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Multiple PCR (Polymerase Chain Reaction) detection method for four food-borne pathogens, detection primer set and kit

A technology for detection of primers and multiplexing, applied in the field of microbial detection, can solve problems such as the difficulty of the PCR system, achieve the effects of reducing detection links, strong specificity, and improving detection efficiency

Inactive Publication Date: 2011-10-19
浙江省质量技术监督检测研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the complex factors affecting multiplex PCR, different primers, templates, primer concentrations, template concentrations, Mg 2+ Concentration, dNTP concentration and its ratio will produce complex comprehensive effects, so the more types of target microorganisms to be detected at the same time, the more difficult it is to establish a PCR system
After searching domestic and foreign literature, there are no relevant reports on the application of multiplex PCR to the rapid detection of Salmonella, Shigella, Staphylococcus aureus and Listeria monocytogenes

Method used

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  • Multiple PCR (Polymerase Chain Reaction) detection method for four food-borne pathogens, detection primer set and kit
  • Multiple PCR (Polymerase Chain Reaction) detection method for four food-borne pathogens, detection primer set and kit

Examples

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Embodiment 1

[0026]Embodiment 1: Salmonella, Shigella, Staphylococcus aureus, Listeria monocytogenes quadruple PCR reaction Mg 2+ concentration test

[0027] The concrete detection method of the present embodiment is:

[0028] 1. Extraction of sample DNA

[0029] 1.1. Inoculate the standard strains of Salmonella, Shigella and Staphylococcus aureus in the nutrient broth respectively, inoculate the standard strain of Listeria monocytogenes in the brain heart infusion broth, and incubate at 36°C±1°C for 18 hours.

[0030] 1.2. Use the QIAGEN Bacterial Genomic DNA Extraction Kit to extract the bacterial DNA from the above-mentioned culture products respectively, and set aside.

[0031] 2. Multiplex PCR reaction

[0032] 2.1. Artificially synthesize upstream and downstream primers for the detection of Salmonella, Shigella, Staphylococcus aureus and Listeria monocytogenes respectively, wherein the upstream primer invAF for the detection of Salmonella has a nucleus as shown in SEQ No.1 Nucleo...

Embodiment 2

[0050] Embodiment 2: Salmonella, Shigella, Staphylococcus aureus, Listeria monocytogenes quadruple PCR reaction annealing temperature test

[0051] The concrete detection method of this embodiment is as follows:

[0052] 1. Extraction of sample DNA

[0053] 1.1. Inoculate the standard strains of Salmonella, Shigella and Staphylococcus aureus in the nutrient broth respectively, inoculate the standard strain of Listeria monocytogenes in the brain heart infusion broth, and incubate at 36°C±1°C for 18 hours.

[0054] 1.2. Use the QIAGEN Bacterial Genomic DNA Extraction Kit to extract the bacterial DNA in the above cultured products respectively.

[0055] 2. Multiplex PCR reaction

[0056] 2.1. Artificially synthesize upstream and downstream primers for the detection of Salmonella, Shigella, Staphylococcus aureus and Listeria monocytogenes respectively, wherein the upstream primer invAF for the detection of Salmonella has a nucleus as shown in SEQ No.1 Nucleotide sequence, downs...

Embodiment 3

[0074] Embodiment 3: The detection sensitivity of fourfold PCR detection to Salmonella

[0075] The concrete detection method of the present embodiment is:

[0076] 1. Extraction of sample DNA

[0077] 1.1. Inoculate the standard strain of Salmonella in nutrient broth and incubate at 36°C±1°C for 18 hours.

[0078] 1.2. Use the QIAGEN Bacterial Genomic DNA Extraction Kit to extract the bacterial DNA in the above cultured products.

[0079] 2. Multiplex PCR reaction

[0080] 2.1. Artificially synthesize upstream and downstream primers for the detection of Salmonella, Shigella, Staphylococcus aureus and Listeria monocytogenes respectively, wherein the upstream primer invAF for the detection of Salmonella has a nucleus as shown in SEQ No.1 Nucleotide sequence, downstream primer invAR has the nucleotide sequence shown in SEQ No.2; The upstream primer ipaHF that is used for Shigella detection has the nucleotide sequence shown in SEQ No.3, and downstream primer ipaH has The nucl...

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Abstract

The invention discloses a multiple PCR (Polymerase Chain Reaction) detection method for four food-borne pathogens, a detection primer set and a kit. The multiple PCR detection method comprises the following steps of: performing multiple PCR reactions on extracted bacterial genome DNA (Deoxyribonucleic Acid) in a sample to be detected in a same reaction system by utilizing rapid detection primer sets of salmonella, shigella, staphylokinase and Listeria monocytogenes, wherein the rapid detection primer sets are obtained through analytic design, and then performing electrophoretic analysis on reaction products to determine whether the sample contains the salmonella, shigella, staphylococcus aureus or Listeria monocytogenes. Each detection primer set has strong specificity and can be used foraccurately detecting the genome DNA of the salmonella, shigella, staphylococcus aureus and Listeria monocytogenes in the same reaction system.

Description

technical field [0001] The invention belongs to the field of microbial detection, and relates to a quadruple rapid detection method for Salmonella, Shigella, Staphylococcus aureus and Listeria monocytogenes, a detection primer set and a detection kit. Background technique [0002] salmonella( Salmonella spp. ) is an important pathogen in the field of public health, belonging to Gram-negative Enterobacteriaceae. According to statistics, in the bacterial food poisoning that occurs in countries all over the world, the food poisoning caused by Salmonella often ranks first, and the bacterial food poisoning that occurs in the inland areas of our country also takes Salmonella as the primary cause. In addition to causing food poisoning, bacteria in this genus can also cause diseases such as gastroenteritis, typhoid fever, and paratyphoid fever. Shigella( Shigella spp ) is a Gram-negative Enterobacteriaceae, the most common pathogen of human bacillary dysentery. In recent years,...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12R1/42C12R1/445C12R1/01
Inventor 姜侃张东雷金燕飞陈小珍
Owner 浙江省质量技术监督检测研究院
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