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Primers, method and kit for detecting carbapenemase blaKPC gene

A carbapenemase and kit technology, applied in the field of molecular biology, can solve the problems of complex operation, high price, and unsuitability for large-scale clinical specimen detection, and achieve the effect of simple operation, high sensitivity, and strong specificity

Pending Publication Date: 2020-03-24
NANFANG HOSPITAL OF SOUTHERN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method is complicated to operate and expensive, and is not suitable for large-scale clinical specimen testing. It is currently limited to the stage of laboratory scientific research.

Method used

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  • Primers, method and kit for detecting carbapenemase blaKPC gene
  • Primers, method and kit for detecting carbapenemase blaKPC gene
  • Primers, method and kit for detecting carbapenemase blaKPC gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Primer design

[0064] Using the complete gene sequence and flanks of the GenBank gene sequence database (http: / / www.ncbi.nlm.nih.gov / nucleotide / ), compared with ClustalX, the conservative region was selected to design the gene subtype of carbapenemase blaKPC Specific primers for whole gene amplification. NCBI Primer-BLAST was used to design primers, and all primers were compared and analyzed by Oligo6, PrimerPremier5.0 and NCBI Primer-BLAST software to exclude potential non-specific amplification fragments. In the case of ensuring the amplification of the full sequence of the blaKPC family, multiple pairs of primers were designed, and after GC content analysis, 3' and 5' complementary sequence analysis, and ordinary PCR agarose experiment verification, it was finally determined that only one The primers are used to specifically amplify the complete sequence and partial flanking sequences of the carbapenemase blaKPC gene. The primers have good specificity and strong an...

Embodiment 2

[0071] Positive template preparation and optimization of PCR amplification conditions

[0072] Collect the positive strains containing the gene sequence of the carbapenemase blaKPC gene subtype, isolate and culture them in the selection medium containing 2 mg / L imipenem, take a single colony, prepare a 2 McFarland bacteria liquid, and take With 500 μl of bacterial liquid, the whole genome DNA of the strain was extracted as a template, and the specific primers obtained in Example 1 for the whole sequence were used to amplify each target gene.

[0073] The amplification system is as follows:

[0074] Premix Ex Taq 12.5μL (contains premixed Taq enzyme, dNTP, 10×PCR Buffer, MgCl 2 ), 1 μL of 10 μM specific upstream and downstream primers, 1.5 μL of DNA template (DNA content 100-300 ng), and 25 μL of nuclease-free deionized water.

[0075] Using gradient PCR amplification, the reaction program is as follows: 94°C for 30s; temperature gradient annealing; 72°C for 30s, 40 cycles. ...

Embodiment 3

[0077] Assembly of kits for detection of carbapenemase blaKPC gene subtypes

[0078] Kit composition:

[0079] QuantiTect SYBR Green PCR Kit: premixed 2×SYBR Green PCR Master Mix, including Taq enzyme, dN(U)TP, 10×PCR Buffer, SYBR Green fluorescent dye, MgCl 2 , a mixture of UNG enzymes;

[0080] Negative control substance: sterile double distilled water;

[0081] Positive control substance: a cloning plasmid carrying the full sequence of blaKPC constructed using the pET28a vector, the positive plasmid was sequenced, and the nucleic acid sequence was 100% consistent with blaKPC-4, GenBank accession numbers: MK816391.

[0082] Standard: pET28a-blaKPC positive clone plasmid 10copies / μL-10 6 copies / μL.

[0083] The upstream and downstream specific primers KPC-F / -R are as shown in Example 1;

[0084] Nuclease-free deionized water.

[0085] 2. How to use the above kit

[0086] (1) Configure the full-sequence fluorescent quantitative PCR reaction system (as shown in Table 1): ...

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Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to primers, a method and a kit for detecting carbapenemase blaKPC gene subtypes. The invention provides a group of primers capable of specifically amplifying all gene subtypes of carbapenemase blaKPC, and a corresponding detection method and a kit are designed on the basis of the primers. The kit and the method for detecting carbapenemase blaKPC gene subtypes have the characteristics of rapidness, accuracy, simplicity in operation and the like, and can detect all the gene subtypes of blaKPC gene family;the detection specificity is high, and cross reaction with other non-target genes or human genes is avoided; the sensitivity is high, and the lowest detection limit determined according to the plasmid copy number is 100 copies per reaction. The kit and the method can be used for quantitatively detecting all genotypes of carbapenemase-resistant blaKPC family in vitro, provide a theoretical basis for clinical determination of infection resistance, and provide a reference method for molecular epidemiological research on carbapenemase.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a primer, a method and a kit for detecting carbapenemase blaKPC gene. Background technique [0002] Carbapenem antibiotics (Carbapenems) are a member of the β-lactam antibiotic family. Cephalosporin enzymes are all highly stable and were once considered an important method for emergency treatment of drug-resistant diseases. However, with the widespread use of this class of antibiotics in recent years, more and more carbapenem-resistant strains have emerged clinically, including Acinetobacter baumannii, Pseudomonas aeruginosa, and bacteria reported in recent years, etc. . In the β-lactam antibiotic family, carbapenem antibiotics can inhibit or resist the hydrolysis of most bacterial β-lactamases. Carbapenemase can be divided into two types according to its amino acid sequence, among which subtype B contains a zinc finger structure, so it is also referred t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12Q1/04C12N15/11
CPCC12Q1/6851C12Q1/689C12Q2600/166C12Q2531/113C12Q2563/107C12Q2545/114
Inventor 胡秀梅王前郑磊芮勇宇颜晓慧杨彪
Owner NANFANG HOSPITAL OF SOUTHERN MEDICAL UNIV
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