Research On Detection Of Mycobacterium Tuberculosis From Animal Tissues By TaqMan Real-time Fluorescent Quantitative PCR

ObjectTuberculosis(TB)is caused by Mycobacterium tuberculosis(M.tb),which can infect many organs and tissues,especially lung and respiration system.TB has become a significant global health issue,because of TB treatment requires long time period,and it becomes a huge economic burden to the society and individuals.There are many methods available now for TB diagnose,however,these methods are mainly used for detecting M.tb in sputum or body fluid specimens.There is little information about methods for M.tb detection in tissues.The purpose of this study is to establish a suitable method TaqMan real-time fluorescent quantitative PCR(TaqMan real-time FQ-PCR)for quantitatively detecting M.tb from tissues in order to provide a valuable tool for research and clinical diagnosis of M.tb in tissues.MethodA 113bp length fragment of M.tb H37Rv(ATCC 25618)IS6110 gene was amplified with conventional PCR,and cloned into pGEM(?)-T vector to construct a plasmid,which was used as standard for M.tb detection by TaqMan real-time FQ-PCR.This method system was optimized using serial dilutions of standards,and the sensitivity,specificity were also evaluated.A lung tissue from a mouse known to be free of TB was homogenized,then was spiked with a suspension of M.tb H37Rv(ATCC 25618),used for testing detection limit of M.tb of the method in tissue.DNA was extracted from a total of 155 animal(Mice,Guinea pigs,Rhesus monkeys)spleen and lung tissue specimens.TaqMan real-time FQ-PCR was used to detect M.tb IS6110 gene.The sensitivity and specificity of the method were evaluated.ResultsThe detection limit of the TaqMan real-time FQ-PCR was 1CFU per ml of tissue homogenate solution.This method has high sensitivity and specificity of 95.58%(95%CI:89.47%-98.36%)and 100%(95%CI:89.56%-100%),respectively.Comparatively the conventional M.tb culture method has sensitivity and specificity of 82.3%(95%CI:73.7%-88.6%)and 100%(95%CI:89.6%-100%),respectively.The kappa coefficient of the two methods was 0.762(95%CI:0.79-0.965).ConclusionThe TaqMan real-time FQ-PCR is a rapid method for M.tb detection in infected animals' tissues,which has high sensitivity and specificity.Thus,this method is suitable for research and clinical laboratory.