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Peptide nucleic acid probe set and kit used for detecting pseudomonas aeruginosa, klebsiella pneumoniae and/or baumanii

A technology of Pseudomonas aeruginosa and Acinetobacter baumannii is applied in the detection field of clinically related microorganisms, which can solve the problems of high false positives, time-consuming and labor-intensive, complicated steps, etc., and achieves reduction of false positives, ensuring environmental sanitation and sensitivity. high degree of effect

Active Publication Date: 2014-09-10
JIANGSU MICRODIAG BIOMEDICINE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The existing detection methods for Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii all need to extract bacterial DNA, and then perform PCR amplification, or amplify the bacterial liquid, and then amplify Product testing is time-consuming and labor-intensive, with cumbersome steps and high false positives, which can easily lead to misjudgments

Method used

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  • Peptide nucleic acid probe set and kit used for detecting pseudomonas aeruginosa, klebsiella pneumoniae and/or baumanii
  • Peptide nucleic acid probe set and kit used for detecting pseudomonas aeruginosa, klebsiella pneumoniae and/or baumanii
  • Peptide nucleic acid probe set and kit used for detecting pseudomonas aeruginosa, klebsiella pneumoniae and/or baumanii

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0109] Example 1, PNA-FISH sensitivity verification

[0110] PNA probe:

[0111] Alexa488-O-CTG AAT CCA GGA GCA (SEQ NO: 1)

[0112] Alexa546-O-CAC CTA CAC ACC AGC (SEQ NO: 2)

[0113] Alexa350-O-GGC CAG ATG GCT GCC (SEQ NO: 3)

[0114] Sensitivity validation was performed on different subspecies of Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii. The experimental procedure is described below.

[0115] bacterial strain

[0116] The bacterial strains were clinical isolates and reference strains obtained from the American Type Culture Collection (ATCC). One drop of each strain culture was added to a glass slide and left to dry at 55°C.

[0117] fixed

[0118] To prevent loss during 16S rRNA hybridization, samples were immersed in 4% paraformaldehyde (wt / vol) and 50% ethanol (vol / vol) solutions for 10 minutes each.

[0119] hybridize

[0120] In this step, a drop containing 10% (wt / vol) dextran sulfate, 10mM NaCl, 50% (v / v) formamide, 0.1% (wt / v...

Embodiment 2

[0137] Example 2 PNA-FISH specific verification

[0138] Select other pathogenic bacteria such as Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii, including common pathogenic bacteria such as Staphylococcus, Enterococcus, Escherichia coli, and the test steps and methods are as in Example 1 mentioned.

[0139] The results showed that the probes SEQ NO: 1-3 could not bind to non-target strains (Table 6). Consistent with the expected results, SEQ NO: 1-3 have good specificity.

[0140] Table 6 PNA probe specificity verification

[0141] species Numbering SEQ NO: 1 SEQ NO: 2 SEQ NO: 3 Pseudomonas aeruginosa ATCC27853 + - - Klebsiella pneumoniae ATCC13884 - + - Acinetobacter baumannii ATCC19606 - - + Staphylococcus epidermidis ATCC12228 - - - Staphylococcus aureus ATCC29213 - - - Staphylococcus aureus ATCC25923 - - - Enterococcus faecalis —— - - - Escherichia coli...

Embodiment 3

[0143] Embodiment 3 drug susceptibility test

[0144] Quality control strains of Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii were selected from different drug-sensitive Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii isolated from the hospital The strains were subjected to a drug susceptibility test, and the test steps and methods were as described in Example 1, and the K-B method was used as a positive control for each test. The results are shown in Tables 7, 8 and 9.

[0145] The results show that the present invention can better detect the drug sensitivity of the target strain.

[0146] Table 7 Pseudomonas aeruginosa drug susceptibility test

[0147]

[0148] Note: A: fluorescence ratio method; B: K-B method; S: sensitive; I: intermediate; R: resistant

[0149] Table 8 Klebsiella pneumoniae drug susceptibility test

[0150]

[0151] Note: A: fluorescence ratio method; B: K-B method; S: sensitive; I: intermedi...

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Abstract

The invention relates to peptide nucleic acid probes used for rapidly detecting pseudomonas aeruginosa, klebsiella pneumoniae and / or baumanii through fluorescence in situ hybridization. The DNA sequences of the peptide nucleic acid probes are shown in SEQ NO: 1 and SEQ NO: 2. The invention further discloses a kit for identifying pseudomonas aeruginosa, klebsiella pneumoniae and / or baumanii and drug susceptibility of pseudomonas aeruginosa, klebsiella pneumoniae and / or baumanii in samples through the probe set, and use steps of the kit. The DNA extraction step is omitted, and the whole identification process generally consumes at most two hours; meanwhile, the false positive is low, a few steps are needed, efficiency is high, sensitiveness is high, and specificity is high. The pseudomonas aeruginosa, klebsiella pneumoniae and baumanii can be rapidly and accurately detected, and the peptide nucleic acid probes and the kit are of great importance in ensuring food safety, ensuring environmental health and preventing spreading of diseases.

Description

technical field [0001] The invention relates to the field of detection of clinically relevant microorganisms, in particular to a peptide nucleic acid probe set for detection of Pseudomonas aeruginosa, Klebsiella pneumoniae and / or Acinetobacter baumannii. Background technique [0002] Pseudomonas aeruginosa, also known as Pseudomonas aeruginosa, is a common conditional pathogen and belongs to Gram-negative bacilli. It is widely distributed in nature and is one of the most common bacteria in soil. All kinds of water, air, normal human skin, respiratory tract and intestinal tract, etc. have their existence. It often causes postoperative wound infection, and can also cause bedsores, abscesses, and suppurative otitis media. Infection lesions caused by this bacteria can lead to hematogenous dissemination, resulting in bacteremia and sepsis. Infection with Pseudomonas aeruginosa after burns can cause bacteremia, which is often a fatal complication of burns. [0003] Many infect...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/385C12R1/22C12R1/01
CPCC12Q1/6841C12Q2543/10C12Q2525/107
Inventor 秦勇何素莉祝茂生张丽
Owner JIANGSU MICRODIAG BIOMEDICINE TECH CO LTD
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