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 the Efficiency of HCV RT-PCR Using Narrowly Spaced Primers / 대한임상병리학회지

BACKGROUND: It is important to choose effective primers to increase the sensitivity of RT PCR for HCV. It was reported that the use of narrowly spaced primers (NSP) resulted in increased sensitivity of HCV PCR because NSP is more effective than widely spaced primers (WSP) in RT or PCR reaction. It will be useful to compare the merits and demerits of PCR using NSP (NSP-PCR), PCR using WSP (WSP-PCR) and anti-HCV EIA. METHODS: We performed NSP-PCR, WSP-PCR, anti-HCV EIA tests and determined sensitivity and specificity of each method with 36 sera from patients with hepatitis C and 14 sera from patients with diseases other than hepatitis C. RESULTS: The sensitivity and specificity for WSP-PCR were 80.6% and 100%, those for NSP-PCR were 86.1% and 78.6%, and those for anti-HCV EIA were 94.4% and 100%. The concordance rate for anti-HCV EIA and WSP-PCR was 82% and that of anti-HCV EIA and NSP-PCR was 80%. The detection limit of WSP-PCR was up to 10(-1) dilution and that of NSP-PCR was up to 10(-2) dilution. The positive bands of WSP-PCR were strong and large. But those of NSP-PCR were weak and small, so it was not easy to differentiate them from primer dimers sometimes. CONCLUSIONS: WSP-PCR seemed to be better than NSP-PCR for clinical laboratory tests. Anti-HCV EIA appears to be a good screening test for liver disease because of the high sensitivity and specificity.