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Kit for detecting klebsiella pneumoniae

A Klebsiella and kit technology, applied in the field of PCR detection of strains, can solve the problems of low detection rate, low accuracy, poor repeatability, etc., and achieve accurate identification, increase the positive rate, and shorten the detection cycle. Effect

Active Publication Date: 2015-09-30
BIOSINO BIO TECH & SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional Klebsiella pneumoniae detection method has the disadvantages of long time consumption, low detection rate, low accuracy and poor repeatability.

Method used

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  • Kit for detecting klebsiella pneumoniae
  • Kit for detecting klebsiella pneumoniae
  • Kit for detecting klebsiella pneumoniae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1 Fluorescence quantitative PCR detects the establishment of the method for Klebsiella pneumoniae

[0041] 1. Primer and probe design

[0042] For the conserved region of the phoE gene of Klebsiella pneumoniae, this embodiment designs 3 pairs of primers and the probes used in conjunction with it. The 3 pairs of primers are all located on the phoE gene, and their nucleotide sequences are as follows:

[0043] (1)

[0044] Upstream primer: 5'-GATGGGCTTTGTGGCTTCAA-3'

[0045] Downstream primer: 5'-CCAGCTTGTTCGCGTTCTTAT-3'

[0046] Probe: 5'-AGCGACGCAGGCAGCGGAA-3'

[0047] (2)

[0048] Upstream primer: 5'-CGATATGTTCCCGGAATTCG-3'

[0049] Downstream primer: 5'-GCTGGCGCGCTTGGT-3'

[0050] Probe: 5'-CGGCGACTCATCCGCTCAGACC-3'

[0051] (3)

[0052] Upstream primer: 5'-GGCCGTTGGGAATCTGAATT-3'

[0053] Downstream primer: 5'-CGAACGCCAGACGAGTCTTT-3'

[0054] Probe: 5'-AACCGAGAGCGACTCCAGCCAGC-3'

[0055] The 5' end of the Taqman probe nucleotide sequence is marked ...

Embodiment 2

[0069] Embodiment 2 Fluorescence quantitative PCR detects the specificity verification of Klebsiella pneumoniae method

[0070] Klebsiella pneumoniae, Enterococcus faecium, Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Acinetobacter baumannii, Enterobacter cloacae, Staphylococcus hemolyticus in LB medium for 37 days Cultivate overnight at ℃, and extract genomic DNA from the bacterial solution. The extracted genomic DNA of the above-mentioned bacteria was used as a template, and sterilized deionized water was used as a negative control, and the Taqman probe method fluorescent quantitative PCR reaction was carried out. The result is as figure 2 , Klebsiella pneumoniae template amplification curve is S line, Ct value is 16, Enterococcus faecium, Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Acinetobacter baumannii, Enterobacter cloacae , Staphylococcus hemolyticus and the negative contr...

Embodiment 3

[0071] Example 3 Sensitivity verification of the method for detecting Klebsiella pneumoniae by fluorescent quantitative PCR

[0072] Klebsiella pneumoniae was cultured overnight at 37°C in LB medium, counted colonies, and then the concentration was 1 × 10 6 CFU / ml bacterial solution was diluted 10 times to 1×10 2 CFU / ml. The genomic DNA of each concentration of bacterial liquid was extracted as a template, and sterilized deionized water was used as a negative control, and the Taqman probe method was used for fluorescent quantitative PCR reaction. The result is as image 3 display, 1 represents 1×10 6 Template for CFU / ml bacterial solution, 2 represents 1×10 5 Template for CFU / ml bacterial solution, 3 represents 1×10 4 Template for CFU / ml bacterial solution, 4 represents 1×10 3 Template for CFU / ml bacterial solution, 5 represents 1×10 2 CFU / ml bacterial liquid template, in a 20μl reaction system under optimal reaction conditions, the minimum detection limit is 1×10 2 Th...

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Abstract

The invention discloses a kit for detecting klebsiella pneumoniae, and belongs to the technical field of PCR detection. The kit comprises specific primers and a probe for detecting klebsiella pneumoniae; the nucleotide sequences of the specific primers and the probe are as shown in SEQ ID NO.1-3; a detected target gene is a sequence of a pho gene, and has a nucleotide sequence as shown in SEQ ID No.4. The kit has the advantages of being accurate in detection, high in sensitivity, strong in specificity, and simple and quick to operate, has a favorable sample detection capacity, can replace a traditional bacterial isolated cultivation and diagnosis method, and the novel kit for quick detection is provided for klebsiella pneumoniae.

Description

technical field [0001] The invention relates to the technical field of PCR detection of bacterial species, in particular to fluorescent quantitative PCR primer pairs and probes for detecting Klebsiella pneumoniae, and the present invention also relates to using the primer pair and probes to detect Klebsiella pneumoniae. Bacteria detection kits. Background technique [0002] Klebsiella pneumoniae is a Gram-negative bacterium that is rod-shaped and encapsulated by a large, viscous polysaccharide capsule. Klebsiella pneumoniae is an important pathogen of respiratory tract infection, often causing severe pneumonia, and can also cause serious diseases such as urinary tract infection, biliary tract infection, sepsis and suppurative meningitis. Infection mostly occurs in debilitated patients who are hospitalized. Pathogens are often inhaled from the upper respiratory tract, or invade the human body through contaminated artificial respirators, nebulizers, or various catheters. Kl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/686C12Q2545/101C12Q2563/107
Inventor 杨君易翔黄茜何犇及思莹
Owner BIOSINO BIO TECH & SCI
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