Detection Of Drug-Resistant Pathogens With Displacing Probes-Based Real-Time PCR

This dissertation consists of three parts for describing the application of the displacing probes to detecting the mutations in hepatitis B virus (HBV) and Mycobacterium tuberculosis.The first part describes the establishment of a single-tube, real-time PCR assay for simultaneous detection of multiple lamivudine-resistance mutations in sera samples. Effective detection of lamivudine-resistant hepatitis B virus (HBV) is extremely important for clinical diagnosis and treatment. By using four allele-specific displacing probes labeled with different fluorophores, a single real-time PCR reaction can tell whether a sample contains any of the following HBV DNA alleles: wild-type, rtM204 mutant, mixtures of wild-type and rtM204 mutant, mixtures of rtM204 and rtL180 mutant, mixtures of wild-type, rtM204 mutant and rtL180 mutant. The assay was evaluated with 50 HBV mutation(s)-containing samples and 36 HBeAg-positive samples. The results of the real-time PCR assay were consistent with the DNA sequencing, but at a much highter sensitivity for detecting a mixture of quasispecies. As low as 102 ~103 copies/ml HBV of all four alleles in pure population and as little as 5% mutant DNA in the presence of wild-type DNA can be detected. Application of this high throughput assay should enable early diagnosis and better treatment of lamivudine-resistant HBV.The second part reports a sensitive single-tube PCR assay that takes less than 3 h and reliably identifies rifampin-resistant M. tuberculosis in DNA extracted directly from sputum. Current clinical assays for determining antibiotic susceptibility in M. tuberculosis require several weeks to be completed due to the slow growth of the bacilli. Ninety-five percent of mutations associated with rifampin resistance occur in an 81-bp core region of the bacterial RNA polymerase gene, rpoB. All mutations that occur within this region result in rifampin resistance. Four different displacing probes are used in the same reaction, each perfectly complementary to a different target sequence within the rpoB gene of rifampin-susceptible bacilli and each labeled with a differently colored fluorophore. Together, their target sequences encompass the mostly region of interests. The generation of all four fluorescent colors during PCR amplification indicates that rifampin-susceptible M. tuberculosis is present. The presence of any mutation in the core region prevents the binding of one of the displacing probes, resulting in the absence of one of the four fluorescent colors. Totally 123 sputum samples infected with tuberculosis was tested. We detected 22 mutation-containing, and the results were consistent with the DNA sequencing. The use of this rapid assay should enable early detection and treatment of drug-resistant tuberculosis in clinical settings.The third part evaluates the feasibility of displacing probes to detect streptomycin-, isoniazid- and ethambutol-related resistance mutations in DNA extracts from sputum samples. We studied three genes: rpsL, katG and embB, which are associated with streptomycin, isoniazid and ethambutol resistance, respectively. Among 118 sputum samples with tuberculosis, we detected in 8 samples to be streptomycin-resistant, 6 samples isoniazid-resistant, 1 sample ethambutol-resistant, 2 samples to be mixture of streptomycin- and isoniazid- resistant, and 8 samples to be mixture of streptomycin-, isoniazid- and ethambutol- resistant. The results of the real-time PCR assay were consistent with the ARMS assay. This assay is more rapid and sensitive compared with classic methods. Our method can be used for high-throughout screening for the mutations of drug resistance in Mycobacterium tuberculosis.