Study On Mycobacterium Tuberculosis And RpoB Gene In Sputum

Abstract:objective:To rapidly identify the category of mycobacterium in sputum with acid-fast positive in luzhou area and to investigate the mutations of rpoB gene of mycobacterium tuberculosis (MTB). Methods:1.The sputa of 60 patients (including 24 patients of initial treatment and 34 patients of retreatment) of pulmonary tuberculosis with positive acid-fast staining were studied in this paper, which come from eleven tuberculosis patients with complications and forty-nine patients with no complications.2. Amplified the insertion sequence 6110(IS6110) that was specific differentiated non-tuberculosis mycobacteria (NTM) from the mycobacterium tuberculosis complex (MTC) groups and the rpoB gene by PCR, and detected target band with agarose gel by electrophoresed.3.Compared the different gene sequence of amplified rpoB gene with the standard strains of mycobacterium tuberculosis H37RV. Results:1. IS6110 gene was not detected in seven samples with positive acid-fast staining.There are three cases and four cases in initial treatment and retreatment group respectively. The ratio of detection of IS6110 gene is no significant difference in two groups.The ratio of detection of IS6110 gene of group with complications is higher than that of group without complications.2. RpoB gene mutations were detected in thirteen samples in fifty-one IS6110-positive sputa. There is no significant difference of ratio of rpoB mutations between initial treatment group and retreatment group, but the ratio of rpoB mutations is higher in group of taking rifampicin over six months than that of taking rifampicin within six months.3. The majority of mutations were single nucleotide mutation, included six cases with 526 point mutatior and codon had CACâ†'AAC, CACâ†'TAC and the CACâ†'CTC; two case were 535 CCCâ†'CTC mutation; two case were 531 TCGâ†'TTG mutation; one cases was 511 CTGâ†'CGG mutation; one case was 513 CAAâ†'CCA mutation; one case was 526CACâ†'TAC and 508ACCâ†'CGC that is two-site co-mutation. All mutations were located in the rifampicin resistance-determining region(RRDR). Conclusion:1.Specific IS6110 of mycobact-erium tuberculosis was not amplified in seven (11.7%,7/60) samples of patients with clinical pulmonary tuberculosis, and which suggested some people may be non-tuberculous mycobacteria infection in this area.2. The ratio of NTM infection of patients who were diagnosed of pulmonary tuberculosis combined with low immunity or long-term use of immunosuppressant is higher than that of tuberculosis patients without complications.3. The mutations of rpoB gene were found existed in thirteen (25.5%,13/51) patients’sputa with pulmonary tuberculosis in this region, and all mutations occurred in the rifampicin resistance-determining region, which suggested some MTB may be resistance to rifampicin in this region. The ratio of mutation is higher in patients with long-time (>6 months) rifampicin therapy than that of patients with short-time (<6 months). Clinicians should pay more attention to the time of rifampicin administration, and adjusted chemotherapy in time.4.Detection of MTB by molecular biology techniques in sputum specimens is a simple and rapid method which can be achieved rapid diagnosis and help us to choose chemotherapy drug and prevent spread of resistance in Mycobacterium tuberculosis。...