Development of a Loop-Mediated isothermal amplification (LAMP) assay for rapid, simple and sensitive detection of Mycobacterium avium subsp. Paratuberculosis

Abstract

Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne’s disease, chronic progressive enteritis in ruminants. The organism has also been isolated from primates, including humans. The disease is economically important in the cattle industry but its control is hampered by the lack of accurate rapid diagnostic tests. Range of diagnostic tests is available, but all have inborn limitations. In the present study, a loop-mediated isothermal amplification (LAMP) assay for the rapid and simple detection of Mycobacterium avium subsp. paratuberculosis (MAP) was developed. Six primers were specially designed for recognizing eight distinct sequences of insertion sequence 900 (IS900). Optimization of LAMP reaction was performed. Time and temperature conditions for amplification of MAP were optimized as 60 min at 63°C. LAMP produces extremely large amounts of amplified products and enables simple detection methods such as visual judgment by the turbidity or fluorescence of the reaction mixture, which is kept in the reaction tube. The visual detection eliminates the need for time-consuming electrophoresis and costly specialized equipment. The performance of LAMP was compared to that of a highly sensitive Nested PCR. The sensitivity of LAMP assay for detection of MAP was 4 fg and the specificity was 100%. The sensitivity was 1000 times greater than the Nested-PCR. Furthermore, the LAMP assay described in this report is simple to use, inexpensive, highly sensitive, and particularly well suited for the early diagnosis of MAP in less well equipped laboratories and in rural settings where resources are limited.