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Kit for detecting klebsiella pneumonia by using nucleic acid constant-temperature amplification technology and amplification method thereof

A Klebsiella pneumoniae, constant temperature amplification technology, applied in the field of pathogen inspection, can solve the problems of protracted course of disease, failure of clinical antibacterial drug treatment, etc., and achieve the effect of improving cost performance, avoiding repeated culture, and accurate detection

Inactive Publication Date: 2013-01-30
ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The rate of nosocomial infection caused by Klebsiella pneumoniae has recently increased year by year, and the continuous increase of multi-drug resistant strains often leads to the failure of clinical antimicrobial treatment and the protracted course of the disease

Method used

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  • Kit for detecting klebsiella pneumonia by using nucleic acid constant-temperature amplification technology and amplification method thereof
  • Kit for detecting klebsiella pneumonia by using nucleic acid constant-temperature amplification technology and amplification method thereof
  • Kit for detecting klebsiella pneumonia by using nucleic acid constant-temperature amplification technology and amplification method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Samples: 1 bacterial culture of Klebsiella pneumoniae standard strain, 2 bacterial cultures of Klebsiella pneumoniae wild strain.

[0033] 1. Sample DNA extraction

[0034] Take 1ml of the sample culture, centrifuge to obtain the precipitated substance, resuspend in 1ml of buffer, add 1ml of lysate, boil in water for 10 minutes, centrifuge at 10,000 rpm at room temperature for 5 minutes, take the supernatant and store it at -20°C.

[0035] 3. The composition ratio of each component in the reaction system in the isothermal amplification system is as follows

[0036]

[0037] The composition of the Bst enzyme buffer is 20mM Tris-HCl, 10mM (NH 4 ) 2 SO 4 , 10mM KCl, 2mM MgSO4, mass percentage 0.1% Triton X-100, 10mM dNTP, and the pH is 8.8.

[0038] The isothermal amplification program was 61°C for 60 minutes.

[0039] A total of 4 tubes were tested for isothermal amplification, and the DNA samples were added as follows:

[0040] Sample 1 is the DNA sample of Kleb...

Embodiment 2

[0046] Sample: A sample to be tested.

[0047] 1. Sample DNA extraction

[0048] A sample is suspected to contain Klebsiella pneumoniae, take 1ml of the sample culture, centrifuge to obtain the precipitated substance, resuspend in 1ml of buffer, add 1ml of lysate, boil water bath for 10 minutes, room temperature 10000 rpm, centrifuge for 5 minutes, Take the supernatant and store it at -20°C.

[0049] 3. The composition ratio of each component in the reaction system in the isothermal amplification system is as follows

[0050]

[0051]

[0052] The composition of the Bst enzyme buffer is 20mM Tris-HCl, 10mM (NH 4 ) 2 SO 4 , 10mM KCl, 2mM MgSO4, mass percentage 0.1% Triton X-100, 10mM dNTP, and the pH is 8.8.

[0053] The isothermal amplification program was 63°C for 60 minutes.

[0054] A total of 3 tubes were tested for isothermal amplification, and the DNA samples were added as follows:

[0055] Sample 1 is a negative control;

[0056] Sample 2 is a positive contr...

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Abstract

The invention discloses a kit for detecting klebsiella pneumonia by using a nucleic acid constant-temperature amplification technology and an amplification method thereof. Sequences of six primer pairs, namely, 5'-TACGCCCCGGTCTGAC, 5'-GCGCTTTCACCCCCAAC, 5'-GTCCCTTTTGCCCGAGGTCGACGGCACGGCCATT, 5'-CGACGGCACGGCCATTTGTCCCTTTTGCCCGAGG, 5'-TGGCAACGACTGGTCTCGCC and 5'-TCGCGTAGGGGGAGGCTGTT, are designed. Aiming at the klebsiella pneumonia, the invention overcomes the defect in the prior art and provides a quick and low-cost detection method based on the nucleic acid constant-temperature amplification technology.

Description

technical field [0001] The invention relates to pathogen detection technology, in particular to the technology of using nucleic acid constant temperature amplification reaction to detect Klebsiella pneumoniae. Background technique [0002] Klebsiella pneumoniae is a Gram-negative bacillus, and the exudate in the lesion is thick and heavy, causing the leaf gap to drop. Bacteria have capsules, and when they grow and multiply in the alveoli, they cause tissue necrosis, liquefaction, and the formation of single or multiple abscesses. When the lesion involves the pleura and pericardium, it can cause exudative or purulent effusion. The fibrous tissue in the lesion proliferates actively and is easy to organize; fibrinous pleural effusions may have early adhesions. In nosocomial sepsis, Klebsiella pneumoniae is an important pathogen with a high mortality rate. [0003] Klebsiella pneumoniae (KNP) is one of the important pathogenic bacteria in clinical isolation and nosocomial inf...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/22
Inventor 张霞郑文杰刘伟张宏伟曲鹏吴冬雪赵良娟刘培高旗利
Owner ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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