Method for detecting staphylococcus aureus by synthesizing DNA (deoxyribonucleic acid) enzyme through magnetic separation RCA (rolling circle amplification)

A staphylococcus, DNA enzyme technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of low sensitivity, high detection limit, complicated operation, etc., to improve sensitivity, accuracy and specificity Effect

Inactive Publication Date: 2017-10-10
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In recent years, many studies have applied the principle of DNA sandwich hybridization combined with nanomaterials to detect pathogenic bacteria, but there are still some problems such as high detection limit, low sensitivity and complicated operation.

Method used

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  • Method for detecting staphylococcus aureus by synthesizing DNA (deoxyribonucleic acid) enzyme through magnetic separation RCA (rolling circle amplification)
  • Method for detecting staphylococcus aureus by synthesizing DNA (deoxyribonucleic acid) enzyme through magnetic separation RCA (rolling circle amplification)
  • Method for detecting staphylococcus aureus by synthesizing DNA (deoxyribonucleic acid) enzyme through magnetic separation RCA (rolling circle amplification)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] (1) Preparation of magnetic nanoparticles functionalized with capture probes

[0055] Add 500 μL of aminated magnetic nanoparticles to 150 μ glutaraldehyde and 1350 μ L PBS solution, mix well, react in the dark for 2 hours, wash with PBS buffer for 3 times, then add 500 μL of a certain concentration of avidin, shake and react in the dark for 6 hours, PBS buffer Wash 3 times to obtain avidin-modified magnetic nanoparticles. Then 240 μL of synthesized avidin-modified magnetic nanoparticles and 10 μL of capture probe were mixed, incubated at room temperature for 30 min, and washed with TE buffer three times to obtain capture probe functionalized magnetic nanoparticles.

[0056] (2) Sandwich structure generated by 16S rDNA sequence and probe

[0057] Take 30 μL of capture probe-functionalized magnetic nanoparticles, add the test solution containing 16S rDNA sequence (that is, target DNA), incubate at room temperature for 30 min, wash with TE buffer for 3 times, then add 3 ...

Embodiment 2

[0067] Embodiment 2: Specific analysis of target DNA (tDNA)

[0068] The specificity is established based on the principle of sandwich hybridization, that is, the specific complementary pairing between the probe and the target sequence.

[0069] Under the condition that the concentration of magnetic nanoparticles is 1.0 mg / mL, the concentration of capture probe is 100 nmol / L, the amplification time is 50 min, and the connection time is 50 min, equal volume and concentration of tDNA (sequence shown in SEQ ID NO:1 ) and base-mismatched DNA sequences (mDNA1, mDNA2, mDNA3, and mDNA4, compared with tDNA, one-base mismatch DNA, two-base mismatch DNA, three-base mismatch DNA, random sequence DNA ; The sequences are shown in SEQ ID NO:6~SEQ ID NO:9, respectively,) as the detection object for evaluating the specificity of this experiment.

[0070] Such as Figure 4 As shown, different relative luminescence values ​​were obtained by detecting different DNA sequences, from which it can...

Embodiment 3

[0071] Example 3: Specific analysis of Staphylococcus aureus

[0072] Based on the principle of specific complementary pairing of nucleic acid probes with 16S rDNA of Staphylococcus aureus.

[0073] Using Staphylococcus aureus, Salmonella, Escherichia coli, Bacillus cereus and Shigella dysenteriae as samples to be tested, the method in Example 1 was used to investigate and compare the detection effects, and TE buffer was used as a blank control.

[0074] Depend on Figure 5 It can be seen that the relative luminescence value of Staphylococcus aureus is much higher than that of Salmonella, Escherichia coli, Bacillus cereus and Shigella dysenteriae, indicating that the method in Example 1 has good specificity for Staphylococcus aureus.

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Abstract

The invention discloses a method for detecting staphylococcus aureus by synthesizing DNA (deoxyribonucleic acid) enzyme through magnetic separation RCA (rolling circle amplification), and belongs to the technical field of food safety. 16S rDNA of the staphylococcus aureus serves as a specific detection target DNA fragment, magnetic nanoparticles modified with a specific probe are used for combining and separating the 16S rDNA of the staphylococcus aureus, the 16S rDNA of the staphylococcus aureus is rapidly separated from complicated components by the aid of the magnetic separation function of the magnetic nanoparticles and the complementary specificity pairing function of the probe and the 16S rDNA of the staphylococcus aureus, and an H2O2-luminol reaction system is effectively catalyzed by the DNA enzyme synthesized by rolling circle amplification technology to realize dual amplification and detection of signals. According to the method, detection accuracy, specificity and sensitivity are improved, and the detection limit of the staphylococcus aureus reaches 4.2*10<1>CFU / mL.

Description

technical field [0001] The invention relates to a method for detecting Staphylococcus aureus by magnetically separating RCA synthetic DNase, which belongs to the technical field of food safety. Background technique [0002] Staphylococcus aureus (Staphylococcus aureus) is a typical spherical Gram-positive bacteria that exists in large quantities in nature. Staphylococcus aureus does not have strict requirements on the growth environment. Most foods such as meat, fish, and dairy products can provide good growth conditions for it. At a suitable temperature, it can produce high-temperature-resistant enterotoxins and cause food poisoning . A large number of food poisoning incidents caused by Staphylococcus aureus occur in my country every year, and the main symptoms are vomiting and diarrhea. Therefore, it is very important to make rapid and accurate detection of S. aureus. [0003] The traditional method of detecting Staphylococcus aureus is to isolate, cultivate and biochem...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/06
CPCC12Q1/6851C12Q2531/125C12Q2521/345C12Q2563/143C12Q2563/155
Inventor 沈晓芳李双双庞月红
Owner JIANGNAN UNIV
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