Detection of Streptococcus uberis in Bovine Milk Using a Simplified DNA Preparation Method and Colorimetric LAMP Assay

Bovine mastitis caused by Streptococcus uberis (S. uberis) remains a major challenge of dairy production system worldwide. This study aimed to develop a colorimetric loop-mediated isothermal amplification (LAMP) assay and a conventional PCR assay targeting the 16S rRNA gene for detection of S. uberis in bovine milk. Both assays were evaluated with a simplified HotSHOT (HS) DNA preparation method that can be completed in under 10 min and was compared with a commercial DNA extraction kit before LAMP and PCR testing. LAMP reactions using HS-extracted DNA required an extended incubation time of 30 min, compared with the standard 60 min, to account for the crude DNA preparation. Using tenfold serial dilutions of purified DNA in nuclease-free water, the limit of detection was 1.84 × 10−4 ng/µL for LAMP and 1.84 × 10−5 ng/µL for PCR. In pasteurised milk spiked with S. uberis DNA and extracted by HS, the limit of detection was 10 ng/mL for LAMP and 1 ng/mL for PCR. Using the commercial kit, both methods achieved 1 ng/mL. Among 17 culture-confirmed clinical milk samples extracted by HS, LAMP detected S. uberis in 14 samples (82.35%; 95% CI: 0.56 to 0.96), while PCR detected 15 samples (88.24%; 95% CI: 0.64 to 0.99), with no significant difference between methods (McNemar’s test, p > 0.05). Both LAMP and PCR showed 100% analytical specificity against 10 non-target bacterial species under the conditions tested. These findings support HS combined with colorimetric LAMP as a practical diagnostic workflow that warrants further validation under field conditions.