植原体TaqMan探针实时荧光PCR检测鉴定方法的建立

 本研究成功建立了植原体分类鉴定和检测的TaqMan探针实时荧光PCR方法,该方法根据植原体16S rDNA保守区设计了1个TaqMan广谱探针和3个植原体组间点突变特异性探针,并对9种植原体和5种细菌以及3个植物样本进行实时荧光PCR。结果表明,用广谱探针可检测到所有植原体产生荧光信号,而细菌不产生荧光信号。当用植原体组间特异性探针检测时,仅能检测到该组植原体产生荧光信号,检测的敏感性比常规的PCR-电泳检测高约100倍、检测速度有较大提高。由于PCR产物是荧光探针检测,本方法特异性强,并可以用组特异探针直接确定植原体种类。实验采用完全闭管检测,降低了污染机会。本研究为其它原核生物、特别是不能培养菌、难培养菌的检测鉴定和分类提供了新方法。

ESTABLISHMENT OF REAL-TIME FLUORESCENT PCR METHOD WITH TaqMan PROBE FOR PHYTOPLASMA DETECTION AND IDENTIFICATION

A real time fluorescent PCR(RTF PCR) method was established to detect and identify phytoplasmas. One universal and three group specific TaqMan probes were designed based on the conserved region of the 16S rDNA of phytoplasmas,and they were used to detect 9 strains of phytoplasmas and 5 species of bacteria and three samples by RTF PCR. The results showed that the universal probe could detect all the phytoplasmas used, while no signal was detected for bacteria. For three group specific probes, each could detect its own group specifically. The method was 100 times more sensitive than normal PCR and more specific and much faster due to fluorescent probe used. It could even identify the phytoplasma species while the group specific probes used. Few contamination would occur because whole detection process was finished in the contained tubes. RTF PCR could be a new technique for detecting other prokaryotes, especially the fastidious bacteria.