荧光偏振和多重聚合酶链技术在肺炎支原体、肺炎衣原体检测中的应用

Application of fluorescence polarization and multiple PCR in the detection of Mycoplasma pneumoniae and Chlamy-dia pneumoniae

目的:应用多重聚合酶链反应和荧光偏振检测技术建立一种方便可靠、简单经济的肺炎支原体、肺炎衣原体DNA同步检测方法.方法:以与肺炎衣原体、肺炎支原体16SRNA序列互补的特异、无交叉反应的两对引物在同一反应体系中行多重聚合酶链反应扩增阴阳性对照及521例患儿咽喉分泌物,将荧光偏振检测技术与模板指导的DNA末端延伸反应(TDI-FP)结合,对样本PCR扩增产物进行进一步检测,并与测序方法结果比较.结果:应用多重聚合酶链反应和TDI-FP技术检测患者521例,肺炎衣原体感染阳性121例,肺炎支原体感染阳性113例,与测序检测方法符合率100%,其中肺炎衣原体、肺炎支原体双重感染阳性10例.结论:建立了基于荧光偏振技术检测肺炎衣原体、肺炎支原体的新方法,具有良好的特异性,方便简单,无需标记探针,可用于临床检测.

AIM： To develop a simple, economical, accurate and practical method for the detection of Mycoplasma pneumoniae （MP） and Chlamydia pneumoniae （CP） using Template-directed dye-terminator incorporation with fluorescence polarization （TDI- FP） and a multiple PCR. METHODS： Two-pair primers of MP and CP were used to amplify DNA of positive and negative controls and 521 samples by a multiple PCR, then the PCR products were identified by TDI-FP： MP and CP specific probe hybridization and special fluorogenic label ddNTP incorporation. A fluorogenic ddNTP was directly added to the end of probe under the direction of template. The results were measured by a fluorescence-polari- zation microplate reader and compared with the results of sequen- cing. RESULTS： There were 113 patients infected with MP, 121 patients infected with CP, 10 with double infections. Compared with the results of sequencing, the results measured by TDI-FP showed an excellent overall agreement when single infection was taken into account. The proposed method could detect single or multiple infection by a multiple PCR, but DNA sequencing method was limited. TDI-FP method reached the detection level （10 copies） and the data of FP values showed excellent repro- ducibility. CONCLUSION： The proposed method allows a sensi- tive, special, simple and economical detection of MP and CP by a multiple-PCR without any use of label probes. It is expected to be an extremely useful tool in clinic.