Technical note: Development of a closed-tube isothermal multiple self-matching-initiated amplification assay for...

Staphylococcus aureus is one of the most common mastitis-causing bacteria in dairy cows. It is associated with reduced production performance in animals and with huge financial losses for the dairy industry worldwide. An accurate and sensitive method for the early diagnosis and identification of Staph. aureus in milk samples is essential. The present study aimed to establish a closed-tube isothermal multiple self-matching-initiated amplification (IMSA) technique for visual confirmation of the presence of Staph. aureus targeting the nuc conserved sequence gene. The specific primers successfully amplified the target sequence within 45 min at 63°C reaction temperature and using the optimal components of the reaction system. The positive amplicon showed bright green fluorescence under UV light when mixed with the chromogenic substrate SYBR Green I, and the negative samples remained orange in color. We observed fluorescence and a ladder-like pattern in the IMSA amplicon for all Staph. aureus strains, and we observed no significant change for the non-Staph. aureus strains. The IMSA assay had high specificity compared with loop-mediated isothermal amplification (LAMP): it confirmed the presence of all 7 Staph. aureus strains, and we found no false-positive results for the 12 non-Staph. aureus strains. The lower limit of detection for the IMSA assay was 1 × 10² cfu/mL, 10-fold more sensitive than the results obtained using LAMP. We also successfully applied the IMSA assay to confirm the presence of Staph. aureus in milk samples of cows with mastitis, and the results were consistent with those of LAMP and real-time PCR. The present study reports the use of IMSA to confirm the presence of Staph. aureus and provides a potentially useful method for rapid preliminary screening for Staph. aureus.