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Composition and method for detecting drug resistance of staphylococcus aureus

A technology of staphylococcus and composition, applied in the direction of biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., to achieve high sensitivity, time-saving and labor-saving results, and simple operation

Active Publication Date: 2013-12-04
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] At present, there are few reports at home and abroad that can quickly, simply, specifically and sensitively detect the antibiotic resistance of Staphylococcus aureus

Method used

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  • Composition and method for detecting drug resistance of staphylococcus aureus
  • Composition and method for detecting drug resistance of staphylococcus aureus
  • Composition and method for detecting drug resistance of staphylococcus aureus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] This embodiment selects Staphylococcus aureus 22011, 22015, Staphylococcus epidermidis 22036, Staphylococcus saparophytics 22007, Salmonella typhi 10593, Listeria monocytogenes 22203, Genomic DNA amplification of Escherichia coli O157 (E.coli) 10102, Vibrio parahaemolyticus (Vibrio parahaemolyticus) 12310, Shigella (Shigella) 11307, Yersinia enterocolitica (Yersinia enterocolitica) 10908 common food pathogens , to verify the specificity of nuc primers. The bacterial strains used in this example are from the Food Safety Microorganism Culture Preservation Management Center of the Chinese Academy of Inspection and Quarantine. The primer sequences used in this example to detect the specific gene nuc of Staphylococcus aureus are:

[0063] nuc-F: GGTCCT A CT TTT TCC CTA CTA GTT G (SEQ ID No. 1)

[0064] nuc-R: AAG ATC TTC AGA ACC ACT TCT ATT A (SEQ ID No. 2)

[0065] Primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0066] The main detection instrum...

Embodiment 2

[0077] This example obtains the sequences of Staphylococcus aureus gene nuc, methicillin resistance gene mecA, aminoglycoside resistance gene aacA-aphD, erythromycin resistance genes ermA and ermC, tetracycline resistance genes tetK and tetM through GenBank , designed primer sequences for each gene, and established a multiplex PCR detection method for Staphylococcus aureus specific genes and drug resistance genes.

[0078] The primer sequences used to detect the specificity and drug resistance genes of Staphylococcus aureus used in this embodiment are:

[0079] nuc-F: GGT CCT ACT TTT TCC CTA CTA GTT G (SEQ ID No. 1)

[0080] nuc-R: AAG ATC TTC AGA ACC ACT TCT ATT A (SEQ ID No. 2)

[0081] aacA-aphD-F: TAT TGC ATG AGC AAT AAG GGC (SEQ ID No. 3)

[0082] aacA-aphD-R: CGG ACA CTA TCA TAA CCA CTA (SEQ ID No. 4)

[0083] ermA-F: ACG CGG TAA ACC GCT CTG A (SEQ ID No. 5)

[0084] ermA-R: TCC GCA AAT CCC ATC TCA AC (SEQ ID No. 6)

[0085]ermC-F: GGT CGT CTA TTC CTG CAT GT (SEQ ID...

Embodiment 3

[0110] Embodiment 3: Utilize DHPLC to detect multiplex PCR amplification products

[0111] For DHPLC detection, a denaturing high-performance liquid chromatograph (DHPLC-4500) from Transgenomic Company was used, the chromatographic column was DNA-99-3510, and the detector was an ultraviolet detector.

[0112] Put the multiple PCR products of Example 2 into the DHPLC sampling chamber, and the detection conditions are: set the double-stranded DNA multi-fragment analysis mode, the detection range is 100-700bp, the detection start time is 0.5min, the detection end time is 15min, and the flow Phase: Liquid A (0.1mol / L TEAA, i.e. triethylamine acetate is 60%, liquid B (0.0mol / L TEAA containing 25% acetonitrile) is 40%, the column temperature is 60°C, the loading The volume is 5 μL, and the flow rate is 0.9ml / min.

[0113] Such as image 3 As shown, 7 pairs of primers showed positive absorption peaks at specific positions, while the blank control only showed elution peaks.

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Abstract

The invention relates to a composition and method for detecting drug resistance of staphylococcus aureus, in particular to a method for using DHPLC for carrying out detection on a multiplex PCR amplification product after multiplex PCR amplification. The determination of the drug resistance of the staphylococcus aureus can be easy, convenient, rapid, and high in sensitivity and specificity through the composition and method for detecting the drug resistance of the staphylococcus aureus.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a composition for detecting drug resistance of Staphylococcus aureus, a detection method using the composition and an application of the composition in actual sample detection. Background technique [0002] Staphylococcus (Staphylococcus) includes at least 20 species, among which Staphylococcus aureus (Staphylococcus aureus) is an important pathogenic bacteria of humans, causing many serious infections. Typical Staphylococcus aureus is spherical, with a diameter of about 0.8 μm, arranged in clusters of grapes under the microscope. Staphylococcus aureus has no spores, flagella, most of them have no capsule, and Gram stain is positive. [0003] Staphylococcus aureus has low nutritional requirements and grows well on ordinary medium, aerobic or facultative anaerobic, with an optimum growth temperature of 37°C and an optimum growth pH of 7.4. The colonies on the plate are thick, shiny, r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/14C12N15/11
Inventor 王娉陈颖胡玥田雪杨海荣赵勇胜赵贵明刘洋
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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