Combination of procedure for ultra rapid extraction (PURE) and loop-mediated isothermal amplification (LAMP) for rapid detection of Mycoplasma bovis in milk

摘要

Polymerase chain reaction (PCR) is typically used for the early detection of mycoplasma in bovine milk; it requires 3 days to obtain results because of the necessary enrichment process. A more rapid, simple, and accurate detection method is required to directly detect the Mycoplasma bovis (M. bovis) gene in milk. In this study, we assess the utility of combining the following two methods to achieve this goal: the loop-mediated isothermal amplification (LAMP), which is more sensitive than PCR, and the procedure for ultra rapid extraction (PURE), which adsorbs and filters components that inhibit DNA amplification/detection. LAMP was examined using DNA extracts obtained by four methods. This showed that PURE had the highest sensitivity and specificity and that the combination of PURE and LAMP was able to detect M. bovis in milk. We then showed that the detection limit of M. bovis was 102 colony-forming units per milliliter of milk using the PURE–LAMP. Finally, the respective sensitivities of the PURE–LAMP and PCR were 57% and 86% for bulk tank milk, 89% and 74% for mature milk, 85% and 92% for colostrum/transitional milk, and 97% and 95% for mastitis milk. The specificity was 100% for all milk samples in both LAMP and PCR. We conclude that PCR was suitable for detecting mycoplasma in bulk tank milk and that the PURE–LAMP could detect mycoplasma within 2 hr and was also effective for mature and mastitis milk.