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LAMP primers for detecting mycobacterium tuberculosis in intraocular fluid and application thereof

A technology of mycobacterium tuberculosis and primer set, applied in the biological field, can solve the problems of easy contamination, long PCR detection time, high false positive rate, etc., and achieve high sensitivity, high specificity, and accurate detection effect

Inactive Publication Date: 2019-10-01
BEIJING CHAOYANG HOSPITAL CAPITAL MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, PCR has the disadvantages of long detection time, easy contamination, and high false positive rate, which limits its application.

Method used

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  • LAMP primers for detecting mycobacterium tuberculosis in intraocular fluid and application thereof
  • LAMP primers for detecting mycobacterium tuberculosis in intraocular fluid and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Embodiment 1, the preparation of the primer set identifying Mycobacterium tuberculosis

[0071] A large number of sequence analyzes and comparisons were carried out to obtain several primers for identifying Mycobacterium tuberculosis. Preliminary experiments were performed on each primer to compare performances such as sensitivity and specificity, and finally a LAMP primer set for identifying Mycobacterium tuberculosis was obtained.

[0072] A primer set for identifying Mycobacterium tuberculosis, including 2 outer primers (F3-1, B3-1), 2 inner primers (FIP-1, BIP-1) and 2 loop primers (LF-1, LB -1), each primer sequence is as follows (5'→3'):

[0073] Primer F3-1 (sequence 1 of the sequence listing): AgTACACCgTTTTCgCC;

[0074] Primer B3-1 (sequence 2 of the sequence listing): gTTgACCATgAggTCgTC;

[0075] Primer FIP-1 (sequence 3 of the sequence listing): gCgTCAgTCTTgAgTTgATCgCCACCAACgCCgCATTC;

[0076] Primer BIP-1 (sequence 4 of the sequence listing): CTCAgCAgCAT...

Embodiment 2

[0080] Embodiment 2, detection method establishment

[0081] The method for detecting Mycobacterium tuberculosis using the primer set for identifying Mycobacterium tuberculosis of Example 1 comprises:

[0082] 1. Extract the genomic DNA of the bacteria to be tested or extract the total DNA of the sample to be tested.

[0083] 2. Take the total DNA obtained in step 1 as a template, and use the primer set prepared in Example 1 to perform LAMP amplification.

[0084] The reaction system for 10μL LAMP amplification includes: 1μL 10×ThermoPol Buffer (Thermo), 0.8M Betaine, 0.5mg / ml BSA, 4mM MgSO 4 , 0.3μL 20×EvaGreen (Hefei Bomei Biotechnology Co., Ltd.), 1.5mM dNTPs, 0.32U / ml Bst DNA polymerase large fragment, primer mix (F3-1, B3-1, FIP-1, BIP-1, LF-1 and LB-1 mixture), 2ng template DNA, balance ddH 2 O. The concentration of each primer in the reaction system is as follows: 0.5 μM F3-1, 0.5 μM B3-1, 2 μM FIP-1, 2 μM BIP-1, 1 μM LF-1, 1 μM LB-1.

[0085] Reaction program for ...

Embodiment 3

[0087] Embodiment 3, identify the specificity of the primer set of Mycobacterium tuberculosis

[0088] Mycobacterium tuberculosis reference plasmid DNA construction: the DNA fragment of Mycobacterium tuberculosis shown in sequence 7 of the sequence listing is inserted between the ApaI and SacI restriction sites of the pGEM-T Easy Vector vector to obtain Mycobacterium tuberculosis Reference plasmid DNA.

[0089] The samples to be tested are: Mycobacterium tuberculosis reference plasmid DNA, Escherichia coli genomic DNA, Staphylococcus aureus genomic DNA, and Candida albicans genomic DNA. Among them, Escherichia coli and Staphylococcus aureus were recorded in "Wu Wei, He Meifeng, Tang Xilan, et al. Analysis of the distribution and drug resistance of pathogenic bacteria in eye bacterial infection [J]. Chinese Journal of Hospital Pharmacy, 2010,30(20):1786- 1788." in the article; Candida albicans was recorded in "Zhang Zhiming, Li Jianping, Zhu Jianwei. Application of 16S rRNA in...

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Abstract

The invention discloses LAMP primers for detecting mycobacterium tuberculosis in an intraocular fluid and application thereof. The LAMP primers are composed of six single-stranded DNA molecules shownin sequences 1-6. The invention further discloses the application of the LAMP primers and a kit containing the LAMP primers. Experiments show that the LAMP primers have high specificity and high sensitivity when used for detecting the mycobacterium tuberculosis, and the applied LAMP primers and method can rapidly and accurately detect the mycobacterium tuberculosis.

Description

technical field [0001] The invention relates to a group of LAMP primers used for detecting Mycobacterium tuberculosis in intraocular fluid and its application in the field of biotechnology. Background technique [0002] Mycobacterium tuberculosis (Mycobacterium tuberculosis), commonly known as Mycobacterium tuberculosis, is the pathogenic bacteria that causes tuberculosis and can invade various organs of the body. Mycobacterium tuberculosis infection is the most common eye lesion is uveitis, can also cause eyelids, conjunctiva, cornea, sclera and episclera, orbital, optic nerve and other lesions. Sudden loss of vision, ciliary congestion, mild turbidity of aqueous humor, suet-like deposits after the cornea, retinal edema, macular yellow exudate lesions, rapid red blood cell sedimentation rate, X-rays showed pulmonary active tuberculosis, skin tuberculosis If the hormone test is positive, the diagnosis is tuberculous retinitis and pulmonary tuberculosis, and anti-tuberculosi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/04
CPCC12Q1/689C12Q1/6844
Inventor 陶勇
Owner BEIJING CHAOYANG HOSPITAL CAPITAL MEDICAL UNIV
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