Detection method of food-borne pathogenic bacteria salmonella

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

Active Publication Date: 2017-06-20
宁波海洋研究院 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented process allows quicker identification of harmful microorganisms from samples like milk or other products that may be contaminated with these organism(s). It uses advanced techniques such as genetic analysis to identify them quickly without causing any health hazards during testing. Overall, it offers an effective way to prevent illness caused by certain types of germs while ensuring their presence remains undetected.

Problems solved by technology

This patented technical problem addressed in this patents relates to finding an effective way to quickly identify or diagnose harmful organisms like Salmonae that may be present within our environment due to their ability to spread easily from person to individual over time without causing any health concerns.

Method used

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  • Detection method of food-borne pathogenic bacteria salmonella
  • Detection method of food-borne pathogenic bacteria salmonella
  • Detection method of food-borne pathogenic bacteria salmonella

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1: Primer and probe design and screening:

[0031] Primers were designed according to the invA gene encoding the Salmonella invasion protein (invasion protein A, invA), and the published invA gene sequence was found through NCBI

[0032] (U43272.1), according to the conserved region, after homology analysis, RPA primers were designed with the gene located at 101-672 as the target fragment.

[0033] Three sets of primer pairs were designed for optimal primer screening. The primer sets and sequences are as follows:

[0034] Group 1:

[0035] Forward primer F1-RPA: 5′-

[0036] TTGTTGTCTTTCTCTATTGTCACCGTGGTCC-3'(30bp)

[0037] Reverse primer R1-RPA: 5′-

[0038] ACTTCATCGCACCGTCAAAGGAACCGTAAA-3′(30bp)

[0039] Product length: 231bp

[0040] Group 2:

[0041] Forward primer F2-RPA: 5′-

[0042] TGTCTTTCTCTATTGTCACCGTGGTCCAGTT-3′(30bp)

[0043] Reverse primer R1-RPA: 5′-

[0044] ACTTCATCGCACCGTCAAAGGAACCGTAAA-3′(30bp)

[0045] Product length: 227bp

...

Embodiment 2

[0076] Detection sensitivity and specificity of embodiment 2 primer probe

[0077] Set 5 groups of Salmonella DNA templates with different concentrations (DNA concentration gradient is 100, 10-1, 10-2, 10-3, 10-4, 0), and carry out nucleic acid amplification under the optimal conditions of RPA.

[0078] The Salmonella DNA extracted according to the instructions of the DNA extraction kit, the original concentration of the extracted DNA template (511.7ng / μL) was diluted into 10-fold gradients to 100ng / μL, 10ng / μL, 1ng / μL, 100pg / μL, 10pg / μL , 1pg / μL, 100fg / μL, 10fg / μL, 1fg / μL, take 1μL respectively as the reaction template. Carry out nucleic acid amplification according to the aforementioned sample loading method, and detect the LFD of the amplified product:

[0079] The results show that the combination of primers and probes designed in the present invention can ensure the sensitivity of detection, and the detection sensitivity is 5 fg / μL of DNA final concentration, which is eq...

Embodiment 3

[0082] Embodiment 3 to the detection application of actual sample

[0083] 1. Sample purchase:

[0084] Aquatic products come from the aquatic product distribution market, including sea melon seeds (Moerella iridescens), razor clams (Sinonovacula constricta), mussels (Mytilidae), clams (Clam), blood cockles (Sanguinolaria) (10g net content / serving).

[0085] 2. Sample Preparation

[0086] 2.1 Non-frozen samples should be stored in a refrigerator at 7°C to 10°C immediately after collection, and tested as early as possible.

[0087] 2.2 Shellfish take all the contents, including shellfish meat and body fluids. For shellfish, 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.

[0088] 2.3 Take 10 g of the sample by aseptic operation, add 90 mL of Buffered Peptone Water medium (BPW), homogenize with a rotary blade homogenizer at 8 000 r / min for 1 min, or beat ...

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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

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Claims

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Application Information

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Owner 宁波海洋研究院
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