Pai S R, Actor J K, Sepulveda E, Hunter R L, Jagannath C
Department of Pathology and Laboratory Medicine, University of Texas Health Sciences Center, Houston, TX, USA.
Microb Pathog. 2000 Jun;28(6):335-42. doi: 10.1006/mpat.2000.0353.
Mycobacterium tuberculosis (MTB) the causative organism of tuberculosis can remain dormant as a non-culturable organism, reactivate and cause disease in man and animals. There is a need for proof of viability of such organisms in order to understand the process of reactivation. PCR for bacterial DNA cannot distinguish between viable and non-viable bacilli. We have tested a previously described two tube directed reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of mRNA of antigen 85B (Ag85B) of MTB that can distinguish between viable and non-viable organisms. Using a set of external and internal primers for Ag85B, a cDNA amplified product (216 bp) was seen among simulated samples containing only viable cfus at a sensitivity of >10 and <100 cfu/ml. Eucaryotic DNA rich normal mouse lung homogenate did not interfere among these samples. The method amplified the 216 bp product also among cfu positive tissues of naturally infected mice. Finally, in a mouse model of dormancy, direct RT-PCR detected a signal among multiple tissues that were negative for cfus and hence non-culturable. Ag85B is abundantly secreted by MTB and hyper-expressed under stress conditions. Thus the method to identify its mRNA message may be useful to detect viable but dormant bacteria.
结核分枝杆菌（MTB）是结核病的病原体，可作为不可培养的生物体保持休眠状态，重新激活并在人和动物中引发疾病。为了了解重新激活的过程，需要证明此类生物体的活力。用于检测细菌DNA的PCR无法区分活菌和死菌。我们测试了一种先前描述的双管定向逆转录聚合酶链反应（RT-PCR），用于检测MTB抗原85B（Ag85B）的mRNA，该方法可以区分活菌和死菌。使用一组针对Ag85B的外部和内部引物，在仅含有活菌的模拟样品中，以>10且<100 cfu/ml的灵敏度观察到一个cDNA扩增产物（216 bp）。富含真核DNA的正常小鼠肺匀浆在这些样品中不产生干扰。该方法在自然感染小鼠的cfu阳性组织中也扩增出了216 bp的产物。最后，在一个休眠小鼠模型中，直接RT-PCR在多个cfu阴性且因此不可培养的组织中检测到了信号。Ag85B由MTB大量分泌并在应激条件下过度表达。因此，识别其mRNA信息的方法可能有助于检测存活但处于休眠状态的细菌。
