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

A Salmonella detection method technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of cloning and sequencing, complex primers, etc., achieve strong specificity, high sensitivity, and strive for epidemic control the effect of time

Inactive Publication Date: 2018-10-26
宁波国际旅行卫生保健中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the Salmonella detection methods established at home and abroad include real-time PCR detection methods, semi-nested PCR detection methods, LAMP detection methods, etc., but the PCR method needs to use complex instruments and well-equipped laboratories, and the LAMP method has more complicated primers. It 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

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

Examples

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Effect test

Embodiment 1

[0056] Embodiment 1, be used to detect the design of the fluorescent RT-RAA primer of Salmonella, probe

[0057] Aiming at the specific conserved region of Salmonella as the target region, design specific primers and fluorescent RAA probes, the sequences of which are:

[0058] Forward primer, such as the nucleotide sequence shown in SEQ ID No. 1 in the sequence listing:

[0059] 5'-ATTGGCGATAGCCTGGCRGTGGGTTTTGTTGTC-3'

[0060] Reverse primer, such as the nucleotide sequence shown in SEQ ID No. 2 in the sequence listing:

[0061] 5'-TACCGGGCATACCATCCAGAGAAAAWCGDGCCGC-3'

[0062] Oligonucleotide probe, such as the nucleotide sequence shown in SEQ ID No. 3 in the sequence listing:

[0063] 5'-CTCKATTGTCACKGTGGTYCAGTTTATCG(FAM-dT)T(THF)T(BHQ-dT)ACCAAAG GTTCA-3';

[0064] in:

[0065] FAM-dT represents a thymidine deoxynucleotide carrying a fluorescein group FAM (6-Carboxyfluorescein);

[0066] THF represents a tetrahydrofuran (tetrahydrofuran) linker;

[0067] BHQ-dT repres...

Embodiment 2

[0074] Embodiment 2, the establishment of a kind of fluorescent RAA method for detecting Salmonella

[0075] (1) Fluorescence RAA reaction

[0076] 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.

[0077] 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:

[0078] 25 μl RAA reaction buffer (provided by the RAA basic kit of Jiangsu Qitian Gene Biotechnology Co., Ltd.);

[0079] 2.1 μL each of the forward primer and reverse primer (10 μM) designed in Example 1;

[0080] 0.6 μL of the probe designed in Example 1 (10 μM);

[0081] 1 μL of the DNA template...

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Abstract

The invention provides a fluorescent RAA primer and a probe as well as a detection method for detecting salmonella in an excrement sample. The fluorescent RAA primer and the fluorescent probe can rapidly and qualitatively detect the salmonella in the excrement sample, can perform the detection at the normal temperature of 39 DEG C, and can obtain a diagnosis result in 20 minutes, so that the detection time is greatly shortened; and the fluorescent RAA primer and fluorescent probe are higher in specificity and higher in sensitivity, can completely satisfy the rapid diagnosis and whole-process monitoring of epidemic situation, and can buy time for early diagnosing and early treating of the epidemic situation, reducing the death rate and controlling the epidemic situation.

Description

technical field [0001] The invention belongs to the field of in vitro nucleic acid detection, and in particular relates to a fluorescent RAA primer and probe for detecting the activity of Salmonella in feces samples and a detection method. Background technique [0002] Salmonellosis, also known as paratyphoid fever, is a general term for various animal diseases caused by Salmonella bacteria. Symptomatic lethal disease that may exacerbate morbidity and mortality, or reduce reproductive productivity of animals. Eggs, poultry and meat products are the main transmission medium of Salmonella, and the infection mainly depends on the serotype of Salmonella and the physical condition of the eater. In 1885, Salmonella and others isolated Salmonella choleraesuis during the epidemic of cholera, so it was named Salmonella. [0003] Salmonella is a Gram-negative non-bacillus that parasitizes in the intestines of humans and animals. Most of them have flagella and can move. They do not r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/10C12N15/11
CPCC12Q1/6844C12Q1/689C12Q2521/507C12Q2563/107C12Q2531/119Y02A50/30
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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