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A kind of Klebsiella pneumoniae carbapenemase genotype detection method

A Klebsiella carbapenem and carbapenemase technology, which is applied in the directions of microorganism-based methods, biochemical equipment and methods, and microbial assay/inspection, etc., and can solve the problem of carbapenemase gene subtypes Problems such as unclear biological function, to achieve the effect of high detection throughput and low cost

Inactive Publication Date: 2017-08-11
海宁市疾病预防控制中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Little work has been done on carbapenemase genotyping and new gene screening of Klebsiella pneumoniae. Biological function is unknown

Method used

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  • A kind of Klebsiella pneumoniae carbapenemase genotype detection method
  • A kind of Klebsiella pneumoniae carbapenemase genotype detection method
  • A kind of Klebsiella pneumoniae carbapenemase genotype detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Selection of typing sites of KPC1-15 and primer design

[0050] NCBI database download KPC 1-15 sequence (KPC-1: AF297554; KPC-2: GU086225; KPC-3: AF395881; KPC-4: FJ473382; KPC-4: AY700571; KPC-5: EU400222; KPC-6: EU555534 ; KPC-7: EU729727; KPC-8: FJ234412; KPC-9: FJ624872; KPC-10: GQ140348; ; KPC-15: KC433553). After the sequences were compared by ClusterX, 6 SNP sites (nt147, nt272, nt308, nt502, nt716 and nt814) were selected.

[0051] A pair of large-fragment primers was designed for template enrichment, and the amplified region contained the six SNP sites to be detected. Snapshot can detect multiple SNPs, select 6 sites to make a combination, and design primers for these target SNP sites. The primers designed for each SNP are extended ddNTPs, designed upstream or reverse downstream of the SNP site. See Table 1 for details. The classification of KPC1-15 is shown in Table 2.

[0052] Table 1. Large fragment amplification primers and SNaPshot PCR pri...

Embodiment 2

[0056] Embodiment 2, the detection of known bacterial strain

[0057] 1. The DNA of six different KPC subtype strains (KPC-2, KPC-3, KPC-4, KPC-5, KPC-8 and KPC-11) was provided by the Zhejiang Provincial Center for Disease Control and Prevention at a concentration of 1ng / μl. The typing results were verified by sequencing.

[0058] 2. Template enrichment, using large fragment primers (KPCF and KPCR) to amplify the template:

[0059] PCR amplification system:

[0060]

[0061] Amplification Thermal Cycling

[0062] (1) PCR amplification tube is placed on thermal cycler;

[0063] (2) Select the program recommended below to amplify;

[0064] (3) The amplified samples should be kept away from light;

[0065] Amplification program for thermal cycler:

[0066]

[0067] Handling of PCR products

[0068] Take 3μL + 0.7μL enzyme mixture (containing 2.5U CIAP (Bao biological alkaline phosphatase) and 5U Exo I) of the PCR amplification products of 6 strains, shake and mix, i...

Embodiment 3

[0083] Example 3 Clinical Sample SNaPshot Typing Detection

[0084] 1. Sample collection

[0085]Clinical isolates of Klebsiella pneumoniae from various hospitals in Haining City from 2008 to 2012 were collected. For further identification in this laboratory, separate and purify on the MacConkey medium plate, and identify as Klebsiella pneumoniae by French BioMérieux Vitek-compact automatic bacterial analysis, scrape the colony to a special magnetic bead storage tube Prepare a uniform bacterial suspension in the medium, blot the liquid, and store it in a low-temperature refrigerator at -70°C. Resuscitate and culture the strains on MacConkey medium plates, use K-B (also known as disc diffusion method), pick a certain amount of bacteria to prepare 0.5 McFarland turbidity bacteria liquid, spread evenly on MH plates, and dry at room temperature for 3~ 5min. Paste three kinds of paper sheets of imipenem, meropenem and ertapenem respectively. The center of each paper sheet should...

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Abstract

The invention discloses a detection method of a carbapenemase gene of Klebsiella pneumonia. The detection method comprises the following steps: carrying out PCR (polymerase chain reaction) amplification reaction by virtue of a primer 1 and a primer 2 by taking a to-be-detected bacterial strain gene as a template, and carrying out SNaPshot PCR reaction by virtue of a mixed primer from a primer 3 to a primer 8 by taking an amplified product as a template; and classifying according to products of the SNaPshot PCR reaction. According to the detection method, the detection method for simultaneously classifying 14 subtypes from KPC1 to KPC15 is provided for the first time, the detection flux is higher than the detection fluxes of traditional quantitative detection methods, the accuracy is equal to that of a sequencing detection method, and the cost is relatively low.

Description

[0001] (1) Technical field [0002] The invention relates to a method for detecting Klebsiella pneumoniae carbapenemase genotype. [0003] (2) Background technology [0004] Klebsiella pneumoniae (Klebsiella pneumoniae) is a common nosocomial pathogenic bacteria, and it is one of the pathogenic bacteria that are mainly monitored in clinical nosocomial infection. With the widespread use of carbapenem antibiotics, multidrug-resistant Klebsiella pneumoniae have emerged. The "Superbug" incident caused by Klebsiella pneumoniae infection in Brazil has brought global attention to "multiple antibiotic resistance" and "Klebsiella pneumoniae". [0005] According to reports, Klebsiella pneumoniae-type carbapenemase was also found in some Enterobacteriaceae bacteria, such as Escherichia coli, Salmonella, Enterobacter cloacae, Klebsiella oxytoca, Serratia, etc. The use of clinical antibiotics to treat bacterial infectious diseases fails or causes the course of treatment to be delayed. Th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/10C12R1/22
Inventor 朱海燕顾建忠葛斌文金大智吴方
Owner 海宁市疾病预防控制中心
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