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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AI Technical Summary

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

Method used

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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

Example Embodiment

[0042] Example 1

[0043] This embodiment provides a kit (PCR-fluorescent probe method) for detecting Mycobacterium tuberculosis based on free nucleic acid in blood, which includes PCR reaction solution, primers, enzymes, internal standards, quality control samples, and detection of MTB target nucleosides Acid fluorescent probe, detection internal standard fluorescent probe, positive quality control product and negative control product, primers include detection MTB target nucleotide primer and detection internal standard primer, and detect the nucleotide sequence of MTB target nucleotide primer 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 as shown in SEQ ID NO.7, and the plasmid concentration is 2×10 4 copies / ml, the nucleotide sequence of the detection internal standard primer is shown in SEQ ID NO.5 and SEQ ID NO.6;

[0045] The PCR reaction solution includes the f...

Example Embodiment

[0082] Example 2

[0083] This embodiment provides a kit for detecting Mycobacterium tuberculosis based on free nucleic acid in blood (PCR-membrane chromatography hybridization method), which includes PCR reaction solution, primers, enzymes, internal standards, hybridization membrane strips, color developing solution, Positive quality control product and negative quality control product, with hybridization probe attached to the hybridization membrane strip;

[0084] The internal standard is a plasmid whose nucleotide sequence is shown in SEQ ID NO.7, and the plasmid concentration 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 to the C3 spacer (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 ...

Example Embodiment

[0122] Example 3

[0123] This example uses the reagent kits of Examples 1 to 2 and the sputum smear method to detect clinical samples. The actual situation of the clinical samples is: 103 cases of tuberculosis, 26 cases of extrapulmonary tuberculosis, 43 cases of negative cases, and a total of 172 cases ; The results of the two kits (PCR-fluorescent probe method and PCR-membrane chromatography hybridization method) in this example are as follows figure 2 As shown, and compared with the sputum smear method, the comparison results are shown in Table 6; Note: Table 6 is the sputum smear test results when viewed vertically, and the test results of the two kits of the present invention when viewed horizontally; the following table views 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), respectively. The positive rates of tuberculosis patients and extra-p...

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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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