Development of a Rapid PCR Test for Identification of Streptococcus agalactiae in Milk Samples Collected on Filter Paper Disks

Streptococcus (Strep.) agalactiae is one of the major pathogens of bovine mastitis and is the main cause of subclinical infection. This study attempted to develop a rapid PCR diagnosis procedure using milk samples collected on filter paper disks. Chromatographic filter paper was employed as the preservation media and kept at room temperature for one to four weeks. The revival rate of Strep. agalactiae kept on dried filter paper disks was affected by the pretreatment preservation time. The revival test suggested that not all the bacteria in artificially contaminated milk samples on the filter paper disks could be recovered. After that, a PCR based on the 16-23S intergenic spacer region of Strep agalactiae was performed. The results distinguished the strep. agalactiae from major pathogens of bovine mastitis at a 2×10 colony forming units (CFU)/ml level, which showed similar sensitivity to the results from liquid milk samples. The results also showed that milk samples collected on filter paper disks could be kept at room temperature for one to four weeks with little negative effect on sensitivity and specificity. The field test showed that the diagnostic sensitivity and specificity was 96.15% and 98.60%, respectively. In conclusion, the protocol will provide a rapid and economic procedure for the detection of bovine mastitis. (