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Rapid detection of IS1081 of Mycobacterium bovis using CRISPR/Cas12a system combined with recombinase polymerase amplification

S H Son(1) J H Lee(1) H Y Lee(1) S S Yoon(1) J S Choi(1)

1:Animal and Plant Quarantine Agency

Mycobacterium tuberculosis variant bovis (M. bovis), a member of Mycobacterium tuberculosis complex, is the most important causative agent of tuberculosis (TB) in cattle. It has a wide host range and can also cause TB in human through zoonotic transmission. Although it is considered less serious in some developed countries, zoonotic TB caused by M. bovis infection is a considerable public health threat in areas where milk is frequently consumed without heat-treatment or close human-animal contact is common. In order to prevent the spread of zoonotic TB, it is necessary to quickly identify cattle infected with M. bovis. Despite the rapid diagnostic platform using sputum for human TB have already been distributed, it has not been evaluated for animal-derived samples and the culture and identification of M. bovis are still the gold standard test for bovine TB diagnosis. For rapid detection of M. bovis, we developed a clustered regularly interspaced short palindromic repeats  (CRISPR)/CRISPR-associated (Cas) 12a system-based assay combined with recombinase polymerase amplification (RPA) detecting insertion sequence element IS1081, one of the widely used genetic markers for M. bovis detection. Using this detection method, the presence of IS1081 in genomic DNA of M. bovis AN5 and BCG strains could be confirmed within 40 min and none of the false-positive reactions for 19 other bacteria including 11 nontuberculous mycobacteria (NTM) strains were shown. Although further evaluation on clinical samples is required, the RPA-CRISPR/Cas12a assay may contribute to zoonotic TB control through more rapid, simple and accurate detection of M. bovis.

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