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Reverse dot blot hybridization kit for detection of mycobacterium tuberculosis and usage method thereof

A technology of mycobacterium tuberculosis and reverse dot hybridization, applied in the field of identification and detection of mycobacterium tuberculosis, can solve the problems of false positives, false negatives, expensive thermal cycle equipment, etc.

Inactive Publication Date: 2015-08-12
CHINA INSPECTION LAB TECH CO LTD
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Polymerase chain reaction (PCR) technology has been successfully used in the detection of clinical specimens due to its good sensitivity and specificity, but the thermal cycle equipment required by PCR technology is expensive, and it is prone to false negatives, false positives, cross-contamination and other problems , so that its application has certain limitations

Method used

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  • Reverse dot blot hybridization kit for detection of mycobacterium tuberculosis and usage method thereof
  • Reverse dot blot hybridization kit for detection of mycobacterium tuberculosis and usage method thereof
  • Reverse dot blot hybridization kit for detection of mycobacterium tuberculosis and usage method thereof

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Experimental program
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Embodiment

[0037] 1. Test materials and methods

[0038] 1.1 Test material

[0039] Mycobacterium tuberculosis H37Rv and DNA extracts from sputum specimens of clinically confirmed tuberculosis patients were provided by the Beijing Tuberculosis Prevention and Control Institute. Five types of non-tuberculous mycobacteria (E. Shigella) were provided by Beijing Entry-Exit Inspection and Quarantine Bureau.

[0040] 1.2 Primer and probe design

[0041] The gene sequence of Mycobacterium tuberculosis H37Rv IS6110 was downloaded from the American NCBI gene database (http: / / www.ncbi.nlm.nih.gov / ) as the target sequence, and Primer5 software was used to design the PCR amplification of Mycobacterium tuberculosis. Increased specific primers (IS1' and IS2') and specific probe Probe-B-3' for reverse dot hybridization, the 5' end of the primer is labeled with biotin, and 10 T tails are added to the 5' end of the probe , the nucleotide sequences of primers and probes are:

[0042]

[0043] 1.3 Ex...

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Abstract

The present invention discloses primers, a probe and a kit for reverse dot blot hybridization detection of mycobacterium tuberculosis and a usage method thereof. The kit is formed by a PCR primer group, a nylon hybridized membrane fixed with a mycobacterium tuberculosis-specific probe and other reagents for reverse dot blot hybridization. The primer group is constituted by an IS1' primer with the sequence of TACGGTGCCCGCAAAGTG and an IS2' primer with the sequence of AGGCGTCGGTGACAAAGG; and the probe comprises a Probe-B-3 'probe with the sequence of GCCTTTGTCACCGACGCCTA. The usage method of the kit is characterized by discriminating mycobacterium tuberculosis from other common pathogens at room temperature. The invention has the advantages of high sensitivity, strong specificity, simple operation and low temperature requirements, and provides technical support for discrimination and diagnosis of mycobacterium tuberculosis.

Description

technical field [0001] The invention relates to a primer, a probe, a kit and a use method thereof for bacterial identification and detection, in particular to a primer, a probe, a kit thereof, and a use method for the identification and detection of Mycobacterium tuberculosis, belonging to tuberculosis The field of mycobacterial detection. Background technique [0002] Tuberculosis (tuberculosis) is one of the diseases with the longest human history, and it is also the disease with the largest number of deaths caused by a single pathogen. Tuberculosis was recorded as early as in ancient Greece and ancient Egypt. German bacteriologist Koch (Koch) discovered and confirmed in 1882 that Mycobacterium tuberculosis (Mycobacterium tuberculosis) is the pathogenic bacteria that causes tuberculosis. Mycobacterium tuberculosis can cause infections throughout the body, the most common of which is tuberculosis. According to the statistics of the World Health Organization (WHO), about 8...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68C12Q1/04
Inventor 邹明强薛强金福姝孙福军郭敏卓张帆
Owner CHINA INSPECTION LAB TECH CO LTD
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