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Loop-mediated isothermal amplification (LAMP) primer and kit for detecting super bacteria
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A loop-mediated isothermal, amplification primer technology is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. Sensitive and specific effects
Inactive Publication Date: 2013-06-05
吴兴海
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[0004] In the existing detection methods, it is generally only through drug sensitivity tests to determine whether microorganisms are resistant to a certain antibiotic, which is time-consuming and cumbersome to operate, and cannot meet the needs of disease prevention and control; It is also required to be able to quickly and sensitively detect whether there are super bacteria in food and water sources, so as to prevent the harm of super bacteria to human body or farmed animals
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Embodiment 1
[0051] Example 1: Detection of artificially synthesized and constructed NDM1 plasmidDNA
[0052] Make the kit for NDM1 gene loop-mediated isothermal amplification reaction according to the following formula
[0054] Each 25 μL contains 2.5 μL 10× Thermopol reaction buffer, 300-500 μmol dNTPs, 2-4 mmol MgSO 4 , 0.8-1.2 μmol upstream internal primer FIP, 0.8-1.2 μmol internal primer BIP, 0.2-0.3 μmol upstream external primer F3, 0.2-0.3 μmol downstream internal primer B3 and 1-1.5 M betaine;
[0055] The 10× Thermopol reaction buffer described therein contains 200mmol of Tris-HCl at pH8.8, 100mmol of KCl, 100mmol of NH 4 2SO 4 , 20mmol MgSO 4 and 1% Triton X-100;
[0056] The primer information described therein is as follows:
[0087] Each 25 μL contains 2.5 μL 10× Thermopol reaction buffer, 300-500 μmol dNTPs, 2-4 mmol MgSO 4 , 0.8-1.2 μmol upstream internal primer FIP, 0.8-1.2 μmol internal primer BIP, 0.2-0.3 μmol upstream external primer F3, 0.2-0.3 μmol downstream internal primer B3 and 1-1.5 M betaine;
[0088] The 10× Thermopol reaction buffer described therein contains 200mmol of Tris-HCl at pH8.8, 100mmol of KCl, 100mmol of NH 4 2SO 4 , 20mmol MgSO 4 and 1% Triton X-100;
[0089] The upstream internal primer, downstream internal primer, upstream external primer, and downstream external primer are the same as above
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Abstract
The invention relates to a loop-mediated isothermal amplification (LAMP) primer and an LAMP kit for detecting New Delhi metallo-beta-lactamase 1 (NDM1) genes of super bacteria. In the primer, an upstream inner primer, a downstream inner primer, an upstream outer primer and a downstream outer primer have the sequences shown as SEQ ID No.1-4 respectively. In the invention, the LAMP primer is designed according to six sequences in basic reserved areas of the NDM1 genes, and the reserved gene sequences are shared by different bacterial strains with super drug resistance, so that the reliability of the NDM1 genes from different sources can be detected at a specific level. In the invention, an LAMP technology is adopted, has high specificity and has high sensitivity which is the same as that ofa polymerasechain reaction (PCR) detection method; expensive PCR instruments are not required, and only a common metal bath or water bath kettle is required; and a fluorescent dye is used for observing a result simply and quickly without the observation by a gel electrophoresis method. The primer and the kit can be used for detecting the NDM1 genes and are particularly suitable for primary medical institutions and farms.
Description
technical field [0001] The invention belongs to the technical field of disease detection reagents, and in particular relates to a loop-mediated isothermal amplification primer and a kit for detecting superbacteria NDM1 gene. Background technique [0002] There are a large number of natural drug resistance genes in nature. These genes are originally in the complex metabolic regulatory network of the original host, and play the role of signal transduction and gene regulation. However, when they enter a new host through horizontal gene transfer, because these drug-resistant genes are difficult to integrate into the metabolic network of the new host, their functions change, showing resistance to antibiotics. The abuse of antibiotics and environmental pollution by humans, like screening pressure, select and evolve these bacteria with integrated drug-resistant genes, making the latter eventually become superbugs. [0003] The NDM1 gene is the key pathogenic gene of superbugs. Und...
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