40][41][42][43]. Majority of the earlier reports of RT-LAMP for ZIKV used primers designed based on the lineage-speci c regions of ZIKV genome [31][32][33][34][35][36][37][38][39][40].…”