| Tuberculosis (TB) is a disease caused by mycobacterium tuberculosis (MTB)with pulmonary tuberculosis as the most frequent chronic infection. In recent years, with the increase of the reappearance of the disease across the globe, it is recognized that tuberculosis remains to be a global serous public health and social problem deserving great attention.Chemotherapy is the foremost means to treat TB patients, control and eradicate the sources of infection. However, on account of the drug-resistant TB bacilli and especially the increase of the TB bacilli with multi-drug resistance, the effect of the chemotherapy for TB is severely affected resulting in great difficulty with the control of the TB. As a result, the drug-sensitive test has become even more mandatory. The conventional drug-sensitive test requires long time and it brings on the drug-resistance tuberculosis to become widely dispersed. Moreover, the positive rate of the sputum cultivation is comparatively low. It is quite hard to use the test as a routine clinical one. So its very meaningful to detect mycobacterium tuberculosis isoniazid-resistance isolates rapidly for the sake ofearly effective chemothe-rapy and spreading control. Along with the development of molecular biology recently, the adoption of the gene technique has opened a new way to carry out the diagnosis of tubercle bacilli and the drug sensitive test with inspiring results achieved. There are a great number of techniques for the detection of drug-resistant genes of tubercle bacilli. In contrast, the single-strand conformation polymorphism (SSCP) may after all be accepted as a better one. SSCP is able to detect the mutation, short-sequence deficiency and insertion at the drug-resistant gene point without any specific instruments. It is rapid and simple in operation and high in sensitivity and specificity.Isoniazid (INH) is one of the basic drugs to treat TB. Therefore, the problem of its resistance to mycobacterium tuberculosis is greatly concerned. It is an urgent clinical problem to be tackled to develop a means of the rapid detection of drug resistance of clinical separate isolates and even clinical samples to work out the mechanism of drug-resistance of isoniazid to tubercle bacilli. Although there were reports about the PCR-SSCP detection of the gene resistant to isoniazid now, failing to detect the majority of the drug-resistant genes at the time. This leads an ineffective treatment with standard chemotherapy. For this, the main research content of the theme is as follows: based on the cultivation and drug-sensitive test of standard isolates and clinical separate isolates of tubercle bacilli, to develop a multi-PCR-SSCP detection technique for aphC promoter. inhA and katG genes in close relation to drug-resistant isoniazid and to deal with the applicability and feasibility of the technique in comparison with the measurement sequence results and in reference to drug-resistant results.Development of the multi-PCR techniqueIt has been verified that the mutation of the katG gene of the encodedhydrogen oxide - peroxidase is the main reason that leads to the resistance ofmycobacterium tuberculosis to isoniazid, but this fails to completely interpret such a phenomenon. The researches of recent years show that the mutation of genes of the inhA and aphC promoter is related to the resistance of mycobacterium tuberculosis to isoniazid and three gene mutations were confirmed in more than 85% of the mutations of the resistance of tubercle bacilli to isoniazid. For this purpose, by employing the multiple primer design software of Primerior Array Company and making the reasonable selection of the extended region and length to cover as much as possible the mutation sites for the drug-resistant genes, the present study has designed 6 primers in an attempt to extend more than 85% of the genes of mycobacterium tuberculosis resistant to isoniazid. The sequences of the primers are as follows: the specific primer of the aphC initiator gene was self-designed according to the Genbank:...
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