Bergh K, Stoelhaug A, Loeseth K, Bevanger L
Norwegian University of Science & Technology, Department of Laboratory Medicine, N-7006 University Hospital of Trondheim, Norway.
Indian J Med Res. 2004 May;119 Suppl:221-3.
BACKGROUND & OBJECTIVES: Implementation of a screening based strategy for the prevention of perinatal group B streptococcus (GBS) disease is anticipated to increase the demand of fast laboratory techniques. The aim of the present study was to develop a real-time PCR method for the specific detection of GBS in vaginal swabs.
Based on partial sequencing of the sip gene, primers and a TaqMan hybridization probe were constructed for a real-time PCR assay. The performance of the assay was tested on 100 consecutive vaginal specimens submitted to the laboratory for culture. Lysis of bacteria and DNA extraction was performed by lysozyme, mutanolysin, proteinase K and Quiagen spin columns. PCR was performed using LightCycler. Highly purified DNA from GBS was used as positive control.
Of the 100 samples investigated, 25 were positive by culture (16 abundant/moderate growth, 6 sparse growth and 3 after enrichment only). At a cut-off of 12 fg per reaction (corresponding to 4 bacterial cells), PCR was positive in 32 samples. A complete concordance was noted between PCR positivity and abundant/moderate and sparse growth by culture. One of 3 samples that were positive by culture only after enrichment was negative in PCR. On the other hand, 8 samples were positive by PCR and negative by culture.
INTERPRETATION & CONCLUSION: The real-time PCR assay was sensitive and rapid and enabled detection of GBS in less than 2 h after collection. Due to the rapidity of the assay by which results could be obtained, the assay harbors the potential for intrapartum detection of GBS.
预计实施基于筛查的策略预防围产期B族链球菌（GBS）疾病会增加对快速实验室技术的需求。本研究的目的是开发一种用于阴道拭子中GBS特异性检测的实时PCR方法。
基于sip基因的部分测序，构建引物和TaqMan杂交探针用于实时PCR检测。在提交至实验室进行培养的100份连续阴道标本上测试该检测方法的性能。通过溶菌酶、变溶菌素、蛋白酶K和Qiagen旋转柱进行细菌裂解和DNA提取。使用LightCycler进行PCR。来自GBS的高度纯化DNA用作阳性对照。
在研究的100份样本中，25份培养阳性（16份生长丰富/中等，6份生长稀疏，3份仅在富集后阳性）。在每个反应12 fg的截断值（相当于4个细菌细胞）时，PCR在32份样本中呈阳性。PCR阳性与培养的丰富/中等及稀疏生长之间存在完全一致性。3份仅在富集后培养阳性的样本中有1份PCR阴性。另一方面，8份样本PCR阳性而培养阴性。
实时PCR检测灵敏且快速，能够在采集后不到2小时检测到GBS。由于该检测方法能够快速获得结果，它具有在分娩期检测GBS的潜力。
