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Specific probe and method thereof for rapid and sensitive quantification of staphylococcus aureus

A staphylococcus, golden yellow technology, applied in the intersection of microbiology and molecular biology, can solve the problem of the research lag of pathogenic bacteria detection method

Inactive Publication Date: 2016-04-20
JIANGSU INST OF POULTRY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Aiming at the current domestic livestock and poultry products and breeding sanitation and safety conditions are severe, and the research on pathogenic bacteria detection methods lags behind, the present invention establishes a set of methods by combining fluorescence in situ hybridization and flow cytometry (FISH-FCM). Quantitative detection method of Staphylococcus aureus

Method used

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  • Specific probe and method thereof for rapid and sensitive quantification of staphylococcus aureus
  • Specific probe and method thereof for rapid and sensitive quantification of staphylococcus aureus
  • Specific probe and method thereof for rapid and sensitive quantification of staphylococcus aureus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Specificity Verification of Nucleic Acid Probes

[0027] 1.1 Strains

[0028] Escherichia coli, Shigella flexneri, Shigella sonii, Salmonella typhimurium, Salmonella choleraesuis, Proteus vulgaris, Bacillus subtilis, Bacillus licheniformis, Staphylococcus aureus, Aeromonas hydrophila Enterococcus faecium

[0029] 1.2 Oligonucleotide probes

[0030] The test uses Staphylococcus aureus specific probe SA-1, the sequence is: 5'-GCCGGTGGAGTAACCTTTTAGGAGC-3'. Among them, the 5' end was labeled with FITC by fluorescein isothiocyanate, and the probe was synthesized and labeled by Dalian Bao Biological Company.

[0031] 1.3 FISH-FCM method to verify probe specificity

[0032] Obtain a single colony of eleven kinds of bacterial strains according to the conventional planning line culture method, cultivate them in liquid medium, and adjust the bacterial concentration to 10 6cells / ml, and the hybridization method is as follows: (1) Take 200 μl of bacterial liquid and fix it wit...

Embodiment 2

[0039] Sensitivity of FISH-FCM to detect the content of Staphylococcus aureus

[0040] 2.1 Nucleic acid probe

[0041] Ditto.

[0042] 2.2 Detection of samples by FISH-FCM method

[0043] Counted under a microscope, the original concentration of Staphylococcus aureus liquid was 5.0×10 6 pieces / ml. 10 times the ratio of diluted bacterial solution to 5.0×10 6 , 5.0×10 5 , 5.0×10 4 , 5.0×10 3 , 5.0×10 2 , 5.0×10 1 Gradient bacterial solution was tested. The detection method is as above 1.3 (1)-(4), adding 50 μl of 1000 / μl fluorescent microspheres (CountBringhtTM, Invitrogeon, USA) to the mixture, and analyzing 5000 objects for each sample by flow cytometry.

[0044] 2.3 Result Analysis

[0045] Analysis was performed with CellQuestPro software (version 5.2.1, Becton Dickinson, USA). On the dotplot graph, the X-axis represents the fluorescence intensity of the target bacteria hybridized with the fluorescent probe, and the Y-axis represents the fluorescence intensity of...

Embodiment 3

[0054] Comparison of FISH-FCM method and traditional culture method in the detection of Staphylococcus aureus in meat samples

[0055] 3.1 Nucleic acid probe

[0056] Ditto.

[0057] 3.2 Detection of samples by FISH-FCM method

[0058] The pretreatment of fresh meat samples is the same as that of SNT0738-1997 to prepare 10 -1 For meat sample liquid, take 1ml of meat sample liquid and centrifuge at 80g for 1min at low speed to remove impurities. The remaining hybridization methods are the same as above, prepare the liquid in step (5) above, add 10 μl propidium iodide (0.5 mg / ml), and incubate at 37°C in the dark for 20 minutes. Add 1000 / μl fluorescent microspheres (CountBringhtTM, Invitrogeon, USA) to 50 μl. The mixed solution was loaded on the flow cytometer for analysis. 10,000 objects were analyzed per sample.

[0059] 3.3 Samples detected by culture method

[0060] According to the national standard GB / T4789.10-2010 method.

[0061] 3.4 Result analysis

[0062] Dit...

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Abstract

The invention relates to a specific probe and a method thereof for rapid and sensitive quantification of staphylococcus aureus. On the basis of the sequence of a 16s-rRNA conservation terminal of the staphylococcus aureus, a specific complementary nucleic acid probe is designed, the probe is named as SA-1 and 5' terminal is labeled with fluorescein isothiocyanate FITC; and the sequence number of the SA-1 is shown as 5'-GCCGGTGGAGTAACCTTTTAGGAGC-3'. According to the invention, the specific complementary nucleic acid probe is designed on the basis of the sequence of the 16s-rRNA conservation terminal of the staphylococcus aureus and is named as SA-1, the 5' terminal is labeled with fluorescein isothiocyanate FITC, and the FIFC-labeled SA-1 can achieve specific combination with the processed staphylococcus aureus so as to excite fluorescent light from the staphylococcus aureus; and on the basis, propidium iodide PI is adopted for overall dyeing treatment on bacteria, sarcoid cells or intestinal contents, and fluorescence parameters are analyzed by virtue of a flow cytometry by taking a fluorescent microsphere with known concentration as internal reference, so that the purpose of quantifying bacteria is achieved.

Description

technical field [0001] The invention relates to the improvement of pathogenic bacteria detection technology using meat food and animal intestinal contents as samples, in particular to the establishment of a nucleic acid probe hybridization method for rapid quantitative detection of Staphylococcus aureus (Staphyloccocusaureus), which belongs to microbiology and molecular Interdisciplinary technical field of biology. Background technique [0002] Staphylococcus aureus (Staphylococcus aureus), a Gram-positive bacterium, is one of the important pathogens causing bacterial food poisoning. The U.S. Centers for Disease Control reports that Staphylococcus aureus is the second-leading infection after E. coli. There are many such poisoning incidents in our country every year. According to reports from research institutes and CDCs in Beijing, Wenzhou, Changsha, Nantong, Changchun and other provinces and cities, golden yellow grapes have been detected in local pigs, chickens, cattle, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/14C12N15/11C12R1/445
CPCC12Q1/689C12Q1/6841C12Q2563/107C12Q2565/626
Inventor 谢鹏卜柱唐梦君周生张安戴鑫
Owner JIANGSU INST OF POULTRY SCI
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