An Efficient Alternative Marker For Specific Identification Of Mycobacterium Tuberculosis And The Construction Of Real-time PCR Method Based On It

ObjectivesTo identify and evaluate a new nucleic acid amplification test target forspecific detection of Mycobacterium tuberculosis (MTB); To establish amtss90-based real-time (TaqMan) PCR assay for the rapid identification ofMTB．MethodsThe MTB-specific sequence was screened by a bioinformaticssoftware. Based on the sequence, a pair of specific primer was designed forthe PCR amplification in266strains of microorganisms and humanumbilical vein endothelial cells. Moreover, the efficiency of theMTB-specific sequence was evaluated compared with16S rDNA(Mycobacterium genus-specific), IS6110[specific identification ofMycobacterium tuberculosis complex (MTBC)], mtp40(MTB-specific) inMycobacterium strains, and traditional bacteriology testing. Base on thegood efficiency of mtss90, a real-time (TaqMan) PCR assay wasestablished for the rapid identification of MTB．ResultsThe mtss90（Mtss1, Mtss2）amplification results showed a goodconservation and specificity. The mtss90（Mtss1, Mtss2）amplificationresults showed a good conservation and specificity. All MTB tested strainswere mtss90positive. No amplification was observed from any other testedstrains with M. microti as an exception. Compared with the traditional PNB/TCH method, the coincidence rate was99.1%（233/235） and thefinal identification results indicate that the mtss90PCR identify the twoinconsistent isolates accurately. All of the mtss90positive strains were16SrDNA and IS6110positive, indicating a100%coincidence rate betweenmtss90and these two genetic markers. Additionally, mtss90may havebetter specificity and conservation than mtp40in the identification of MTB.The mtss90PCR identify an mtp40absent MTB isolate. A mtss90-basedreal-time PCR assay was successfully established for the rapid identificationof MTB.ConclusionsThe mtss90may be an efficient alternative marker for species-specificidentification of MTB and could be used for the diagnosis or identificationof tuberculosis singly or combined with other genetic markers.