PCR法制备地高辛标记探针原位杂交在人结核杆菌Edman株基因检测中的应用

Detection of Mycobacterium Tuberculosis Edman Strain by Situ Hybridization with Digoxigenin Labeled Probe by PCR

目的建立一种用于临床上检测人感染结核杆菌Edman株的快速敏感的PCR探针原位杂交方法。方法根据NCBI报道的人结核杆菌编码序列，以结核杆菌Edman株Ag85A基因150～841bp之间的基因序列为靶序列，采用PCR法制备对人结核杆菌特异性高的地高辛标记DNA探针（690bp），并对探针进行电泳和测序鉴定。分离培养结核杆菌，通过原位杂交检测所制备探针的特异性；对结核杆菌做10倍比稀释，运用探针对其进行敏感性检测。结果所制备的探针对结核杆菌的杂交反应呈阳性，以dNTP代替地高辛标记探针作为对照组的杂交反应为阴性；通过基因序列测定证实了PCR产物的特异性；PCR法制备地高辛标记探针原位杂交法检测结核杆菌Ag85A基因下限为1．0×10^2du／ml。结论本研究建立的地高辛标记探针原位杂交法对结核杆菌Edman株Ag85A基因的检测具有快速、特异、敏感的特点，适合结核分枝杆菌的临床检测。

Objective To establish a sensitive and quick method of situ hybridization with digoxigenin labeled probe by PCR for determining the mycobacteriurn tuberculosis infection in clinic. Methods According to the sequence of the myco- bacterium tuberculosis gene of Ag85A reported in NCBI, the sequence from 150bp to 841bp was chosen as the target gene fragment, the digoxigenin labeled DNA probe was prepared by PCR, the length of the probe was 690bp. Then, the electro- phoresis and sequence analysis were carried out to identify the probe. After culturing and detecting mycobacterium tuberculosis, the specificity of the probe was detected by situ hybridization. The probe＇ s sensitivity was detected after the mycobacterium was diluted with 10 multi - proportion. Results The hybridization results of the probe to the mycobacterium tuberculosis was positive and the results were negative when the probe was replaced by dNTP. The specificity of the probe was certified by gene sequence analysis; the lower limit of the detection was 1. 0×10^2 cfu/ml by the method of hybridization to the my- cobacterium tuberculosis. Conclusions The probe labeled with digoxigenin by PCR has specific, sensitive and quick char- acters for detecting the mycobacterium tuberculosis infection in clinic.