Establishment of fluorescence quantitative PCR assay in detection of Mycobacterium tuberculosis

Objective To establish and assess a rapid real-time quantifiable method for the detection of Mycobacterium tuberculosis(Mtb).Methods The specific primers targetting 16S rDNA gene in Mtb were designed and the fluorescence quantitative PCR(FQ-PCR)assay was performed with SYBR Green I.After extraction of Mtb genomic DNA,the 16S rDNA fragment was amplified by conventional PCR and used to construct recombinant pMD-TB16S plasmid.Then,the serial dilutions of pMD-TB16S plasmid were subjected to the quantitation standard curve in FQ-PCR assay.The sputum samples from 11 patients with pulmonary tuberculosis were detected by the same FQ-PCR,with various genomic DNAs of Streptococcus viridans,Staphylococcus epidermidis,Staphylococcus aureus,and Escherichia coli used as the negative control to confirm specificity of FQ-PCR assay.The stabiity was analyzed with inter and intra FQ-PCR test by using the same Mtb genomic DNA,and the coefficient of variation(CV)values of threshold cycle(Ct)were calculated.Results The primers targetting Mtb 16S rDNA gene were specific to the amplification of 16S rDNA gene of Mtb but not other bacteria in control group by FQ-PCR,with the sensitivity of 1.2 pg/μL of Mtb genomic DNA or 38.9±3.54 copies/μL of 16S rDNA,and the CV values of Ct were 1.26% and 0.27% for inter and intra FQ-PCR assay.Conclusion The FQ-PCR assay targetting Mtb 16S rDNA gene is of rapid,sensitive,specific,and quantifiable in Mth detection.