title: Identification of viable and non-viable Mycobacterium tuberculosis in mouse organs by directed RT-PCR for antigen 85B mRNA.
abstract: 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.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Texas Health Sciences Center, Houston, TX, USA.
authors.forename: S R
authors.initials: SR
authors.lastname: Pai
authors.forename: J K
authors.initials: JK
authors.lastname: Actor
authors.forename: E
authors.initials: E
authors.lastname: Sepulveda
authors.forename: R L
authors.initials: RL
authors.lastname: Hunter
authors.forename: C
authors.initials: C
authors.lastname: Jagannath
mesh_terms: D000217:Acyltransferases; D000818:Animals; D000942:Antigens, Bacterial / Q000032:analysis* / Q000235:genetics; D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics; D015169:Colony Count, Microbial; D004195:Disease Models, Animal; D005260:Female; D051379:Mice; D008810:Mice, Inbred C57BL; D009169:Mycobacterium tuberculosis / Q000254:growth & development / Q000276:immunology* / Q000302:isolation & purification; D012329:RNA, Bacterial / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014376:Tuberculosis / Q000188:drug therapy / Q000382:microbiology*; D014781:Viscera / Q000382:microbiology
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D000217:Acyltransferases; C121433:antigen 85B, Mycobacterium tuberculosis