Mycobacterium tuberculosis gene rapid diagnostic kit based on loop-mediated isothermal amplification technology and detection method thereof
A technology for rapid diagnosis of Mycobacterium tuberculosis, which is applied in the directions of microorganism-based methods, biochemical equipment and methods, and determination/examination of microorganisms. The effect of significant color difference, high yield and low detection cost
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Embodiment 1
[0040] The preparation of embodiment 1 kit
[0041] (1) Synthesize oligodeoxynucleic acid primers by DNA synthesizer according to the following sequence:
[0042] Outer primer F3: GGCTGGTCTCTGGCGTT, as shown in SEQ ID NO: 1;
[0043] Outer primer B3: GGCCTATACAAGACCGAGCT, as shown in SEQ ID NO: 2;
[0044] Internal primer FIP: GCCTCTACCAGTACTGCGGCTTTTGAGCGTAGTAGGCAGCCT, as shown in SEQ ID NO: 3;
[0045] Internal primer BIP: GTTGAACCAGTCGACCCAGCGTTTTAACCCGGCAAGCCCT, as shown in SEQ ID NO: 4;
[0046] (2) Purchasing DNA polymerase: Bst DNA polymerase is placed in the container.
[0047] (3) Prepare the reaction solution: the reaction solution contains 2mmol dNTP, 25mmol Tris-Cl, 12.5mmol potassium chloride, 12.5mmol ammonium sulfate, 10mmol magnesium sulfate, 1.25ml TritonX-100, 1mol betaine, and internal primer FIP per 1L 2 mol of each BIP and 0.25 mol of each outer primer F3 / B3 were prepared and placed in a container.
[0048] (4) Preparation of Lysis Solution 1: Lysis S...
Embodiment 2
[0060] The preparation of embodiment 2 kit
[0061] The reaction solution contains 1.6mmol dNTP, 20mmol Tris-Cl, 10mmol potassium chloride, 10mmol ammonium sulfate, 8mmol magnesium sulfate, 1ml TritonX-100, 0.8mol betaine, 1.6mol each of internal primer FIP / BIP and external primer per 1L Prepare 0.2mol each of F3 / B3 and place in the container.
[0062] Lysis solution 1 contains 20mmol of Tris-HCl with pH8.3, 100mmol of KCl, 5mmol of MgCl per 1L 2 , 10ml Triton X-100, 10ml Tween-20 and 0.2g gelatin preparation.
[0063] The chromogenic solution is EvaGreen.
[0064] Others are the same as embodiment 1.
Embodiment 3
[0065] Example 3 Application of Mycobacterium tuberculosis Gene Rapid Diagnostic Kit
[0066] (1) Purpose of the experiment
[0067] The sensitivity, specificity and accuracy of the Mycobacterium tuberculosis LAMP gene rapid detection kit (Example 1) developed by Guangzhou Huafeng Biotechnology Co., Ltd. were evaluated. Understand the impact of the processing of clinical samples on the test results of the kit, and provide a basis for evaluating the practicability of the product.
[0068] (2) Selection of reference reagents
[0069] The conventional reagents used in the daily detection of Dongguan Infectious Disease Hospital and the Mycobacterium tuberculosis fluorescent quantitative PCR detection kit produced by Shenzhen Piji Bioengineering Co., Ltd. were used as controls.
[0070] The tested reagent was the Mycobacterium tuberculosis LAMP gene rapid detection kit (Example 1) developed by Guangzhou Huafeng Biotechnology Co., Ltd.
[0071] (3) Selection of research objects ...
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