A tailored reverse transcription loop‐mediated isothermal amplification for sensitive and specific detection of serotype A foot‐and‐mouth disease virus circulating in pool 1 region countries

• Published in: Transboundary and emerging diseases Vol. 65; no. 6; pp. 1898 - 1908
• Main Authors: Lim, Da‐Rae, Kim, Hye‐Ryung, Park, Min‐Ji, Chae, Ha‐Gyeong, Ku, Bok‐Kyung, Nah, Jin‐Ju, Ryoo, Soyoon, Wee, Sung‐Hwan, Park, Choi‐Kyu
• Format: Journal Article
• Language: English
• Published: Germany Hindawi Limited 01.12.2018
• Subjects: Animal health; Animals; Assaying; Capsid Proteins - genetics; Cell Line; Developing countries; Diagnosis; Diagnostic systems; Disease control; Disease Outbreaks - veterinary; DNA Primers; Foot & mouth disease; Foot-and-Mouth Disease - diagnosis; Foot-and-Mouth Disease - epidemiology; Foot-and-Mouth Disease - virology; Foot-and-Mouth Disease Virus - genetics; Foot-and-Mouth Disease Virus - isolation & purification; foot‐and‐mouth disease virus; Genotypes; LDCs; Nucleic Acid Amplification Techniques - methods; Nucleic Acid Amplification Techniques - veterinary; Nucleic acids; Outbreaks; Polymerase chain reaction; Real-Time Polymerase Chain Reaction - methods; Reverse Transcriptase Polymerase Chain Reaction - veterinary; Reverse transcription; RT‐LAMP; Sensitivity analysis; Sensitivity and Specificity; Serogroup; serotype A; Serotypes; Strains (organisms); Viruses; VP1 protein; RT-LAMP; serotype A; foot-and-mouth disease virus
• ISSN: 1865-1674; 1865-1682
• DOI: 10.1111/tbed.12971

Rapid and accurate diagnosis of foot‐and‐mouth disease viruses (FMDV) is essential for the prompt control of FMD outbreaks. Reverse transcription polymerase chain reaction (RT‐PCR) and real‐time quantitative RT‐PCR (qRT‐PCR) are used for routine FMDV diagnosis as World Organisation for Animal Health‐recommended diagnostic assays. However, these PCR‐based assays require sophisticated equipment, specialized labour, and complicated procedures for the detection of amplified products, making them unsuitable for under‐equipped laboratories in developing countries. In this study, to overcome these shortcomings, a simple, rapid, and cost‐effective reverse transcription loop‐mediated isothermal amplification (RT‐LAMP) assay was developed for the sensitive and specific detection of serotype A FMDV circulating in the pool 1 region. The amplification could be completed in 40 min at 62°C, and the results could be visually detected by the naked eye without any additional detection systems. The assay specifically amplified the VP1 gene of the Sea‐97 genotype of serotype A FMDV, but it did not amplify other viral nucleic acids. The limit of detection of the assay was 102 TCID50/ml, which is 10 times more sensitive than RT‐PCR and is comparable to the sensitivity of qRT‐PCR. Evaluation of the assay using different FMDV strain serotypes showed 100% agreement with the results of RT‐PCR. Surprisingly, the previously reported RT‐LAMP assay did not detect all eight tested strains of serotype A FMDVs, due to multiple mismatches between primer and template sequences, demonstrating that it is not suitable for detecting serotype A FMDVs circulating in pool 1‐region countries. Conversely, the newly developed RT‐LAMP assay using improved primers can rapidly and accurately diagnose the genotype of Sea‐97 strains of serotype A FMDVs from the pool 1 region. The established RT‐LAMP assay in this study is a simple, rapid, specific, sensitive, and cost‐effective tool for the detection of serotype A FMDV in the resource‐limited pool 1‐region countries.