| Summary: | 碩士 === 國立臺灣大學 === 醫事技術學研究所 === 92 === Tuberculosis, a predominant disease in the nineteenth century, remains one of the most life-threatening diseases at the beginning of the new millennium. It causes two million deaths per year and eight million new case of TB infection. The M. tuberculosis complex comprises M. tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. microti and M. canneti. The major pathogen for tuberculosis among M. tuberculosis complex is M. tuberculosis. The major syndrome is pulmonary tuberculosis and the most often clinical specimens are sputum. Traditional diagnosis of tuberculosis are culture and acid-fast stain. Culture need 3~8 weeks and the sensitivity of acid-fast stain is low. We want to develop a molecular diagnosis method to shorten time and has high sensitivity and specificity.
IS6110 is the specific DNA fragment exist the M. tuberculosis complex. We use PCR for IS6110 to amplify the fewer mycobacteria DNA in clinical sputum specimen. PCR method can amplify the DNA and raise the concentration of DNA for easy detection. Traditionally electrophoresis and EtBr stain is the method to detect PCR product. It is a trouble for clinical diagnosis that making a electrophoresis agarose gel and EtBr staining. We use a lateral-flow platform to detect PCR product. Advantages of lateral-flow are economic and easy to use. Use of biotin and FITC labeled primers in PCR, the amplicon contain the biotin and FITC. The anti-FITC antibody captures FITC and biotin linked with colloidal gold conjugated streptavidin. The test strip show a red line in test zone if amplicon was amplified in PCR procedure. Combine the high sensitivity and specificity of PCR and the rapid and easy of lateral-flow, we can shorten test time and have good accuracy. In clinical respiratory specimens, culture as standard, the sensitivity and specificity are 93.3% and 96.2%. The sensitivity and specificity are 65.0% and 98.2% when resolved results as standard.
In some studies report there are some IS6110 homologs exist in NTM chromosome DNA and make false positive results in IS6110 PCR. In the other way, we want to separate which species among MTB complex in clinical diagnosis. Former study reported that compare of M. tuberculosis, M. bovis and M. bovis BCG and shows there were 16 RD extra region in M. tuberculosis DNA. Another study shows RD9 region seem to be the most specific region for M. tuberculosis. We try to design primers for RD9 PCR and showed good results in reference strains PCR. But the sensitivity of RD9 PCR is very poor in clinical specimen PCR. According to the test showed that the sensitivity of RD9 PCR is 100~1000 fold lower than IS6110 PCR. In this study showed nest PCR lateral flow is work in identification of M. tuberculosis, although the sensitivity and specificity need to be approved.
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