Comparison of Three Radiolabeled Probes for PCR-Hybridization to Detect Mycobacterium tuberculosis / 대한진단검사의학회지

BACKGROUND: Three homemade radiolabeled probes to detect DNA of Mycobacterium tuberculosis by PCR-hybridization (PCRH) assay were compared in order to select the most sensitive and economic probe with the longest lifespan. METHODS: One full length probe, probe 1, prepared by the random priming method and two oligonucleotide probes, probes 2 and 3, prepared by the 5' end-labeling method were designed and assessed for sensitivity, specificity, and life span. The detection limit of each probe was determined on sample membranes containing serially diluted M. tuberculosis DNA from 5 ng to 5 fg on weekly intervals. To assess the specificity of each probe, DNA samples from 4 species of nontuberculous mycobacteria (NTM) and 9 species of bacteria other than mycobacteria were also tested. RESULTS: Each probe with PCRH showed the same detection limits of 50 fg of M. tuberculosis DNA after a 48-hr film exposure time. There were no nonspecific reactions to bacteria when tested for specificity. When we defined the life span of each probe as the longest period for detecting the lowest detection limits of M. tuberculosis DNA, the life spans of probes 1, 2, and 3 after a 3-hour film exposure were 7, 0, and 0 weeks, respectively. For probes 2 and 3, no band was visible even on the day of preparation. The life spans after a 48-hour film exposure were 9, 3, and 2 weeks for probes 1, 2 and 3, respectively. CONCLUSIONS: Probe 1, a full length probe prepared by the random priming method, was more sensitive and was a cheaper probe with a longer life span compared to probes 2 and 3, oligoprobes prepared by the 5' end-labeling method.

Evaluation of Two PCR-Hybridization Methods for the Detection of Mycobacterium tuberculosis / 대한진단검사의학회지

BACKGROUND: We evaluated two homemade polymerase chain reaction-hybridization (PCRH) assays for the detection of M. tuberculosis. METHODS: Two PCRH assays, using a digoxigenin-luminescent probe (DL-PCRH) and using a radioactive probe (R-PCRH) were developed and compared. To determine the detection limit of each assay, 10-fold serial dilution samples of M. tuberculosis DNA were tested. To determine the specificity of each assay, 4 nontuberculous mycobacteria (NTM) and 13 bacteria other than mycobacteria were tested. Sputum samples from 50 patients with tuberculosis and 26 patients with nontuberculous diseases were tested by DL-PCRH, R-PCRH, culture, AFB stain and PCR assay methods, respectively. RESULTS: Both of the DL-PCRH and R-PCRH methods showed the same detection limit of 100 fg of M. tuberculosis DNA which was a log higher than that for the PCR. Sensitivity of both methods for the detection of M. tuberculosis in sputum specimens was 84%, which were slightly inferior to that of the culture (90%) and similar to that of the PCR (82%). However, both DL-PCRH and R-PCRH methods correctly identified M. tuberculosis for all six specimens that showed weakly-positive or negative PCR results. No false-positive results were obtained for patients with nontuberculous diseases (n=26), NTM (n=4) and other bacteria (n=13). CONCLUSIONS: Although the diagnostic efficiency of the two PCRH assays were similar to that of the PCR, both methods have greater objectivity in reading results than the PCR. This suggests that the PCRH is suitable for use in large number of clinical samples for the rapid detection of M. tuberculosis.