Fluorescent detection kit and detection method for Klebsiellapneumoniae carbapenamase

A technology of carbapenemase pneumonia and Klebsiella, applied in the field of fluorescent detection kits, to achieve the effects of reduced sensitivity, high sensitivity, and increased sensitivity

Inactive Publication Date: 2011-04-06
ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The KPC-2 enzyme gene in my country is mainly found in

Method used

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  • Fluorescent detection kit and detection method for Klebsiellapneumoniae carbapenamase
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  • Fluorescent detection kit and detection method for Klebsiellapneumoniae carbapenamase

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Embodiment 1

[0034] 1. Materials:

[0035] Bacterial genomic DNA extraction reagents were purchased from Dalian Bao Biological Engineering Co., Ltd.; restriction endonucleases were purchased from LTI / Gibco in the United States, pGEM-T-Easy cloning system and Taq DNA polymerase were purchased from Promega in the United States; sequencing reagents, type 377 Sequencer, Bio-Radicycler PCR instrument, Rotor Gene Q 5-plex quantitative PCR instrument (Qiagen Company).

[0036] 2. Primer and probe design and synthesis:

[0037] Using the gene sequence of carbapenemase Klebsiella pneumoniae (registration number AY034847) as a template, use Primer Express TM (V2.0, American ABI Company) software analyzes TaqMan primer and probe site, selects the optimal combination therefrom.

[0038] Standard PCR sequence:

[0039] Upstream primer: 5'-GGCACGGCAAATGACTATGC-3',

[0040] Downstream primer: 5'-CCCTCGAGCGCGAGTCTAGC-3',

[0041] The PCR sequence for detection is:

[0042] Upstream primer: 5′-ACTGGG...

Embodiment 2

[0062] Embodiment 2: Detection of clinical samples by fluorescent quantitative PCR method

[0063] 1. Specimen detection:

[0064] Genomic DNA was extracted using genomic DNA extraction reagents from 5 artificial simulated specimens, and 1.0 μL was taken as a template, and PCR amplification was carried out on a Rotor Gene Q 5-plex quantitative PCR instrument (Qiagen Company) with upstream and downstream primers for detection.

[0065] The composition of the PCR reaction solution is as follows:

[0066] 2×PCR buffer 10.0μL

[0067] Detection upstream primer (10μM) 1μL

[0068] Downstream primer for detection (10μM) 1μL

[0069] Fluorescent probe for detection (10μM) 0.5μL

[0070] DNA polymerase (5U / μL) 0.2μL

[0071] dNTPs (250mM each) 1.60μL

[0072] (dATP, dTTP, dCTP, dGTP substance ratio 1:1:1:1)

[0073] Template DNA (50ng / μL) 1μL

[0074] Make up to 20 μL with water.

[0075] The PCR reaction conditions were: pre-denaturation at 95°C for 2 minutes, 40 cycles of a...

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Abstract

The invention provides a fluorescent detection kit and a detection method for Klebsiellapneumoniae carbapenamase. The sequences of specific primers and a fluorescent probe in the kit are as follows: a forward primer: 5'-ACT GGG CGC GCACCTA-3'; a reverse primer: 5'-TCG CTGTGC TTG TCATCC TT-3'; and the fluorescent probe: 5'-FAM-CCG TCT ACACCCGGG CGC C-TAMRA-3', wherein the FAM is a fluorescent reporter, and the TAMRA is a fluorescent quencher. The invention has the advantages that: the detection kit has high sensitivity and good specificity for the Klebsiellapneumoniae carbapenamase, and reduces the false positive rate of the conventional polymerase chain reaction (PCR) amplification; and quick, accurate and specific detection for the Klebsiellapneumoniae carbapenamase can be realized.

Description

(1) Technical field [0001] The invention relates to a carbapenemase Klebsiella pneumoniae fluorescence detection kit and a detection method. (2) Background technology [0002] The drug-resistant bacterium KPC is Klebsiella pneumoniae (Klebsiellapneumoniae carbapenamase), which was first discovered in an imipenem-resistant Klebsiella pneumoniae, belonging to the 2f group of the Bush classification, Ambler Class A of the classification can hydrolyze antibiotics such as carbapenems, penicillins, cephalosporins and aztreonam. At present, 5 subtypes of KPC have been discovered by domestic and foreign studies, and carbapenemase Klebsiella pneumoniae belongs to type 2. Carbapenems are usually used to treat bacterial infections that other antibiotics cannot cope with. They have a very wide range of antibacterial activities. Because they can resist the hydrolysis of most lactamases, they are often used to produce extended-spectrum-lactamase (ESBL) and / or derepressed AmpC-lactamase ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/06G01N21/64
Inventor 金大智罗芸张政程苏云韩建康
Owner ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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