Rapid diagnostic reagent kit of legionella pneumophilia genes based on loop-mediated isothermal amplification technique and detecting method thereof
A technology for rapid diagnosis of Legionella pneumophila, which is applied in the field of biological detection reagents, can solve the problems of no gene rapid diagnosis kit for detection of Legionella pneumophila, and achieves significant color difference, high yield, and low detection cost Effect
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Embodiment 1
[0038] The preparation of embodiment 1 kit
[0039] (1) Synthesize oligodeoxynucleic acid primers by DNA synthesizer according to the following sequence:
[0040]Outer primer F3: TCTATTGAAATAGCACTTAAGCT, as shown in SEQ ID NO: 1;
[0041] Outer primer B3: TGGATTCCGATCGGGATG, as shown in SEQ ID NO: 2;
[0042] Internal primer FIP: GCTGTTCCATAACTGCTCACAATTTttttAATTTAGAGTCTTCATACGACTGA, as shown in SEQ ID NO: 3;
[0043] Internal primer BIP: TTAGAAGTAAGTCGAAATGCGAGTGttttGTATTTCATTGCCAGCCTG, as shown in SEQ ID NO: 4;
[0044] (2) Purchase DNA polymerase: Bst DNA polymerase (large fragment) and place it in a container.
[0045] (3) Preparation of reaction solution: The formula of 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, 2 mol each of primers FIP / BIP and 0.25 mol each of outer primers F3 / B3 were prepared and placed in containers.
[0046] (4) Pre...
Embodiment 2
[0057] The preparation of embodiment 2 kit
[0058] The formula of the reaction solution is: each 1L reaction solution contains 1.6mmol dNTP, 20mmol Tris-HCl, 10mmol potassium chloride, 10mmol ammonium sulfate, 8mmol magnesium sulfate, 1ml TritonX-100, 0.8mol betaine, internal primer FIP / BIP each 1.6 mol and 0.2mol each of outer primer F3 / B3;
[0059] The formula of the sample pretreatment solution is: every 1L of the sample pretreatment solution contains 10mmol of Tris-HCl with pH8.0, 1mmol of EDTA and 10ml of Triton X-100.
[0060] The chromogenic solution is EvaGreen.
[0061] Others are the same as embodiment 1.
Embodiment 3
[0062] Example 3 Application of Legionella pneumophila Gene Rapid Diagnostic Kit
[0063] (1) Purpose of the experiment
[0064] The sensitivity, specificity and accuracy of the Legionella pneumophila LAMP gene rapid detection kit (Example 1) developed by Guangzhou Huafeng Biotechnology Co., Ltd. were evaluated.
[0065] (2) Selection of reference reagents
[0066] The routine reagents used in the daily detection of Guangzhou Center for Disease Control and Prevention, the Health Industry Standard of the People's Republic of China: Legionnaires' Disease Diagnostic Criteria and Treatment Principles WS 195--2001 were selected as the control.
[0067] The reagent for assessment was the Legionella pneumophila LAMP gene rapid detection kit developed by Guangzhou Huafeng Biotechnology Co., Ltd. (Example 1).
[0068] (3) Selection of research objects
[0069] Eight Legionella isolation cultures and two interfering strains provided by the Guangzhou Center for Disease Control and Pre...
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