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LAMP primer group for salmonella typhimurium, kit and use method of kit

A technology for Salmonella and typhimurium, applied in the field of Salmonella typhimurium LAMP primer set and detection kit, can solve the problems of high requirements for equipment and instruments, rapid detection and grass-roots popularization, cumbersome operation, low accuracy rate, etc., to avoid the risk of aerosol pollution , Improve the amplification efficiency, and the effect of simple result judgment

Inactive Publication Date: 2017-01-04
中华人民共和国广州机场出入境检验检疫局 +2
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the detection of Salmonella is mainly based on traditional methods such as bacterial isolation, serotype experiment and biochemical identification, which have defects such as cumbersome operation, low sensitivity and low accuracy, and cannot meet the needs of timely and effective quality monitoring and disease control.
With the development of molecular biology technology, new diagnostic techniques such as ELISA, immunofluorescence, PCR and other methods to detect biological macromolecules such as pathogenic nucleic acid and protein are widely used in the detection of Salmonella, but the specificity is poor and the false positive is high. The high requirements for equipment and instruments cannot meet the needs of on-site rapid detection and grassroots popularization

Method used

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  • LAMP primer group for salmonella typhimurium, kit and use method of kit
  • LAMP primer group for salmonella typhimurium, kit and use method of kit

Examples

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Embodiment 1

[0040] Example 1: Design and synthesis of a LAMP primer for Salmonella typhimurium and establishment of a detection kit

[0041] (1) Design and synthesis of LAMP primers

[0042] According to literature reports, using the invA gene of Salmonella typhimurium as the target gene, six LAMP primers including loop primers were designed using the primer design software PrimerExplorer 4.0. The primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., and the sequences of the six primers are as follows:

[0043] SEQ NO.1: SainvA-F3: GGTCGTTTCTACATTGACAGAA

[0044] SEQ NO.2:SainvA-B3:CGACACGTTCTGAACCTT

[0045] SEQ NO.3: SainvA-FIP: CGGCATCGGCTTCAATCAAGGGTACTGTTAATTACCACGCT

[0046] SEQ NO.4: SainvA-BIP:

[0047] GTGAAATTATCGCCACGTTCGGGGTGACAATAGAGAAGACAACA

[0048] SEQ NO.5: SainvA-LF: TGGTACTGATCGATAATGCCAG

[0049] SEQ NO.6: SainvA-LB: TATTGGCGATAGCCTGGC

[0050] (2) In addition to the above-mentioned LAMP primer set, the Salmonella typhimurium LAMP detection kit...

Embodiment 2

[0056] Embodiment 2: LAMP sensitivity experiment

[0057] The standard strain of Salmonella typhimurium was inoculated in nutrient broth, and cultured at 36°C±1°C for 18 hours. Bacterial DNA in cultured products was extracted using Magen Bacterial Gene DNA Extraction Kit. The DNA extracted from the standard strain of Salmonella typhimurium was serially diluted 10 times, and the DNA and the negative control (sterilized ultrapure water) as a template to establish a detection method according to the reaction system and conditions of Example 1 to determine the sensitivity of the detection method of the kit.

[0058] see figure 1 , the results showed that: after the Salmonella typhimurium genomic DNA was serially diluted 10 times, the established Salmonella typhimurium LAMP detection kit could detect 1pg / μl of Salmonella typhimurium DNA in the sample.

Embodiment 3

[0059] Embodiment 3: LAMP specific experiment

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Abstract

The invention discloses a LAMP primer group for salmonella typhimurium, a kit and a use method of the kit. The LAMP primer group comprises a pair of outer primers, a pair of inner primers and a pair of loop primers. According to the LAMP primer group, loop-mediated isothermal amplification primers are designed according to the sequence of a gene conserved region of salmonella typhimurium invasion protein A (invA), a specific primer group is designed by virtue of PrimerExplorer V4 online, a specific region of a target gene is amplified by virtue of an LAMP technique, and the rapid detection of salmonella typhimurium is realized in a molecular level, and an effectively and rapid nucleic acid screening detection method is provided for the detection of salmonella typhimurium.

Description

[0001] Technical field: [0002] The invention belongs to the field of molecular biology detection methods for food safety, in particular to a Salmonella typhimurium LAMP primer set and a detection kit. [0003] Background technique: [0004] Salmonella spp. is a large group of Gram-negative bacilli that parasitize the intestinal tract of humans and animals with similar biochemical reactions and antigenic structures. It is the most common zoonotic pathogenic bacteria in the Enterobacteriaceae family. There are currently more than 2,000 known species of Salmonella, and almost all of them can cause serious food poisoning incidents. The World Health Organization (WTO) lists Salmonella as food-borne pathogens with serious and moderate hazards. Food transmission is the main way for humans to be infected with Salmonella. In my country, 70%-80% of bacterial food poisoning is caused by Salmonella, and about 90% of the food that causes Salmonella poisoning is meat, eggs, milk and other ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12R1/42
CPCC12Q1/689C12Q1/6844C12Q2531/119C12Q2563/107C12Q2547/107
Inventor 柏建山戴金邓艳何淑华谢会陈洵石磊
Owner 中华人民共和国广州机场出入境检验检疫局
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