快速检测细菌并革兰分型的半巢式聚合酶链反应及其初步应用

目的以半巢式聚合酶链反应(PCR)检测细菌及作革兰阴、阳性分型,并与细菌培养法比较。方法以细菌16SrRNA基因为靶序列,采用一对通用引物(pm1,pm3)和一条革兰阴性型特异性引物(pm2),以半巢式PCR方法扩增实验室保留菌株的DNA并作出革兰染色分型;以人类外周血白细胞基因组DNA、HBVDNA阳性血清以及白假丝酵母菌为对照,检测此方法的特异性;采用倍比稀释菌液作敏感性实验;与细菌培养法比较,验证此方法检测临床标本的敏感性。结果对17个实验室保留菌株进行检测,以通用引物对作第1次PCR均得到371bp长度的DNA片段;再以革兰阴性菌特异引物对(pm2,pm3)作第2次PCR,9种阴性菌均得到353bp的DNA片段,而8种阳性菌未被扩增。特异性实验表明,此通用引物与人类基因组DNA、真菌及病毒无交叉反应。敏感性实验表明,采用半巢式PCR可检测出3个CFU的细菌。对120份临床标本检测,半巢式PCR检测阳性率(29.2%)显著高于细菌培养法检测阳性率(17.5%)。结论此半巢式PCR检测细菌方法,具有特异、敏感、快速的特点,并能对细菌进行革兰阴性、阳性分型,可用于临床感染性疾病的初步诊断。

Semi-nested polymerase chain reaction used for the rapid detection of bacteria and the Gram stain typing and its preliminary applications

In the present study, the semi-nested polymerase chain reaction(PCR) was used for the rapid detection of bacteria and for the Gram stain typing and the result of this method was compared with that of bacterial culturing method. The bacterial 16S rRNA gene was used for the target sequence, and a pair of universal primers (pm1 and pm3) and a Gram-negative (G-)type-specific primer (pm2) were used to amplify the DNA of the laboratory stored strains of bacteria by semi-nested PCR. The human genomic DNA from peripheral blood white cells, HBV-DNA positive sera and C.albicans DNA were used as control for comparison to test the sensitivity of this method to detect the organisms in clinical specimens. The same clinical specimen was examined with two methods of the semi-nested PCR and the bacterial culture method used in clinics in order to compare the accuracy for clinical specimens. The experimental results showed that a 371 bp DNA fragment was amplified from all the 17 species of bacteria stored in our laboratory by using the universal primers(pm1 and pm3),while a 353 bp fragment was obtained from 9 species of Gram-negative bacteria by using G-specific primers (pm2 and pm3), but no positive result was obtained from 8 species of G+ bacteria. With the templates of human genomic DNQA, C.albicans-DNA and HBV-DNA positive sera, no amplified signal was observed when the PCR was operated by using the universal primers.The sensitivity of the semi-nested PCR was as high as 3 CFU of bacteria, and the positive rate of the semi-nested PCR as demonstrated by the detection of bacteria in 120 clinical specimens was 29.2%, while that obtained by the bacterial culture method was 17.5%. In addition, there was significantly statistical difference in the positive rates between these two methods. It is concluded that the semi-nested PCR method possesses the advantages of high specificity and sensitivity, simple operation and rapid detection of bacteria in clinical specimens, and can be used for the preliminary diagnosis of bacterial infections.