Primer, probe, method and kit for detecting KPC (Klebsiella Pneumoniae Carbapenemases) antibiotic gene

A technology of Klebsiella pneumoniae and carbapenems, applied in the field of molecular biology, can solve the problems of inability to understand the molecular biological mechanism of resistance and easy error detection, and achieve high accuracy, high sensitivity, and accurate detection Effect

Inactive Publication Date: 2016-09-21
宁波基内生物技术有限公司 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The invention provides a primer, a probe, a method and a kit for detecting carbapenem-resistant genes of Klebsiella pneumoniae, so as to solve the problem that conventional drug susceptibility tests in the prior art detect KPC enzyme-producing bacteria are easy to be misdetected, and Technical issues that fail to understand the molecular biological mechanisms underlying resistance

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  • Primer, probe, method and kit for detecting KPC (Klebsiella Pneumoniae Carbapenemases) antibiotic gene
  • Primer, probe, method and kit for detecting KPC (Klebsiella Pneumoniae Carbapenemases) antibiotic gene
  • Primer, probe, method and kit for detecting KPC (Klebsiella Pneumoniae Carbapenemases) antibiotic gene

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Embodiment 1

[0023] The embodiment of the present invention provides a primer and probe for detecting Klebsiella pneumoniae carbapenem-resistant gene, and the nucleotide sequence of the primer for detecting Klebsiella pneumoniae outer membrane phosphoprotein gene phoE is as SEQ ID NO: 2 As shown in ~3, the nucleotide sequence of the Taqman probe for detecting Klebsiella pneumoniae outer membrane phosphoprotein gene phoE is shown in SEQ ID NO: 4; the primer nucleotide sequence for detecting carbapenem-resistant antibiotic gene KPC is shown in As shown in SEQ ID NO: 6-7, the nucleotide sequence of the Taqman probe for detecting the carbapenem-resistant antibiotic gene KPC is shown in SEQ ID NO: 8; the nucleotide sequence of the primer for detecting the internal standard gene int is shown in SEQ ID NO: 10- As shown in 11, the nucleotide sequence of the Taqman probe for detecting the internal standard gene int is shown in SEQ ID NO: 12;

[0024] Wherein, the sequence of the specific primer des...

Embodiment 2

[0041] In the embodiment of the present invention, a method for detecting Klebsiella pneumoniae gene resistant to carbapenem antibiotics is provided, including:

[0042] Step 101, sample nucleic acid preparation to obtain a nucleic acid template;

[0043] Step 102, designing specific primers and fluorescently labeled probes for Klebsiella pneumoniae outer membrane phosphoprotein gene phoE, carbapenem-resistant antibiotic gene KPC, and internal standard gene int, respectively;

[0044] Wherein, the nucleotide sequence of the primer for detecting Klebsiella pneumoniae outer membrane phosphoprotein gene phoE is shown in SEQ ID NO: 2-3, and the nucleotide sequence of the Taqman probe for detecting Klebsiella pneumoniae outer membrane phosphoprotein gene phoE As shown in SEQIDNO: 4; The nucleotide sequence of the primer for detecting the carbapenem-resistant gene KPC is shown in SEQIDNO: 6-7, the nucleotide of the Taqman probe for detecting the carbapenem-resistant gene KPC The se...

Embodiment 3

[0056] The embodiment of the present invention provides a kit for detecting carbapenem-resistant gene of Klebsiella pneumoniae, comprising nucleic acid extract, first primer-probe mixed solution, second primer-probe mixed solution, PCR reaction Enzyme system, internal standard, negative control substance, positive control substance and the packing box that separate and pack these reagent bottles or tubes collectively, wherein, the first primer-probe mixed solution is made of deoxyribonucleoside triphosphate dN(U)TP , the upstream and downstream primers of the phoE gene and a fluorescent labeling probe, the upstream and downstream primers of the int gene and a fluorescent labeling probe, and the second primer probe mixture is composed of deoxyribonucleoside triphosphate dN(U) TP, KPC gene upstream and downstream primers and a fluorescent labeling probe, int gene upstream and downstream primers and a fluorescent labeling probe;

[0057] Wherein, the nucleotide sequence of the pr...

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Abstract

The invention relates to the technical field of molecular biology, and discloses a primer, a probe, a method and a kit for detecting a KPC (Klebsiella Pneumoniae Carbapenemases) antibiotic gene. Primers and fluorescent labeled probes are respectively designed aiming at a conservative region of a phoE gene, a KPC gene and an interior standard (int) gene by adopting a Taqman probe real-time fluorescence PCR (Polymerase Chain Reaction) method; the 5' ends of phoE gene probes and KPC gene probes are all labeled by a fluorescent report group FAM, and 3' ends are labeled by a fluorescent quenching group TAMRA; the 5' ends of int genes are labeled by a fluorescent report group JOE, and the 3' ends are labeled by a fluorescent quenching group BHQ1. After the primer and the probe are prepared into a PCR detection mixed solution, enzyme and sample nucleic acid are added, an FAM channel on a fluorescent PCR instrument is selected to amplify the phoE gene and the KPC gene, a JOE channel is selected to amplify the int gene, and detection on a target gene is realized through change of a fluorescent signal. The primer, the probe, the method and the kit, disclosed by the invention, have the characteristics of high accuracy, strong specificity ad high sensitivity, and KPN (Klebsiella Pneumoniae) and KPC in sputum and a throat swab sample can be rapidly and accurately detected.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a primer, a probe, a method and a kit for detecting a carbapenem-resistant gene of Klebsiella pneumoniae. Background technique [0002] Klebsiella pneumoniae carbapenemases (KPC) is a newly discovered carbapenemase, which can hydrolyze various β-lactam antibiotics including carbapenem antibiotics, And the KPC gene is located on the transferable plasmid, so that the KPC can be transmitted between strains. KPC is a potential risk factor for pulmonary infection in patients with alcoholism, diabetes and chronic obstructive pulmonary disease, which can cause pneumonia, urinary tract infection, meningitis, systemic sepsis, etc. The emergence and spread of KPC-type carbapenemase-producing bacteria have seriously weakened the clinical efficacy of β-lactam antibacterial drugs (including carbapenem antibacterial drugs), making clinical anti-infection treatment face severe challe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/686C12Q1/689C12Q2600/166C12Q2545/101C12Q2545/113C12Q2561/101C12Q2563/107
Inventor 王伟建倪剑锋孙小燕翁毅
Owner 宁波基内生物技术有限公司
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