A detection method for food-borne pathogen Salmonella

A technology for food-borne pathogenic bacteria and salmonella, which is applied in the field of microbial screening, can solve the problems of easy misjudgment, long detection cycle, and large workload, and achieve simple detection, effective salmonellosis outbreaks, and inhibition of salmonellosis. The effect of the outbreak

Active Publication Date: 2020-11-06
宁波海洋研究院 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because there are too many serotypes of Salmonella, the national food safety standard method needs to go through five steps: pre-enrichment, selective enrichment, isolation and culture, biochemical identification, and serological typing identification. The workload is heavy, the detection cycle is long, and it is subject to detection Restricted by various factors such as the professionalism and experience of personnel, misjudgment is prone to occur

Method used

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  • A detection method for food-borne pathogen Salmonella
  • A detection method for food-borne pathogen Salmonella
  • A detection method for food-borne pathogen Salmonella

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Design and screening of primers and probes:

[0029] Primers were designed based on the Salmonella invasion protein (invasion protein A, invA) encoding gene invA gene, and the public invA gene sequence (U43272.1) was found through NCBI. According to the conservative region, after homology analysis, it was located at 101-672 RPA primer design for the target fragment.

[0030] Design 3 sets of primer pairs for optimal primer screening, the primer sets and sequences are as follows:

[0031] Group 1:

[0032] Forward primer F1-RPA: 5′-TTGTTGTCTTCTCTATTGTCACCGTGGTCC-3′ (30bp)

[0033] Reverse primer R1-RPA: 5′-ACTTCATCGCACCGTCAAAGGAACCGTAAA-3′ (30bp)

[0034] Product length: 231bp

[0035] Group 2:

[0036] Forward primer F2-RPA: 5′-TGTCTTCTCTATTGTCACCGTGGTCCAGTT-3′ (30bp)

[0037] Reverse primer R1-RPA: 5′-ACTTCATCGCACCGTCAAAGGAACCGTAAA-3′ (30bp)

[0038] Product length: 227bp

[0039] Group 3:

[0040] Forward primer F3-PRA: 5′-GGCGATAGCCTGGCGGTGGGTTTTGTTGTCTT-3′ (32bp)

[0041]...

Embodiment 2

[0063] Example 2 Detection sensitivity and specificity of primer probes

[0064] Set 5 groups of different concentrations of Salmonella DNA templates (the DNA concentration gradient is 100, 10-1, 10-2, 10-3, 10-4, 0) to perform nucleic acid amplification under the optimal conditions for RPA.

[0065] Refer to the Salmonella DNA extracted from the DNA extraction kit instructions, and the original concentration of the extracted DNA template (511.7ng / μL) was diluted 10-fold to 100ng / μL, 10ng / μL, 1ng / μL, 100pg / μL, 10pg / μL , 1pg / μL, 100fg / μL, 10fg / μL, 1fg / μL, respectively take 1μL as the reaction template. Perform nucleic acid amplification according to the aforementioned sample loading method, and detect the amplified product LFD:

[0066] The results show that the primer and probe combination designed in the present invention can ensure the sensitivity during detection, and the detection sensitivity is 5 fg / μL of final DNA concentration, which is equivalent to 5 CFU / mL. When the templ...

Embodiment 3

[0069] Example 3 Detection and application of actual samples

[0070] 1. Sample purchase:

[0071] Aquatic products come from the aquatic product circulation market, including sea melon seeds (Moerella iridescens), razor clams (Sinonovacula constricta), mussels (Mytilidae), clams (Clam), and blood cockles (Sanguinolaria) (10g net content per serving).

[0072] 2. Sample preparation

[0073] 2.1 Non-frozen samples should be stored in a refrigerator at 7℃~10℃ immediately after collection, and tested as early as possible.

[0074] 2.2 Shellfish take all contents, including shellfish meat and body fluids. For shellfish, first wash the shell in tap water and dry the surface moisture, then open the shell with aseptic operation and take the corresponding part according to the above requirements.

[0075] 2.3 Take a sample of 10g aseptically, add 90mL of Buffered Peptone Water (BPW), homogenize with a rotating blade homogenizer at 8 000r / min for 1 min, or slap with a slap homogenizer for 2 min ...

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Abstract

The invention provides a detection method of food-borne pathogenic bacteria salmonella. A detection kit comprises recombinase polymerase amplification primer pairs and probes used for detection of food-borne pathogenic bacteria salmonella, and the sequences of the primer pairs are represented by SEQ ID NO:1-3. The detection method used for rapid detection of food-borne pathogenic bacteria salmonella is invented based on molecular biology, is capable of realizing safe, specific, rapid, sensitive, simple, on-site detection of salmonella, avoiding problems of conventional detection technology, is especially suitable for on-site detection, can be used for inhibiting salmonellosis epidemic situation effectively in time, and improving food safety guarantee system.

Description

Technical field [0001] The invention belongs to the technical field of microbial screening, and specifically relates to a detection method of food-borne pathogenic bacteria Salmonella. Background technique [0002] Salmonella is classified into Bacteria, Proteobacteria, Gammaproteobacteria, Bacillales, Enterobacteriaceae, and is an important intestinal tract Nearly 1,000 species (or strains) of pathogenic bacteria have been found. Salmonella infection can cause typhoid fever, enteric fever, infectious diarrhea, sepsis and gastroenteritis in humans, and cause salmonellosis in animals. It is a common zoonotic pathogen. Eggs, poultry, meat, and aquatic products are the main vectors of salmonellosis. According to relevant data, my country’s annual food poisoning incidents caused by Salmonella account for 70% to 80% of all food poisoning incidents, ranking first in bacterial food poisoning. The World Health Organization (WHO) has listed Salmonella as having serious harm and Medium-h...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/10C12N15/11C12R1/42
CPCC12Q1/6844C12Q1/689C12Q2521/507C12Q2521/101C12Q2565/625
Inventor 朱鹏高威芳黄海龙严小军
Owner 宁波海洋研究院
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