Primer set, kit and detection method for rapid detection of carbapenemase genes
A carbapenemase and primer set technology, applied in biochemical equipment and methods, recombinant DNA technology, resistance to vector-borne diseases, etc., can solve the problem of not detecting genes at the same time, and shorten the detection time and save the detection. Cost and effect of fully closed reaction process
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[0047] Example 1
[0048] Composition and detection method of a kit for rapid detection of carbapenemase gene
[0049] 1. The composition of the kit
[0050] (1) Primer set: Amplification primer pairs and molecular beacon probes for detecting Enterobacter carbapenemase resistance genes KPC, NDM, OXA48, VIM and IMP;
[0051] Among them, the primer set of KPC gene is:
[0052] KPC-F (SEQ ID NO:1): CAGCTCATTCAAGGGCTTTC;
[0053] KPC-R (SEQ ID NO: 2): CGTCATGCCTGTTGTCAGAT;
[0054] Molecular beacon probe KPC-probe (SEQ ID NO: 3): cgcgaACACACCCATCCGTTACGGCA tcgcg;
[0055] The primer set of NDM gene is:
[0056] NDM-F (SEQ ID NO: 4): ATATCACGTTGGGATCGAC;
[0057] NDM-R (SEQ ID NO: 5): TAGTGCTCAGTGTCGGCATC;
[0058] Molecular beacon probe NDM-probe (SEQ ID NO: 6): cgcgaGCCTGATCAAGGACAGCAAGG tcgcg;
[0059] The primer set of OXA48 gene is:
[0060] OXA48-F (SEQ ID NO: 7): GTGGCATCGATTATCGGAAT;
[0061] OXA48-R (SEQ ID NO: 8): AGAGCACAACTACGCCCTGT;
[0062] Molecular beacon probe OXA48-probe (SEQ ID NO: ...
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[0081] Example 2
[0082] Method for rapidly detecting carbapenemase gene using the kit described in Example 1
[0083] 1. Sample
[0084] The CRE strain of Klebsiella pneumoniae ST11 was isolated from random collection of clinical samples, and then the isolated CRE strains were cultured at 37°C for 24 hours, and then the DNA was extracted using the method described in Example 1. The specific primers of the Enterobacter carbapenemase resistance genes KPC, NDM, OXA48, VIM and IMP were screened by PCR and sequenced to identify the corresponding positive samples, and then a single carbapenemase resistance gene KPC, DNA samples of NDM, OXA48, VIM and IMP were mixed to produce DNA samples containing five common enterobacteria carbapenemase resistance genes KPC, NDM, OXA48, VIM and IMP. The clinical sample used in this embodiment is a blood sample, but it is not limited to this. In addition, respiratory tract samples such as urine and sputum containing the CRE strain of Klebsiella pneumo...
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[0089] Example 3
[0090] Sensitivity analysis of constant temperature amplification melting curve detection kit for CRE strains isolated from clinical samples based on molecular beacon technology
[0091] 1. Method
[0092] Sample processing: The CRE strain of Klebsiella pneumoniae ST11 isolated from clinical samples is adjusted to 1.8×10 with normal saline 8 CFU / mL, then perform a gradient dilution (dilute by 100μL bacterial solution + 900μL physiological saline), then centrifuge at 10000rpm, discard the supernatant, and then break it on a shaker for 10 minutes, take 300μL of the supernatant and add To the 96 deep well plate, extract the DNA on the nucleic acid extractor to 1.5×10 7 CFU / mL-1.5×10 2 DNA extracted from CFU / mL bacterial solution was used as a template, and each concentration gradient was 5μL for amplification.
[0093] 2. Use the detection method described in Example 1 for detection. After the reaction is completed, observe the melting curve peak Tm value, analyze the...
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