The performance of a commercial, real-time PCR assay was compared with traditional bacterial culture for the identification of @entity8211 and @entity794 in @entity291 milk collected at different stages of lactation. Initial validation tests using fresh and frozen quarter milk samples identified factors that affected the success of the PCR. Therefore, the standard protocol was adjusted for samples collected at the first milking postpartum (colostrum) and from clinical @entity1271 cases. The adjustment involved PCR testing both undiluted and diluted (1 in 10 with sterile water) DNA extracts. The performance comparison between culture and the PCR assay used milk samples collected aseptically from individual quarters of mixed-age spring-calving dairy @entity291 , during early, mid, and late lactation. Bacterial culture results were used to select a subset of samples for PCR testing (n = 315) that represented quarters with a current or prior Strep. uberis or Staph. @entity281 . Compared with culture, PCR had a sensitivity of 86.8% and specificity of 87.7% for detecting Strep. uberis (kappa = 0.74) and 96.4% and 99.7%, respectively, for detecting Staph. aureus (kappa = 0.96). The dilution of DNA extracts for colostrum and clinical samples increased the relative sensitivity from 79.2% to 86.8% for Strep. uberis detection and from 92.9% to 96.4% for Staph. aureus, presumably through diluting unidentified PCR inhibitors. The sensitivity for detecting Strep. uberis using PCR, relative to culture, was similar throughout lactation (85-89%), whereas relative specificity was lowest immediately postcalving (64%) but improved in mid and late lactation (98%). Specificity estimates for samples collected in early lactation can be optimized by reducing the cutoff cycle threshold (Ct) value from the recommended value of 37 to 34. Although using this value improved specificity (77%), it reduced test sensitivity (77%). The PCR assay lacked agreement with culture in early lactation, specifically for diagnosing Strep. uberis. Thus, PCR should not be used as the only tool for diagnosing @entity1271 in early lactation.

Evaluating a commercial PCR assay against bacterial culture for diagnosing @entity8211 and XXXX throughout lactation.

@entity794 :: ('1280', 'Species') :: ['Staphylococcus aureus'] @entity281 :: ('MESH:D007239', 'Disease') :: ['aureus infection'] @entity8211 :: ('1349', 'Species') :: ['Streptococcus uberis'] @entity1271 :: ('MESH:D008413', 'Disease') :: ['mastitis'] @entity291 :: ('9913', 'Species') :: ['cows', 'bovine']
@entity794 :: (1280,Species) :: ['Staphylococcus aureus']
