应用荧光PCR检测土壤、脏器中的炭疽芽孢杆菌

Detection of Bacillus anthracis from environmental samples by real-time fluorescence polymerase chain reaction

目的用荧光定量PCR方法检测土壤、动物脏器和血液中炭疽芽孢杆菌,评价并优选目标核酸纯化方法。方法采用TaqMan荧光定量PCR技术,以炭疽芽孢杆菌pagA、rpoB2引物探针和相应外标、内标为手段,比较4种从土壤中检测炭疽菌及3种从感染脏器及血液中检测炭疽菌的方法,探讨环境和组织样品中抑制因素对荧光定量PCR检测的影响,优选并确定纯化检验手段。结果应用NaI裂解glassmilk纯化炭疽菌核酸并用荧光定量PCR检验,检出底限为2·5万拷贝/g土壤(pagA),从血液样本的检出限为1000拷贝/ml。结论荧光定量PCR技术和NaI裂解glassmilk纯化制备模板的联合应用可灵敏、特异地从土壤、血液中检测炭疽芽孢杆菌。

To establish and assess different methods for detecting Bacillus anthracis from environmental samples by real-time fluorescence polyrnerase chain reaction, different methods for purification of nucleic acids from soil and tissues contaminated by B. anthracis vaccine strain were established. TaqMan Primers and probes designed from pagA （pXO1） and rpoB2 genes （chromatosome DNA） of B. anthracis were used to assess these methods by real-time fluorescence polyrnerase chain reaction. The most effective systems were confirmed by comparing the amplification results. It was found that the method of NaI-glassmilk was the most effective way under these conditions, positive results could be obtained at 1 000 copies/ml or more in crude blood specimens mingled vaccine strain, and same as 25 000 copies or more in one gram soil. The NaI-Glassmilk method and the real-time fluorescence polyrnerase chain reaction could directly and rapidly detect B. anthracis in contaminated issues or soil.