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RPA primer, probe and detection method for detecting salmonella

A Salmonella and probe technology, applied in the directions of microorganism-based methods, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of unsatisfactory target gene specificity, no application of Salmonella detection and application, etc., to facilitate promotion The effect of using and eliminating the investment of expensive equipment

Inactive Publication Date: 2018-09-28
深圳市计量质量检测研究院(国家高新技术计量站国家数字电子产品质量监督检验中心)
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

At present, RPA technology has been applied to the rapid detection of viruses, bacteria, mycoplasma, parasites, etc. However, the primers, probes and detection methods based on the Salmonella iroB gene have not been applied to the detection of Salmonella.
[0005] At present, a variety of Salmonella genes are used as target genes in the detection of Salmonella, but the specificity of most target genes is not ideal

Method used

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  • RPA primer, probe and detection method for detecting salmonella
  • RPA primer, probe and detection method for detecting salmonella
  • RPA primer, probe and detection method for detecting salmonella

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Selection of target genes and design and screening of RPA primers and probes

[0034] Use bioinformatics knowledge and related analysis software to analyze the common specific genes of Salmonella, such as fimA, invA, iroB, etc. By comparing these genes of Salmonella with other microorganisms, iroB is used as the target gene to be selected, and Select sequence fragments with higher specificity. Design specific primers and probes according to the design requirements of RPA for primers and probes, and then compare the sequences of the primers and probes with the species with high homology of Salmonella, and select the primers and probes with high specificity . Afterwards, a combination with high specificity and high amplification efficiency is selected through comparative analysis of the primers and probes to be selected. The sequences of the primers and probes obtained by screening are shown respectively (SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3). The probe needs to be...

Embodiment 2

[0039] Establishment of RPA method for Salmonella

[0040] Inoculate Salmonella ATCC14028 into nutrient broth, place it on a shaker, and culture overnight according to the optimum growth temperature of each bacterium, draw 1mL culture solution into a 1.5mL centrifuge tube dropwise, centrifuge at 12000r / min for 2min, and discard the supernatant solution, add 500 μL sterile saline, suspend and mix well, centrifuge at 12 000 r / min for 1 min, discard the supernatant, add sterile saline repeatedly, centrifuge again, and discard the supernatant. Add 50 μL of normal saline, put in a water bath at 100°C for 10-15min, centrifuge at 12000r / min for 1min after cooling down to room temperature, and store the supernatant at -20°C for later use.

[0041]Using the DNA of Salmonella ATCC14028 as a template, set a blank control at the same time, utilize the RPA primers and probes in Example 1 to carry out RPA amplification, and use the fluorescence collection device to collect fluorescence, the...

Embodiment 3

[0045] Specific test analysis of the established RPA method

[0046] The standard strains used in this example were all purchased by Guangzhou Huankai Microbiology Co., Ltd., and the specific information is shown in Table 1.

[0047] Table 1 Strains used for RPA specificity analysis

[0048] bacteria

Numbering

Source of bacteria

salmonella

ATCC14028

American Type Culture Collection

Salmonella Paratyphi A

CMCC(B)50093

China Medical Microbiology Culture Collection Management Center

Salmonella paratyphi b

CMCC(B)50094

China Medical Microbiology Culture Collection Management Center

Salmonella paratyphi C

CICC21512

China Industrial Microbiology Culture Collection Management Center

Salmonella typhi

CMCC(B)50071

China Medical Microbiology Culture Collection Management Center

Salmonella Enteritidis

CMCC(B)50335

China Medical Microbiology Culture Collection Management Center

...

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Abstract

The invention provides a RPA primer, probe and detection method for detecting salmonella. A primer sequence of the RPA primer is as follows: for an iroB gene: an upstream primer SEQ ID No.1:5'-GGAATGTCATACTTAGCGGGTTTGACACGAGC-3', and a downstream primer SEQ ID No.2:5'-GGTGGTATTTGACGCTGGCGGTGCAAACCT-3'. According to the invention, an RPA technology is combined with fluorescence detection, the salmonella can be quickly and accurately detected. By adopting the method, the salmonella can be specifically detected from common pathogenic bacteria, thereby providing a technical support for rapid detection of the salmonella, and at the same time, the investment of expensive instruments can be eliminated, and the method is convenient for popularization and use at the grassroots level.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, and in particular relates to a primer, a probe and a detection method for detecting Salmonella. Background technique [0002] Salmonella is one of the most common food-borne pathogens. As a zoonotic pathogen, it spreads widely around the world every year, causing patients to experience symptoms such as gastroenteritis and typhoid fever, and bringing huge to the world. Economic losses. Therefore, the development of a rapid detection method for Salmonella is of great significance for ensuring social food safety and people's health. [0003] The current detection methods for Salmonella are traditional culture methods, methods based on immunological principles, and methods based on molecular biology. The traditional culture method does not have high requirements for experimental equipment, but the detection period is long, usually more than five days, and the method requires high operatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/04C12Q1/6844C12N15/11C12R1/42
CPCC12Q1/6844C12Q1/689C12Q2521/507C12Q2522/101C12Q2563/107
Inventor 陈佳平陈晶黄静敏陈血建郭正洋肖承荣蔡琳施棣欣张永鑫马芳草杨国武
Owner 深圳市计量质量检测研究院(国家高新技术计量站国家数字电子产品质量监督检验中心)
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