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Kit for detecting Klebsiella pneumoniae based on LAMP (Loop-mediated isothermal amplification) method

A Klebsiella pneumoniae and kit technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/inspection, can solve the problems of being difficult to be observed, weak turbidity, etc., and achieve a detection cycle Short, highly specific effects

Inactive Publication Date: 2019-08-02
南京广方生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] For the detection of LAMP amplification products, there are mainly the following methods: 1. Turbidity method, the pyrophosphate radical produced by the amplification process combines with magnesium ions in the reaction system to form a precipitate, indicating that an amplification reaction has occurred in the system, and its disadvantages The turbidity caused by the white precipitate is usually weak and difficult to be observed; 2. Color change, such as adding hydroxynaphthol blue (HNB) to the reaction system, the pyrophosphate ion generated by the reaction competes with HNB and magnesium ions Combined, HNB changes from violet to sky blue to identify the occurrence of the reaction

Method used

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  • Kit for detecting Klebsiella pneumoniae based on LAMP (Loop-mediated isothermal amplification) method
  • Kit for detecting Klebsiella pneumoniae based on LAMP (Loop-mediated isothermal amplification) method
  • Kit for detecting Klebsiella pneumoniae based on LAMP (Loop-mediated isothermal amplification) method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Add the reaction reagents in the following table to the sterile EP tube, add 2.5 μL Klebsiella pneumoniae DNA to the positive control group, and replace it with sterile water to the negative control group.

[0030] Negative control group / μL Positive control group / μL Klebsiella pneumoniae DNA 0 2.5 10xThermobol Buffer 2.5 2.5 MgSO 4 (100mM)

[0031] Mix the above reaction tubes and place them in a 65°C water bath for 60 minutes. The results show that the negative control is violet, and the positive reaction tubes are sky blue, indicating that the primers used in the present invention can specifically bind to the Klebsiella pneumoniae target DNA above, yielding a positive amplification result.

Embodiment 2

[0033] Klebsiella pneumoniae and Staphylococcus aureus were cultured separately on sterile broth agar medium. Pick a single clone separately, extract DNA by boiling method, and add each reaction reagent according to the following table:

[0034]

[0035]

[0036] Mix the above reaction tubes and place them in a 65°C water bath for 60 minutes to incubate. As a result, the reaction tubes of the Klebsiella pneumoniae group are sky blue, and the Staphylococcus aureus group is violet, indicating that the primers used in the present invention have good specificity and sensitivity, other bacteria such as Staphylococcus aureus will not produce positive amplification results, and can be used for rapid diagnosis of Klebsiella pneumoniae.

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Abstract

The invention discloses a kit for detecting Klebsiella pneumoniae, and belongs to the technical field of molecular detection. The kit comprises outer primers F3 and B3 and inner primers FIP and BIP used for LAMP (Loop-mediated isothermal amplification), and buffer solutions of dNTP, enzymes and the like which are used for amplification reaction; the sequences of the outer primers and the inner primers are respectively shown as SEQ ID NO. 1 to SEQ ID NO. 4. The kit has the characteristics that the specificity of the primers is good; the accuracy is high; no special instrument is needed; the operation is simple and convenient; the kit can be used for fast detecting the Klebsiella pneumoniae.

Description

[0001] Technical field: [0002] The invention relates to a detection kit, in particular to a loop-mediated isothermal amplification (LAMP) technology combined with quantum dot luminescence to detect Klebsiella pneumoniae. [0003] Background technique: [0004] Klebsiella pneumoniae, a member of the Enterobacteriaceae family and the genus Klebsiella, is an important Gram-negative, zoonotic opportunistic pathogen. The bacterium mainly causes pneumonia, respiratory tract infection, enteritis, liver abscess, peritonitis, meningitis, diarrhea, and even sepsis in humans and various animals. The emergence of multidrug-resistant and highly pathogenic Klebsiella pneumoniae strains has caused huge economic losses to the aquaculture industry, and has also posed a serious threat to human food safety. Therefore, it is of great significance to establish an early and rapid detection method for the prevention and control of Klebsiella pneumoniae infection. [0005] The traditional pathogen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/10C12R1/22
CPCC12Q1/689
Inventor 陈佳鑫郑珩
Owner 南京广方生物科技有限公司