Development of conventional and real-time PCR assays for the rapid detection of group B streptococci

Background: Group B streptococci (GBS), or Streytococcus agalactiae, are th e leading bacterial cause of meningitis and bacterial sepsis in newborns. C urrently available rapid methods to detect GBS from clinical specimens are unsuitable for replacement of culture methods, mainly because of their lack of sensitivity. Methods: We have developed a FCR-based assay for the rapid detection of GBS . The cfb gene encoding the Christie-Atkins-Munch-Petersen (CAMP) factor wa s selected as the genetic target for the assay. The PCR primers were initia lly tested by a conventional PCR method followed by gel electrophoresis. Th e assay was then adapted for use with the LightCycler(TM). For this purpose , two fluorogenic adjacent hybridization probes complementary to the GBS-sp ecific amplicon were designed and tested. In addition, a rapid sample-proce ssing protocol was evaluated by colony-forming unit counting and PCR. A tot al of 15 vaginal samples were tested by both standard culture method and th e two PCR assays. Results: The conventional PCR assay was specific because it amplified only GBS DNA among 125 bacterial and fungal species tested, and was able to dete ct all 162 GBS isolates from various geographical areas. This PCR assay all owed detection of as few as one genome copy of GBS. The real-time PCR assay was comparable to conventional PCR assay in terms of sensitivity and speci ficity, but it was more rapid, requiring only similar to 30 min for amplifi cation and computer-based data analysis. The presence of vaginal specimens had no detrimental effect on the sensitivity of the PCR with the sample pre paration protocol used. All four GBS-positive samples identified by the sta ndard culture method were detected by the two PCR assays. Conclusion: These assays provide promising tools for the rapid detection an d identification of GBS. (C) 2000 American Association for Clinical Chemistry.