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

Jang, Sook Jin; Kang, Jeong Suk; Kim, Jin Hee; Cho, Seong Sig; Park, Geon; Moon, Dae Soo; Park, Young Jin

Abstract

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.