Salmonella LAMP (loop-mediated isothermal amplification) detection method, and special primer and kit thereof

A detection kit and Salmonella detection technology, applied in the biological field, can solve the problems of Salmonella LAMP-specific primers and kits, and achieve the effect of being suitable for large-scale promotion and application, broad market prospects, and simple operation

Active Publication Date: 2012-04-25
INST OF PLA FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But so far, there are no LAMP-specific primers

Method used

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  • Salmonella LAMP (loop-mediated isothermal amplification) detection method, and special primer and kit thereof
  • Salmonella LAMP (loop-mediated isothermal amplification) detection method, and special primer and kit thereof
  • Salmonella LAMP (loop-mediated isothermal amplification) detection method, and special primer and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1. Primer Design for LAMP Detection of Salmonella

[0037] Obtain the Salmonella sequence (GenBank No.: NC_003197.1) from the U.S. Gene Database, perform homology analysis by BLAST software, and obtain the specific conserved target sequence invA gene of Salmonella (sequence 1 in the sequence table), and then based on the conserved target DNA Sequence, using the software Primer design V4 to design primers for LAMP detection of Salmonella, as a result, three sets of primer pairs were optimized, F3 and B3 were the first set, FIP and BIP were the second set, LF and LB were the third set, The specific primer sequences are listed in Table 1. In the application, the three sets of primer pairs can be mixed and used in any proportion, and a specific optimized combination is given in the reaction system of Example 2 as an example.

[0038] Table 1 Primers used for LAMP detection of Salmonella

[0039] Primer name

Embodiment 2

[0040] Embodiment 2, establishment of the LAMP detection method of Salmonella of the present invention

[0041] Carry out LAMP detection to Salmonella enteritidis (from the Infectious Disease Control Center of the Chinese People's Liberation Army Institute of Disease Control and Prevention) using the six primers obtained in Example 1 for the LAMP detection of Salmonella to obtain the best reaction system and reaction conditions. The specific method is as follows :

[0042] 1. Determination of the best response system

[0043] Under the same reaction conditions (set at 63°C for 50 minutes), different concentrations of Mg were added to the reaction system 2+ , to determine the best reaction system, including the following steps:

[0044] 1) Using the Salmonella genomic DNA (using the Wizard Genomic DNA purification Kit commercial DNA extraction kit of Promega to extract the nucleic acid in the sample to be tested) as a template, carry out LAMP amplification under the guidance ...

Embodiment 3

[0052] Embodiment 3, the specificity and sensitivity detection of the LAMP detection method of Salmonella of the present invention

[0053] One, the specific detection of the LAMP detection method of Salmonella of the present invention

[0054] Salmonella enteritidis, Salmonella paratyphi A, Vibrio parahaemolyticus, Shigella sonnei, enteroinvasive Escherichia coli, pathogenic Escherichia coli, enterotoxigenic Escherichia coli, Acinetobacter baumannii, maltophilic oligo The genomic DNA of Trotrophomonas and Shigella flexneri (all bacterial strains all come from the Infectious Disease Control Center of the Chinese People's Liberation Army Institute for Disease Control and Prevention) is a template, and double-distilled water is a negative control, and the best result obtained in Detection Example 2 is used. Specificity of the LAMP detection method for Salmonella.

[0055] Salmonella LAMP detection method specific turbidimeter detection result of the present invention is as foll...

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Abstract

The invention discloses a salmonella LAMP (loop-mediated isothermal amplification) detection method, and a special primer and kit thereof. The special primer is designed according to a salmonella specific conservative target sequence invA gene shown in sequence 1 in a sequence list, and is used for qualitatively detecting salmonella in a sample to be detected. The invention can realize the fast, convenient, efficient, specific and sensitive detection of salmonella under isothermal conditions without using complicated instruments, thereby providing a new technical platform for salmonella detection. The invention can be used for salmonella screening and detection in livestock farming and production units, grass-root medical health units and various disease prevention and control centers, has broad market prospects and great economic and social benefits, and is suitable for wide popularization and application.

Description

technical field [0001] The invention belongs to the molecular biology detection method of bacteria in the field of biotechnology, in particular to the LAMP detection method of Salmonella and its special primers and kit. Background technique [0002] Bacterial food poisoning is the most common among all kinds of livestock products, and food poisoning cases caused by Salmonella are in the forefront of food poisoning. Animal products contain a variety of rich nutrients, which are very suitable for the growth and reproduction of Salmonella. Once people ingest animal products containing a large amount of Salmonella, they will cause bacterial infections and food poisoning under the action of toxins. Salmonella not only endangers human health, but also brings huge economic losses to animal husbandry. Diseases caused by Salmonella are mainly divided into two categories: one is typhoid and paratyphoid, and the other is acute gastroenteritis. Salmonella typhimurium, Salmonella chole...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12R1/42
Inventor 刘威黄留玉袁静邹大阳尹志涛尚伟李云梅郭晶
Owner INST OF PLA FOR DISEASE CONTROL & PREVENTION
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