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Mycobacterium tuberculosis TB detection kit

A technology of Mycobacterium tuberculosis and detection kit, which is used in material excitation analysis, microbial determination/inspection, fluorescence/phosphorescence, etc., can solve the problem of low detection sensitivity, and achieve reliable experimental basis, high detection sensitivity and fast operation. Effect

Active Publication Date: 2013-05-01
湖南圣维尔医学检验所有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, kits for quantitative detection of TB-DNA based on real-time fluorescent quantitative PCR technology have been used in clinical detection at home and abroad, but most of these kits use the boiling method to extract nucleic acid, and the detection sensitivity is not high, about 1-10 TB Bacteria around

Method used

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Examples

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Effect test

Embodiment 1

[0023] This embodiment provides a specific Mycobacterium tuberculosis detection kit, which includes the following components:

[0024] ①Nucleic acid release agent: contains surfactin 0.1mM / L, potassium chloride 100mM / L, sodium dodecyl sulfonate (SDS) 0.1%, 0.1% ethanol and solvent TE buffer.

[0025] ②Internal standard (positive internal control): It is a recombinant of 97 base pairs of synthetic DNA sequence inserted into pUC18T vector, namely plasmid, with a concentration of 2.00E+05copies / ml, and its sequence is: 5'-GTGTCTGCGGCGTTTTATCATCTTCCTCTGTCATCCAGTGCAAGTCTTGATCCTGTCGTTGGTTCTTCTGACTGCCAGTGTGT -3'.

[0026] ③PCR reaction solution: including 5μl of 10×PCR reaction buffer, 0.2mmol / L dNTP, the upstream and downstream primers used for target polynucleotide amplification are 0.3μmol / L, and the probe used for target polynucleotide detection It is 0.3μmol / L, the upstream and downstream primers used for the amplification of internal standard fragments are both 0.3μmol / L, and the pro...

Embodiment 2

[0032] This example provides the operating steps of the kit described in Example 1 for detecting TB-DNA in unknown samples such as sputum:

[0033] 1. Reagent preparation

[0034] According to the quantity of test sample, TB negative control, TB positive control and TB quantitative reference A~D, take the corresponding amount of PCR reaction solution (38μl / person), enzyme mixture (2μl / person) and internal Mark 1.0μl / person and mix thoroughly to form PCR-mix. For example, when the sample to be tested is 3 persons, a total of 9 persons (the number of persons for the above four are 3, 1, 1, and 4) PCR-mix; reserve after short-term centrifugation.

[0035] 2. Nucleic acid extraction

[0036] Add 2 to 3 times the volume of 4% NaOH solution to the sample, shake and mix, and let it stand for 30 minutes to liquefy. Take 500μl of the liquefied sample in a 1.5ml centrifuge tube (to avoid aspiration of obvious solid impurities), centrifuge at 12000r / min for 3min, aspirate the supernatant; add ...

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Abstract

The invention provides a mycobacterium tuberculosis TB detection kit. The kit comprises a nucleic acid releasing agent and polymerase chain reaction (PCR) liquid, wherein the nucleic acid releasing agent comprises surfactin of 0.01-0.05 mM / L, potassium chloride of 20-300 mM / L, 0.01 to 2 percent of sodium dodecyl sulfate and 0.05 to 1 percent of ethanol; the PCR liquid comprises an upstream primer, a downstream primer and a probe; the upstream primer and the downstream primer are applied to amplification of target polynucleotide; and the probe is applied to detection of target polynucleotide. No significant difference exists between the detection result of the method for releasing nucleic acid by using the nucleic acid releasing agent in the kit and that of a boiling method. The nucleic acid is extracted by a strong protein denaturing agent, the shell protein structure of the pathogene is quickly damaged, and the nucleic acid of the pathogene is released, release and extraction of DNA can be finished without heating. According to the kit, the TB detection sensitivity can reach 400 copies / ml and the quantitative linear range is 400-4.00E+09 copies / ml. With the application of the kit, the TB-DNA of unknown samples such as sputum can be detected quickly and accurately, and reliable test basis is provided for diagnosis of TB infection.

Description

Technical field [0001] The present invention provides a detection kit for Mycobacterium tuberculosis (TB), specifically a TB-DNA detection kit based on fluorescent PCR. Background technique [0002] Mycobacterium tuberculosis is the causative bacterium of tuberculosis. It can invade all tissues and organs of the body, but it is most common in lung infections. At present, there are about 8 million new tuberculosis cases worldwide each year and cause about 3 million deaths; China dies every year The number of people suffering from tuberculosis is about 250,000, which is more than twice the total number of deaths from various infectious diseases; therefore, tuberculosis has become a global health problem threatening human health, and it has become some developing countries and regions, especially The leading cause of death among people in high-risk areas of AIDS. [0003] At present, the clinical diagnosis of tuberculosis in my country is mainly through sputum smear microscopy and ba...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/06G01N21/64
Inventor 戴立忠邓中平吴康
Owner 湖南圣维尔医学检验所有限公司
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