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Fluorescent RAA primer, probe and detection method for detecting activity of staphyloccocusaureus

A staphylococcus and detection method technology, applied in microorganism-based methods, biochemical equipment and methods, DNA/RNA fragments, etc., can solve problems such as complex primers, achieve strong specificity, ensure reliability, and meet rapid epidemic diagnosis. and the effect of full monitoring

Inactive Publication Date: 2018-09-14
宁波国际旅行卫生保健中心
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the established detection methods of Staphylococcus aureus at home and abroad include real-time PCR detection method, semi-nested PCR detection method, LAMP detection method, etc., but the PCR method requires the use of complex instruments and well-equipped laboratories, and the LAMP method primers It is more complex and must be designed with special software, and the amplification products obtained by the LAMP method are some fragments of different sizes, which cannot be directly cloned and sequenced, and can only be used to determine the existence of the target gene

Method used

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  • Fluorescent RAA primer, probe and detection method for detecting activity of staphyloccocusaureus
  • Fluorescent RAA primer, probe and detection method for detecting activity of staphyloccocusaureus

Examples

Experimental program
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Effect test

Embodiment 1

[0055] Embodiment 1, be used to detect the design of the fluorescent RT-RAA primer of Staphylococcus aureus activity, probe

[0056] Aiming at the specific conserved region of Staphylococcus aureus as the target region, design specific primers and fluorescent RAA probes, the sequences of which are:

[0057] Forward primer, such as the nucleotide sequence shown in SEQ ID No. 1 in the sequence listing:

[0058] 5'-CCTGCGACATTAATTAAAGCGATTGATGGTGATACG-3'

[0059] Reverse primer, such as the nucleotide sequence shown in SEQ ID No. 2 in the sequence listing:

[0060] 5'-CAAGCCTTGACGAACTAAAGCTTCGTTTAC-3'

[0061] Oligonucleotide probe, such as the nucleotide sequence shown in SEQ ID No. 3 in the sequence listing:

[0062] 5'-GTCAAACAATGACATTYAGACTATTAT(FAM-dT)G(THF)T(BHQ-dT)GATACACCTGAAACA-3';

[0063] in:

[0064] FAM-dT represents a thymidine deoxynucleotide carrying a fluorescein group FAM (6-Carboxyfluorescein);

[0065] THF represents a tetrahydrofuran (tetrahydrofuran) l...

Embodiment 2

[0068] Embodiment 2. Establishment of a fluorescent RAA method for detecting Staphylococcus aureus

[0069] (1) Fluorescence RAA reaction

[0070] 1) Extraction of DNA in the stool sample: DNA extraction in the stool sample can be carried out using a DNA extraction kit according to the instructions of the kit.

[0071] 2) Using the forward primer, reverse primer and probe designed in Example 1, and the RAA reaction kit (this example specifically uses the RAA basic kit of Jiangsu Qitian Gene Biotechnology Co., Ltd.), with this example Step 1) DNA in the extracted stool sample is used as a template to perform an amplification reaction, wherein the reaction system is as follows:

[0072] 25 μl RAA reaction buffer (provided by the RAA basic kit of Jiangsu Qitian Gene Biotechnology Co., Ltd.);

[0073] 2.1 μL each of the forward primer and reverse primer (10 μM) designed in Example 1;

[0074] 0.6 μL of the probe designed in Example 1 (10 μM);

[0075] 1 μL of the DNA template ...

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Abstract

The invention provides a fluorescent RAA primer, probe and detection method for detecting the activity of staphyloccocusaureus in a stool sample. The fluorescent RAA primer and the fluorescent probe can rapidly and qualitatively detect the activity of staphyloccocusaureus in the stool sample, detection can be conducted at the normal temperature of 39 DEG C, a diagnosis result can be obtained within 20 minutes, and the detection time is greatly shortened; the fluorescent RAA primer and the fluorescent probe are higher in specificity and sensitivity, the requirements for rapid diagnosis and whole-process monitoring of epidemics are completely met, and the time is saved for early diagnosing and treating the epidemics, reducing the mortality and controlling the epidemics.

Description

technical field [0001] The invention belongs to the field of in vitro nucleic acid detection, in particular to a fluorescent RAA primer and probe for detecting the activity of Staphylococcus aureus in feces samples and a detection method. Background technique [0002] Staphylococcus aureus (Staphyloccocus aureus) is one of the main pathogenic bacteria that cause food poisoning. It exists widely in nature, especially in major food categories such as meat and meat products, milk and dairy products, and belongs to Gram-positive bacteria. 0.8-1.5 microns in diameter, without flagella, spores, a few capsules, immobile, aerobic or facultative anaerobic bacteria. The optimum growth temperature is 37°C and the pH value is 7.4. Staphylococcus aureus has a high degree of salt tolerance and can decompose lactose, maltose, sucrose, ribose, turanose, mannose to produce acid without producing gas. It was first discovered in 1884. Rosenbach cultivated staphylococcus on solid medium , and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/14C12N15/11C12R1/445
CPCC12Q1/689C12Q1/6844C12Q2521/507C12Q2531/119C12Q2563/107
Inventor 周冬根罗洁应清界俞雪钧
Owner 宁波国际旅行卫生保健中心
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