新型MGB探针特异性检测变形链球菌

A novel TaqMan~ MGB probe for specifically detecting Streptococcus mutans

目的:设计一种新型TaqManMGB探针,利用实时荧光定量PCR检测变形链球菌,提高PCR法检测变形链球菌的特异性,减少检测的假阳性。方法:提取6种不同链球属细菌的DNA,分别进行巢式PCR和TaqManMGB实时荧光定量PCR,比较两种PCR方法检测变形链球菌的特异性。巢式PCR第1轮扩增的引物是细菌16S rRNA基因通用引物,第2轮扩增使用变形链球菌16S rRNA基因可变区序列的特异性引物。TaqManMGB实时荧光定量PCR的引物与第2轮巢式PCR特异性引物相同,设计的MGB探针序列与变形链球菌16S rRNA基因中特异性序列相匹配,不与其他细菌的基因序列匹配。将变形链球菌标准株的DNA样本从2.5 mg/L至0.16μg/L按5倍梯度稀释,制备出实时荧光定量PCR的标准曲线。结果:变形链球菌和格氏链球菌在巢式PCR中均扩增出282 bp的DNA片段,出现假阳性结果。TaqManMGB实时荧光定量PCR能定量检出变形链球菌标准株和临床株,不检出其他链球菌,比巢式PCR特异性更好。TaqManMGB实时荧光定量PCR对变形链球菌DNA的最低检出浓度为20μg/L。结论:本研究设计出一种针对变形链球菌的TaqManMGB探针,建立利用TaqManMGB实时荧光定量PCR特异性检测口腔中变形链球菌的方法。

Objective：To design a new TaqMan（R） MGB probe for improving the specificity of Streptococ- cus mutans＇s detection. Methods： We extracted six DNA samples from different streptococcal strains for PCR reaction. Conventional nested PCR and TaqMan（R） MGB real-time PCR were applied independently. The first round of nested PCR was carried out with the bacterial universal primers, while a second PCR was conducted by using primers specific for the 16S rRNA gene of Streptococcus mutans. The TaqMan（R） MGB probe for Streptococcus mutans was designed from sequence analyses, and the primers were the same as nes- ted PCR. Streptococcus mutans DNA with 2.5 mg/L was sequentially diluted at 5-fold intervals to 0.16 μg/ L. Standard DNA samples were used to generate standard curves by TaqMan（R） MGB real-time PCR. Results： In the nested PCR, the primers specific for Streptococcus mutans also detected Streptococcus gordo- nil with visible band of 282 bp, giving false-positive results. In the TaqMan（R） MGB real-time PCR reaction, only Streptococcus mutans was detected. The detection limitation of TaqMan（R） MGB real-time PCR for Strep- tococcus mutans 16S rRNA gene was 20 μg/L. Conclusion： We designed a new TaqMan（R） MGB probe, and successfully set up a PCR based method for detecting oral Streptococcus mutans. TaqMan（R） MGB real- time PCR is a both specific and sensitive bacterial detection method.