Primers, probe and kit used for detecting Klebsiella pneumoniae carbapenemase (KPC) genes
A Klebsiella carbapenem and enzyme gene technology, applied in the biological field, can solve problems such as inaccurate detection of Klebsiella pneumoniae carbapenemase, achieve reliable detection results, avoid subjectivity, and improve sensitivity Effect
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Embodiment 1
[0033] Example 1. A primer and probe for the quantitative detection of Klebsiella pneumoniae carbapenemase gene nucleic acid
[0034] The embodiment of the present invention provides a kind of primer and probe for detecting Klebsiella pneumoniae carbapenemase gene, the primer and probe include: for detecting Klebsiella pneumoniae carbapenemase gene A forward primer, a reverse primer for detecting the Klebsiella pneumoniae carbapenemase gene and a probe for detecting the Klebsiella pneumoniae carbapenemase gene, wherein,
[0035] The forward primer for detecting Klebsiella pneumoniae carbapenemase gene is shown in SEQ ID NO.1 in the sequence listing;
[0036] The reverse primer for detecting Klebsiella pneumoniae carbapenemase gene is shown in SEQ ID NO.2 in the sequence listing;
[0037] The probe for detecting Klebsiella pneumoniae carbapenemase gene is shown in SEQ ID NO.3 in the sequence listing;
[0038] The 5' end of the probe is connected with a fluorescent group FAM, ...
Embodiment 2
[0044] Example 2. A kit for detecting Klebsiella pneumoniae carbapenemase gene
[0045]The embodiment of the present invention provides a kit for the detection of Klebsiella pneumoniae carbapenemase nucleic acid quantification, the kit includes the primers and probes provided in Example 1 of the present invention, and the kit also includes: nucleic acid Release agent, PCR reaction solution, critical positive quality control, positive quality control, negative quality control and working standard.
[0046] Specifically, each component of the PCR reaction solution includes in the PCR amplification reaction system: Taq enzyme with a final concentration of 0.01U / μL-0.05U / μL, dNTPs with a final concentration of 0.2-0.6mM, 10×PCR buffer and A solution containing Mg ions at a final concentration of 1.5-5.0 mM. In this example, the ratio of the contents of each component of the PCR reaction solution is: 0.3 μL of Taq enzyme at a concentration of 5 U / μL, 2 μL of dNTPs at a concentrati...
Embodiment 3
[0077] Example 3. A kit for detecting Klebsiella pneumoniae carbapenemase gene
[0078] The embodiment of the present invention provides a kit for detecting Klebsiella pneumoniae carbapenemase nucleic acid in a sample, and the components of the kit differ from those in the kit provided in Example 2 in that:
[0079] The ratio of the contents of each component of the PCR reaction solution is: 0.1 μL of Taq enzyme at a concentration of 5 U / μL, 1 μL of dNTPs at a concentration of 10 mmol / L, 5 μL of 10×PCR Buffer and MgCl at a concentration of 25 mmol / L 2 Solution 3 μL.
[0080] Add 0.25 μL each of forward primer and reverse primer with a concentration of 10 μmol / L, and add 0.25 μL of probe with a concentration of 10 μmol / L.
[0081]During practical application, primers and probes can be added together into the PCR reaction solution, and then sterile water can be added to a volume of 10 μL.
[0082] The DNA extraction solution included chloroform at a final concentration of 0.15...
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