Peptide nucleic acid probe set for fluorescence in-situ hybridization detection of enterococcus faecalis and/or other enterococcocci, reagent kit, using method and applications

A fluorescence in situ hybridization, Enterococcus faecalis technology, applied in the detection field of clinically relevant microorganisms, can solve the problems of easy misjudgment, cumbersome steps, time-consuming and labor-intensive, etc., and achieves increased penetration, high sensitivity, and high sensitivity. Effect

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

AI Technical Summary

Problems solved by technology

[0007] Existing detection methods for enterococcus all need to extract the bacterial DNA, then perform PCR amplification, or amplify the bacterial liquid, a...

Method used

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  • Peptide nucleic acid probe set for fluorescence in-situ hybridization detection of enterococcus faecalis and/or other enterococcocci, reagent kit, using method and applications
  • Peptide nucleic acid probe set for fluorescence in-situ hybridization detection of enterococcus faecalis and/or other enterococcocci, reagent kit, using method and applications
  • Peptide nucleic acid probe set for fluorescence in-situ hybridization detection of enterococcus faecalis and/or other enterococcocci, reagent kit, using method and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0108] Example 1, PNA-FISH sensitivity verification

[0109] PNA probe:

[0110] Alexa488-O-TTA TCC CCC TCT GAT GGG (SEQ NO: 1)

[0111] Alexa546-O-TAC TGA TCG GCA GCT (SEQ NO: 2)

[0112] Sensitivity validation of different subspecies of Enterococcus faecalis and other enterococci.

[0113] The experimental procedure is described below.

[0114] bacterial strain

[0115] 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.

[0116] fixed

[0117] 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.

[0118] hybridize

[0119] In this step, a drop containing 10% (wt / vol) dextran sulfate, 10mM NaCl, 50% (v / v) formamide, 0.1% (wt / vol) sodium pyrophosphate, 0.2% (wt / vol) A hybridization solution of polyvinylpyrrol...

Embodiment 2

[0135] Example 2 PNA-FISH specific verification

[0136] Select Enterococcus faecalis, Enterococcus faecium and other pathogenic bacteria, including Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella pneumoniae and Acinetobacter baumannii and other common pathogenic bacteria, the test procedure And method is described with embodiment 1.

[0137] The results showed that only the positive control (DAPI) could bind to all bacteria, while the probes SEQ NO: 1 and 2 could only bind to the target strain (Table 5). Consistent with the expected results, SEQ NO: 1 and 2 have good specificity.

[0138] Table 5 PNA probe specificity verification

[0139] species

[0140] Note: ——is isolated from hospitals, food, and the environment, without the description of the strain number.

Embodiment 3

[0141] Embodiment 3 drug susceptibility test

[0142] Select the quality control strain of Enterococcus number and different drug-sensitive Enterococcus faecalis and other Enterococcus bacterial strains isolated in hospital, food, environment to carry out drug susceptibility test, test procedure and method are as described in Example 1, each test At the same time, the K-B method was used as a positive control. The results are shown in Table 6.

[0143] The results show that the present invention can better detect the drug sensitivity of the target strain.

[0144] 1 Table 6 Enterococcus drug susceptibility test

[0145]

[0146] Note: A: fluorescence ratio method; B: K-B method; S: sensitive; I: intermediate; R: resistant

[0147] 2 The present invention uses PNA probe combined with in situ fluorescence hybridization (FISH) technology, compared with traditional biochemical identification, it does not need to extract DNA from bacteria, does not need cell membrane permeabi...

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Abstract

The invention relates to a peptide nucleic acid probe for rapid fluorescence in-situ hybridization detection of enterococcus faecalis and/or other enterococcocci. The DNA sequence of the probe is shown as the SEQ NO:1 or the SEQ NO:2. The invention further discloses a reagent kit for appraising the enterococcus faecalis and/or other enterococcocci in samples and the drug susceptibility of the enterococcus faecalis and/or other enterococcocci through the probe and provides a using method of the reagent kit. The probe and the reagent kit can be used for detecting the enterococcus faecalis and/or other enterococcocci in the samples and the drug susceptibility of the enterococcus faecalis and/or other enterococcocci. The DNA extraction step is avoided, and the time for a whole appraising process is generally less than two hours; besides, the false positive rate is low, the number of steps is small, efficiency is high, sensitivity is high, and specificity is high. The enterococcus faecalis and other enterococcocci can be rapidly and accurately detected, and important significance to food safety guaranteeing, environmental sanitation guaranteeing and pathophoresis preventing is achieved.

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 Enterococcus faecalis and / or other enterococci. Background technique [0002] Enterococcus (Enterococcus) is Gram-positive, cocci arranged in pairs or short chains, oval, without spores, without capsule, and some Enterococcus have sparse flagella. High nutritional requirements, aerobic and facultative anaerobic bacteria, the optimum growth temperature is 35°C. [0003] Enterococcus faecalis (Enterococcus faecalis) and Enterococcus faecium (Enterococcus faecium) are commonly seen clinically, which are currently one of the most important pathogenic bacteria for nosocomial infection. Enterococcus durans), Enterococcus casseliflavus, etc. [0004] Enterococcus is a normal flora of the human intestinal tract and also exists in the external environment. This kind of bacteria can cause nosocomial infectio...

Claims

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Application Information

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