The diagnosis and treatment of drug resistant tuberculosis especially MDR/XDR-TB are the critical and difficult factors in the prevention and control of tuberculosis. Currently, the conventional culture-based techniques have long turnaround times, we cannot offer timely and effective treatment programs for tuberculosis patients especially those combined with HIV patients . In 2010, WHO endorsed the GeneXpert MTB/RIF assay (Cepheid, Sunnyvale, CA)  that used real-time PCR to identify M. tuberculosis complex DNA and the mutations associated with RFP resistance directly from sputum specimens. But Xpert MTB/RIF assay cannot detect mutations associated with other anti-tuberculosis drug such as INH, EMB, SM, AM, FQs. With the deeply research on the molecular mechanism of drug resistance, the establishment of a simple, fast and accurate method for the detection of drug resistant mutations become more and more significant to improve the cure rate, reduce the occurrence of drug resistance, decrease the risk of recurrence and death rate.
PCR fluorescence probe technology is to use double PCR technology and the Taqman probe technique to detect mycobacterium tuberculosis and drug resistance by monitoring the fluorescence signal of different fluorescent channels. This technique has strong specificity, sensitivity, and easy to operate, etc. Our previous study  showed that PCR fluorescence probe technology is important clinical value in the rapid diagnosis of tuberculosis in sputum specimens. However, there are few studies on exploratory development of PCR-fluorescent probes in rapid detection of mutations associated with XDR-TB.
This study is based on the PCR-fluorescent probe method which has a low cost, simple operation and only need 1.5 hours to the detect the nucleic acid from the specimens. Besides, this method can significantly shorten the detect cycle comparing with Phenotypic DST. In this study, we established and evaluated the detection system of multi-drug resistance and extensively drug resistance in mycobacterium tuberculosis, which reflects mainly on: (1) The drug resistance of TB was detected by fluorescence PCR detection technology, which covered six anti-tuberculosis drugs with seven drug resistance genes. Additionally, a multidrug- and extensively drug-resistant mutation detection system was established, and the results were statistically analyzed for 900 clinical sputum samples, and had high the specificity and sensitivity values; (2) The clinical diagnostic performance of PCR detection system was evaluated by testing 900 clinical specimens of mycobacterium tuberculosis, and the results were compared with the absolute concentration method of DST. In total coincidence rate, all of the six drugs were larger than 88%, five of which drugs were larger than 90%.
Compared with phenotypic DST, the coincidence rates of rpoB(RFP), katG/inhA(INH), embB(EMB), gyrA(FQs), rpsL(SM) and rrs(AM) detected by fluorescent probe method were 95.89%, 91%, 92.11%, 90.89%, 88.11% and 93.22%, respectively in this study. The coincidence rate of two methods of RIF, INH, EMB, FQs, AM resistance testing is higher than 90%, only SM coincidence rate was 88.11%, the mainly reason was that rpoB, katG/inhA, embB, gyrA and rrS gene mutations of Mycobacterium tuberculosis were the main resistance mechanism of RFP, INH, EMB, FQs, AM. In addition to rpsL gene mutation (50–78%), rrs gene mutation (20–30%) is also the main molecular mechanism of drug resistant to SM [26, 27]. However, this study had not detect the rrs gene mutation locus of drug resistant to SM of Mycobacterium tuberculosis, which may be one of the reasons for the low consistency of SM drug resistance detected by PCR fluorescent probe method compared with phenotypic DST.
In This study, phenotypic DST was used as the standard, the detection rate of MDR-TB by phenotypic DST was 47.44% (427/900), and the detection rate of rpoB and katG/inhA mutation was 42.22% (398/900) by PCR fluorescence probe method. The sensitivity, specificity, coincidence rate of PCR fluorescent probe method for detection of MDR-TB were 90.16%, 97.25%, 93.89%, respectively. The detection rate of XDR -TB by phenotypic DST was 16.22%(146/900), and the detection rate of rpoB, katG/inhA, rpsL, rrS, embB, and gyrA by PCR fluorescence probe method was 15.22% (137/900), the sensitivity and specificity of PCR fluorescent probe method for detection of XDR-TB were 87.67%, 98.81%, and the coincidence rate was 97.00%.
The studies demonstrate that drug-resistant gene mutations are a major form of TB resistance. As for PCR fluorescence probe method, the positive detection rate was lower than DST, because drug-resistant gene mutation was just a form of drug resistance. Reducing cell's membrane vulnerability and efflux pump and inactivated enzymes changes, were also the causes of TB resistance. On the other hand, only a few loci of 7 common drug-resistant genes of 6 drugs were detected, while other drug-resistant genes or other drug-resistant loci were not developed in this study. For example, many genes reported in the literature such as ndh, efpA, kasA, iniABC operon (for INH resistance), rpoC (for RFP resistance ) , embA, embC, ubiA (for EMB resistance) , and gyrB (for FQs resistance ). These may be reasons for lower detection rate of PCR-fluorescent probe method than phenotypic DST.