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Kit for detecting nucleic acid of mycobacterium tuberculosis and method thereof

A technology of Mycobacterium tuberculosis and a detection kit is applied in the detection field of Mycobacterium tuberculosis nucleic acid, which can solve the problems of difficulty in popularization, long time for reporting results, low sensitivity and the like

Active Publication Date: 2012-03-14
USTAR BIOTECHNOLOGIES (HANGZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] There are many techniques currently used in the diagnosis of Mycobacterium tuberculosis. Although these routine inspection methods are technically mature, there are still some problems: the internationally recommended smear and culture methods still have poor specificity, low sensitivity, and poor results reporting. However, the internationally recognized tuberculosis rapid culture system has greatly improved the reporting time of culture results, but it is difficult to popularize due to expensive equipment and high detection costs.
However, the defect of this technology is that the culture process is too long and needs to be cultured for 7-14 days, which is not suitable for clinical use.

Method used

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  • Kit for detecting nucleic acid of mycobacterium tuberculosis and method thereof
  • Kit for detecting nucleic acid of mycobacterium tuberculosis and method thereof
  • Kit for detecting nucleic acid of mycobacterium tuberculosis and method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Composition and preparation of embodiment 1 kit of the present invention

[0100] a) DNA extraction reagent: our company's instrument-free nucleic acid extraction kit;

[0101] b) Reaction solution: two peripheral primers (0.05 μmol), two probes (0.5 μmol), and two cross primers (0.5 μmol), 10×Thermol buffer, MgSO 4 (6mmol), dNTPs solution (0.4mmol), Bst DNA polymerase (10U) and sterile double distilled water, the total reaction volume is 16μl. in:

[0102] The forward peripheral primer sequence is 5'-AGGACCACGATCGCTGATC-3';

[0103] The reverse peripheral primer sequence is 5'-TGGCCATCGTGGAAGCGA-3';

[0104] The sequences of the two probes are respectively:

[0105] Forward 5' Biotin labeled probe 5-biotin-TAGCAGACCTCACCTATGTGTC

[0106] Reverse 3' end fluorescein isothiocyanate (FitC) labeled probe 5-CTGGGCAGGGTTCGCCT-FitC;

[0107] The amplification cross primers are respectively:

[0108] Amplification reverse primer 5-CTCGTCCAGCGCCGCTTCGGTTCGGTGACAAAGGCCACG ...

Embodiment 2

[0115] Embodiment 2 detects the concrete method of mycobacterium tuberculosis nucleic acid with kit of the present invention

[0116] a) Using a DNA extraction kit to extract DNA from the specimen to be tested. For the specific protocol, see the DNA extraction kit.

[0117] b) Take the sample DNA as a template and add it to the PCR tube containing the reaction solution, and carry out the amplification reaction at 60°C for 90 minutes, including 4 μl of the sample DNA and 16 μl of the reaction solution; add the positive control template and the negative control template to the control PCR tube respectively .

[0118] c) Put the reacted PCR tube into the nucleic acid anti-pollution detection device for detection, and interpret the result after 15 minutes. When the sample contains Mycobacterium tuberculosis nucleic acid, the detection line of the test strip is positive.

[0119] The experiment was repeated 3 times, and there was no significant difference in the test results, in...

Embodiment 3

[0120] Embodiment 3 uses kit of the present invention to detect the specificity of Mycobacterium tuberculosis

[0121] Detect mycobacterium marinum according to the method of embodiment 2; ; Mycobacterium vaccae; Mycobacterium fortuitously; Mycobacterium chelonis; Mycobacterium chelonis; Image 6 .

[0122] from Image 6 It can be seen from the test results that the detection of Mycobacterium tuberculosis nucleic acid by the kit of the present invention has strong specificity.

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Abstract

The invention relates to a rapid detecting technology for detecting mycobacterium tuberculosis through nucleic acid constant-temperature amplification and a kit for qualitative detection. The kit comprises an instrument-free DNA (Deoxyribonucleic Acid) extraction kit, a mycobacterium tuberculosis nucleic acid constant-temperature amplification reaction liquid, a contamination-prevention nucleic acid amplification product detecting device, mycobacterium tuberculosis positive control, and mycobacterium tuberculosis negative control. The kit disclosed by the invention has the advantages of high specificity, high sensitivity and high reaction speed, wherein 1-1.5 hours are only needed from the step of processing a single sample to the step of completing the detection; and the kit can simultaneously meet requirements on sample detection of medium flux and low flux and is particularly suitable for being used in field detection and primary hospitals, and one constant-temperature instrument is only needed in the entire reaction process.

Description

technical field [0001] The invention relates to a detection technology for mycobacterium tuberculosis nucleic acid, in particular to a rapid detection kit for nucleic acid of mycobacterium tuberculosis and a method thereof, which are suitable for qualitative detection of mycobacterium tuberculosis. Background technique [0002] Tuberculosis (TB) is an infectious disease that seriously endangers human health. It is a major problem that hinders the country's economic and social development. In recent years, the recovery of the global tuberculosis epidemic has attracted great attention from the international community. The World Health Organization has regarded tuberculosis as one of the key infectious diseases to be controlled, and declared the global tuberculosis a state of emergency. Improving testing can boost international TB control efforts and address huge market demand, WHO said in its tuberculosis report, calling for industrial investment in new diagnostic tools target...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
Inventor 胡林尤其敏徐高连王宏莹钟华燕
Owner USTAR BIOTECHNOLOGIES (HANGZHOU) CO LTD
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