A method for genotyping Mycobacterium tuberculosis spacer oligonucleotides encoded by melting point

Abstract

The invention discloses a mycobacterium tuberculosis spacer oligonucleotide typing method based on melting point coding and relates to mycobacterium tuberculosis. The method comprises the following steps: (1) designing a pair of universal primers on a direct repeat sequence to amplify all spacers, and designing a pair of primers in a GyrA conserved region; (2) designing four groups of double-tagging fluorescent probes specifically hybridized therewith according to 43 spacer sequences and GyrA sequences, and manually controlling the welding point value of each probe by adjusting the probe length and / or a base composition, wherein each probe is provided with the same fluorescent group; (3) distributing the fluorescent probes in three reaction tubes, wherein by the combination of the reaction tubes, the fluorescent groups and the welding point values, each spacer forms a three-dimensional code, and software automatically reads a result. The method has the advantages of simple and convenient one-step operation, high specificity, high repeatability, low time consumption and relatively low price, and a powerful tool is provided for exploration and research of the prevalence and propagation rule of tuberculosis in China.

Description

technical field [0001] The invention relates to mycobacterium tuberculosis, in particular to a typing method for mycobacterium tuberculosis spacer oligonucleotides coded by melting point. Background technique [0002] Mycobacterium tuberculosis is the pathogenic bacterium that causes tuberculosis, which can invade the whole body organs, among which tuberculosis is the most common, and has become a major global public health problem. The M. tuberculosis complex includes M. tuberculosis hominis, M. bovis, M. africanum, and M. microti. Traditionally, the classification of Mycobacterium tuberculosis complex is based on the differences in growth, morphology, and biochemical characteristics of different species of mycobacteria, but this classification method is far from meeting the needs of modern tuberculosis prevention and control work. With the advancement of science and technology and the completion of the genome sequencing of Mycobacterium tuberculosis, people have a deep un...

AI Technical Summary

Problems solved by technology

Although the traditional solid-phase membrane hybridization technology has the advantages of mature detection process and low reagent cost, it requires more manual operations, long detection time, and the sample detection throughput cannot meet clinical needs.

These two technical bottlenecks have seriously hindered the clinical promotion of Spoligotyping. For this, scientists have proposed the use of gene chips [6,7] , encoded microspheres [8,9] and mass detection [10,11] and other technologies to replace the solid-phase membrane hybridization technology. Although the reported improved Spoligotyping technology has increased the throughput and greatly shortened the detection time, these technologies have not got rid of the detection mode of post-processing of PCR tubes, which may easily cause contamination of PCR products. , and due to the need for some large instruments, it is difficult to be widely used clinically

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