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Kit for detecting mycobacterium tuberculosis based on blood free nucleic acid

A technology of Mycobacterium tuberculosis and free nucleic acid is applied in the field of test kits for detecting Mycobacterium tuberculosis, which can solve the problems of low diagnosis rate of sputum samples, delayed patient treatment time, low sensitivity, etc., so as to avoid crossover between multiple pathogens. The effect of reaction, avoiding the generation of non-specific amplification products, and reducing the safety risk factor

Active Publication Date: 2019-02-19
广州市宝创生物技术有限公司
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Problems solved by technology

[0003] At present, the clinical diagnosis of tuberculosis is mainly based on medical history, CT, sputum smear, sputum culture and immunodiagnosis. These diagnostic methods have the following shortcomings: 1) The traditional "gold standard" (sputum culture) can distinguish different types of tuberculosis Mycobacterial infection, but its long cycle and low sensitivity often delay the best treatment time for patients; 2) CT examination can usually only find patients with existing lung lesions, and cannot perform early diagnosis, and it cannot be compared with other lung diseases 3) Sputum smear is a commonly used pulmonary tuberculosis screening technique, which is simple and fast, but its positive rate is extremely low, and more than 60% of tuberculosis patients are diagnosed as smear-negative; although smear-negative patients The content of tuberculosis is low, but it is still one of the important sources of infection of tuberculosis, which is easy to cause missed diagnosis; 4) immunodiagnosis is based on the detection method of specific antigen and antibody, which is simple to operate, but the specificity and sensitivity are not enough
Molecular diagnostic products for Mycobacterium tuberculosis, currently on the market mainly include fluorescent PCR products and constant temperature amplification products, the detection sensitivity of which is between 1 and 10 bacteria / ml, and the detection results can usually be obtained within 3 to 4 hours, which is simple and convenient. Fast, but it also has some shortcomings: 1) Most of them are qualitative or drug resistance detection for fresh sputum samples, and the diagnostic rate of sputum samples in children, patients with extrapulmonary tuberculosis and HIV patients is low; 2) Sampling is difficult , The difference between samples is large, and the accuracy of the test results obtained is low

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  • Kit for detecting mycobacterium tuberculosis based on blood free nucleic acid
  • Kit for detecting mycobacterium tuberculosis based on blood free nucleic acid
  • Kit for detecting mycobacterium tuberculosis based on blood free nucleic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The present embodiment provides a kind of test kit (PCR-fluorescence probe method) based on blood free nucleic acid detection Mycobacterium tuberculosis, it comprises PCR reaction solution, primer, enzyme, internal standard, quality control sample, detect MTB target nucleoside Acid fluorescent probes, detection internal standard fluorescent probes, positive quality controls and negative quality controls, primers include detection of MTB target nucleotide primers and detection of internal standard primers, detection of the nucleotide sequence of MTB target nucleotide primers As shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 (2 pairs of primers);

[0044] The internal standard is a plasmid whose nucleotide sequence is shown in SEQ ID NO.7, and the concentration of the plasmid is 2×10 4 copies / ml, detect the nucleotide sequence of the internal standard primer as shown in SEQ ID NO.5, SEQ ID NO.6;

[0045] The PCR reaction solution includes the following compo...

Embodiment 2

[0083] The present embodiment provides a test kit (PCR-membrane chromatography hybridization method) for detecting Mycobacterium tuberculosis based on blood free nucleic acid, which includes PCR reaction solution, primers, enzymes, internal standard, hybridization membrane strip, chromogenic solution, Positive quality control and negative quality control, with hybridization probes attached to the hybridization membrane strip;

[0084] The internal standard is the plasmid whose nucleotide sequence is shown in SEQ ID NO.7, and the concentration of the plasmid is 2×10 4 copies / ml, the nucleotide sequence of the detection internal standard primer is shown in SEQ ID NO.5, SEQ ID NO.6, and the 5' end of the upstream primer of the detection internal standard primer is also connected with the C3 spacer arm (C3spacer ) and the first tag sequence, the first tag sequence is shown in SEQ ID NO.15; the 5' end of the downstream primer of the detection internal standard primer is also connec...

Embodiment 3

[0123]In this embodiment, the clinical samples are detected by the kits of Examples 1-2 and the sputum smear method. The actual situation of the clinical samples is: 103 cases of pulmonary tuberculosis, 26 cases of extrapulmonary tuberculosis, 43 negative cases, and a total of 172 cases. Two kinds of kits (PCR-fluorescent probe method and PCR-membrane chromatography hybridization method) measurement results of the present embodiment are as follows: figure 2 As shown, and compared with the sputum smear method, the comparison results are as shown in Table 6; Note: Table 6 is the test result of the sputum smear when viewed vertically, and the test results of the two kits of the present invention when viewed horizontally; the views of the following tables similar.

[0124] Table 6

[0125]

[0126]

[0127] As shown in Table 6, the overall positive and negative rates of the sputum smear method were 20.93% (36 / 172) and 79.07% (136 / 172), and the positive rates of pulmonary t...

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Abstract

The invention discloses a kit for detecting mycobacterium tuberculosis based on blood free nucleic acid. The kit comprises PCR (polymerase chain reaction) solution, a primer, an enzyme and an interiorlabel, the primer includes a primer for detecting MTB target nucleotide and a primer for detecting the interior label, and a nucleotide sequence of the primer for detecting the MTB target nucleotideis as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4. A detection method of the kit includes a PCR-fluorescent probe method and a PCR-membrane chromatography hybridization method. According to the kit, high-sensitivity detection based on blood free nucleic acid can be achieved, the positive rate of tubercular patients with negative sputum smears, extra pulmonary tuberculosis patients and HIV (human immunodeficiency virus) concurrent tuberculosis infection patients is remarkably increased, the screening accuracy of tuberculosis infection patients is improved, transmission routescan be effectively controlled by detecting blood samples, safety risks of medical staff and inspection staff are reduced, and the kit has ground-breaking clinical values to guiding pharmacy of tuberculosis and control and prevention of diseases.

Description

technical field [0001] The invention belongs to the technical field of biomedical detection, in particular to a kit for detecting mycobacterium tuberculosis based on blood free nucleic acid. Background technique [0002] Mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB), referred to as Mycobacterium tuberculosis or Mycobacterium tuberculosis, is the main pathogenic bacteria that causes tuberculosis (Tuberculosis, TB). Mycobacterium tuberculosis is a slender, slightly curved bacillus that is a non-spore-forming, immobile, single-celled bacterium. Tuberculosis is a chronic infectious disease that is airborne. Tuberculosis is an important disease that seriously endangers human life and health: about 35% of the world's population is infected with tuberculosis, with more than 8 million new cases every year, and more than 2 million people die from this type of disease. my country is also one of the countries with a high burden of tuberculosis in the world. There are c...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/6848C12Q1/04C12R1/32
CPCC12Q1/6848C12Q1/686C12Q1/689C12Q2600/166C12Q2545/101C12Q2563/107C12Q2521/531
Inventor 肖慧杨祥胜
Owner 广州市宝创生物技术有限公司
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