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Loop-mediated isothermal amplification primer set, kit and detection method for Klebsiella pneumoniae

A Klebsiella pneumoniae, ring-mediated isothermal technology, applied in the biological field, can solve problems such as complexity, poor repeatability of results, and high cost, and achieve the effects of preventing cross-contamination, requiring no equipment, and preventing false positives

Active Publication Date: 2021-03-30
FOSHAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The microbial culture method is time-consuming and the culture conditions are complicated, and the interpretation of physiological and biochemical test results depends on the subjective judgment of the operator, resulting in poor repeatability of the results and easy misjudgment; histopathological observation relies on sectioning techniques, which is time-consuming; microscopy techniques rely on smears Technology; Molecular detection such as PCR, real-time fluorescent PCR technology requires expensive equipment and high technical requirements
Due to their respective defects, most of the conventional pathogen detection technologies are not suitable for on-site rapid detection at the grassroots level

Method used

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  • Loop-mediated isothermal amplification primer set, kit and detection method for Klebsiella pneumoniae
  • Loop-mediated isothermal amplification primer set, kit and detection method for Klebsiella pneumoniae
  • Loop-mediated isothermal amplification primer set, kit and detection method for Klebsiella pneumoniae

Examples

Experimental program
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Effect test

Embodiment 1

[0018] Example 1: LAMP nucleic acid test strip detection kit for Klebsiella pneumoniae specificity test nucleic acid test strip detection.

[0019] Bacterial strains used in the test: fish-pathogenic Klebsiella pneumoniae, coded as a1; Aeromonas victoria, coded as a2; Aeromonas sobria, coded as a3; Aeromonas hydrophila, coded as a4 ; Aeromonas caviae, coded a5; Citrobacter fischeri, coded a6; Flavobacterium columnar, coded a7; Pseudomonas putida, coded a8; Pseudomonas aeruginosa, coded a9; Pleuromonas Shigella, coded a10; Edwardsiella tarda, coded a11; Shewanella putrefaciens, coded a12; Vibrio anguillarum, coded a13; Vibrio alginolyticus, coded a14; Vibrio parahaemolyticus , numbered a15; Vibrio cholerae, numbered a16.

[0020] Culture of bacterial strains: Klebsiella pneumoniae, Aeromonas victoria, Aeromonas sobria, Aeromonas hydrophila, Aeromonas caviae, Citrobacter fischeri, Pseudomonas putida, Pseudomonas aeruginosa Monomonas, Pseudomonas shigellai, Edwardsiella tarda, ...

Embodiment 2

[0023] Example 2: LAMP nucleic acid test strip detection kit for Klebsiella pneumoniae sensitivity test nucleic acid test strip detection.

[0024] The concentration of the Klebsiella pneumoniae template was measured by a micro-nucleic acid analyzer, and then a 10-fold serial dilution was performed. After LAMP amplification of templates with different concentrations, the detection of nucleic acid test strips showed that the template concentration was 2×10 -6 At μg / μl, two clear red bands can still be observed, one in the quality control area (C line), one in the detection area (T line), the template concentration is 2×10 -7 At μg / μl, 2 red bands can also be observed, indicating that the lowest concentration of the template that can be detected by the LAMP reaction is 2×10 -7 μg / μl, where 1 is DNA Marker, and the concentrations of Klebsiella pneumoniae templates in samples b1-b10 are 20 μg / μl, 2 μg / μl, 2×10 -1 μg / μl, 2×10 -2 μg / μl, 2×10 -3 μg / μl, 2×10 -4 μg / μl, 2×10 -5 μg...

Embodiment 3

[0025] Example 3: The LAMP nucleic acid test strip detection kit for Klebsiella pneumoniae was used to detect the nucleic acid test strips of 18 strain samples.

[0026] Collection of strain samples: isolate and collect 18 samples from diseased fish, respectively c1-c18.

[0027] Using the LAMP method and nucleic acid test strip detection method described in Example 1, 18 isolated bacterial strains were detected, and the T vector clone containing the FimK gene sequence (GenBank accession number EU044788.1) was used as a positive quality control product numbered c19, sterile ddH 2 O was used as the negative quality control product numbered c20.

[0028] Containing the preparation of FimK gene sequence T vector clone: ​​take Klebsiella pneumoniae as template, utilize FimK primer pair (SEQID NO:1 and SEQ ID NO:2) to carry out PCR amplification, obtain the amplified product of 265bp, through sequencing and The sequence of the GenBank accession number EU044788.1 was consistent, a...

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Abstract

The invention discloses a loop-mediated isothermal amplification primer group of klebsiella pneumoniae, an LAMP nucleic acid strip detection kit using the primer group and a detection method of the kit. The loop-mediated isothermal amplification primer group of klebsiella pneumoniae, the LAMP nucleic acid strip detection kit using the primer group and the detection method of the kit has the advantages that nucleic acid amplification products are placed in a physically-closed environment by a nucleic acid strip for detection, through the nucleic acid detection strip, by adoption of a principle of chromatographical double-antibody sandwich method, fast detection of the nucleic acid amplification products is conducted, operation is simple (1-2 minutes), and result interpretation time is short (10-15 minutes); compared with traditional agarose gel electrophoresis, the method has the advantages that cross contamination of a laboratory and the false positive are prevented, operation time is short, toxic substances do not exist, and any instruments or equipment is needed.

Description

technical field [0001] The present invention relates to the field of biotechnology. Background technique [0002] Klebsiella pneumoniae (Klebsiella pneumoniae), belonging to the enterobacteriaceae (Enterobacteriaceaespp.) Klebsiella genus, is a class of opportunistic pathogenic bacteria with strong pathogenicity, widely present in human and animal intestinal tract, respiratory tract and water, In soil and grain, it often causes pneumonia, meningitis, suppurative inflammation, urinary system diseases, sepsis, etc. According to the data from the China Antimicrobial Resistance Surveillance Network, the clinical isolation rate of Klebsiella bacteria ranks second among Gram-negative bacteria, and it is also one of the drug-resistant bacteria regularly monitored by my country's CHINET antimicrobial resistance detection system. . In addition to causing human infection, this bacterium has been isolated from various animals such as poultry, amphibians and fish, and has been reported...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6804C12Q1/10C12N15/11C12R1/22
CPCC12Q1/6804C12Q1/689C12Q2531/119C12Q2565/625
Inventor 彭钟琴于辉杨映刘华吴勇亮苗鹏飞谢佳芳
Owner FOSHAN UNIVERSITY
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