골수천자액에서의 결핵균 검출과 Competitive PCR에 의한 정량방법의 소개

Abstract

The conventional methods for the detection of Mycobacterium tuberculosis are the AFB staining and cultivation method. But cultivation method spends 2 to 8 weeks to identify and in the case of sputum specimens, only 28 to 70% numbers of overall bacteria is able to be cultivated due to the loss of viability resulting in pretreatment of sputum specimen. And the bacteria is rarely cultivated in BM aspirate, peripheral blood , or many kinds of body fluids as a specimens. In order to overcome these problems, we introduced PCR method to detect M. tuberculosis. In this study, we reported an example of the detection of this bacteria in BM aspirate of a patient and described semi -quantitative method using competitive PCR. We used the cloned PCR product as a positive control element and the newly developed DNA fragment as an internal control that is derived from v-erbB gene and has the same primer binding regions as positive control at the 5’ and 3’ end and about 120bp size difference. ln the competitive PCR, we put the various known amounts of internal control and the constant amounts of the genomic DNA extracted from BM aspirate in one tube and amplified the IS6110 fragment of M. tuberculosis with thermal cycler. And then the PCR products were electrophoresed and stained with EtBr. After photographed, we scanned the photographs using the Image Master program of Phamacia Biotech to compare the amounts of intensity of amplified target DNA and the amplified internal control. We found the region that represented the equal amount of intensity of target DNA and internal control. At that region , we could calculate the amount of target DNA from the amount of internal control. As a result, it was estimated that 2ml of BM aspirate of a patient contained about 37 cells of M. tuberculosis. The detection of M. tuberculosis by PCR is very useful in BM aspirates, bloods, or several kinds of body fluids as specimens. This method could be used f or semi - quantitation and it could quantitate more accurate if we applied intimately controlled amounts of internal control. We might also apply to EL1SA system to estimate the amounts of amplified products using each specific probes of target DNA and internal control.