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Primer group, detection method and kit for quickly detecting salmonella typhimurium by utilizing LAMP technology

A technology of Salmonella typhi and detection kit, which is applied in the field of microbial detection, can solve the problems of high experimental technical requirements, incompetence and high detection cost, and achieves the effects of high sensitivity, easy results and simple operation.

Active Publication Date: 2021-04-30
武汉海关技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Various PCR techniques mainly analyze pathogenic bacteria through DNA extraction, PCR amplification, electrophoresis observation, or fluorescence curves. The detection is sensitive, accurate, and rapid, but it still relies on expensive PCR instruments, which has high detection costs and high requirements for experimental techniques. Competent on-site portable rapid detection

Method used

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  • Primer group, detection method and kit for quickly detecting salmonella typhimurium by utilizing LAMP technology
  • Primer group, detection method and kit for quickly detecting salmonella typhimurium by utilizing LAMP technology
  • Primer group, detection method and kit for quickly detecting salmonella typhimurium by utilizing LAMP technology

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The establishment of the specific LAMP detection method of embodiment 1 Salmonella

[0044] 1. DNA extraction from purely cultured bacteria

[0045] Three kinds of Salmonella were inoculated in LB liquid medium overnight at 37°C under aerobic conditions. Take 1 mL of the bacterial liquid and use a commercial bacterial genome extraction kit to extract genomic DNA strictly according to the steps, and store the samples at -20 °C.

[0046] 2. Fecal mock sample preparation and DNA extraction

[0047] Mix 300 μL of freshly cultured bacteria with 2 g of sterile normal mouse feces with PBS buffer, inoculate 100 μL of the suspension in LB liquid medium at 37 °C overnight, take 1 mL of the culture solution to extract genomic DNA according to the method in 1, and store the samples in at -20°C.

[0048] 3. Comparison of the specificity of primers in the 4 groups of SirA gene

[0049] The gene sequence of the Salmonella target gene SirA (U88651.1) is shown in SEQ ID NO.1; the se...

Embodiment 2

[0073] Embodiment 2 kit sensitivity test

[0074] Using the genome of Salmonella ATCC 14028 as a template, PCR amplification was carried out with primers SirA-3F3 and SirA-3B3, and the obtained product was recovered by commercial methods. -1 ng / μL, 10 -2 ng / μL, 10 -3 ng / μL, 10 -4 ng / μL, 10 -5 ng / μL, 10 -6 ng / μL. Use this as a template to test the sensitivity of LAMP reaction with primers such as SirA-3F3, SirA-3B3, SirA-3FIP, and SirA-3BIP. See the real-time fluorescence amplification curve and color changes in Figure 5 . Its result shows that the LAMP detection sensitivity of primer of the present application is 10 -6 ng / μL, the detection time is 50min.

Embodiment 3

[0075] Embodiment 3 preferred Mg 2+ concentration

[0076] Magnesium ion is an important component that affects the activity of DNA polymerase, and its concentration must be optimized. Set seven magnesium ion concentrations from 6mM to 20mM with 2mM as a gradient, and investigate the influence of different magnesium ion concentrations on the reaction Ct value (magnesium ion concentration is 2×buffer concentration). The result is as follows Figure 6 shown. Preferably the magnesium ion concentration is 6-8 mM.

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Abstract

The invention belongs to the technical field of microbiological detection, and particularly relates to a primer group, a detection method and a kit for quickly detecting salmonella typhimurium by utilizing an LAMP (Loop-mediated Isothermal Amplification) technology. The salmonella virulence gene SirA (U88651.1) is selected as a main regulating gene for controlling the intestinal virogenic function of salmonella typhimurium and has the function of enhancing expression of hilA and prgH virulence genes in a salmonella pathogenic island, so that four specific primer groups capable of amplifying the gene at 62-68 DEG C are designed by selecting the salmonella virulence gene SirA as a specific target; the detection result is indicated through the HNB dye, violet (negative) is changed into sky blue (positive), and the method for detecting salmonella is good in specificity, easy to operate, free of expensive instruments and higher in sensitivity compared with conventional PCR.

Description

technical field [0001] The invention belongs to the technical field of microbial detection, and in particular relates to a primer set, a detection method and a kit for rapidly detecting Salmonella typhi using LAMP technology. Background technique [0002] Salmonella typhimurium (Salmonella typhimurium) is a common food-borne pathogen, including a variety of common serotypes, and is the most virulent pathogen among Salmonella. The symptoms of Salmonella typhi poisoning are mainly acute gastroenteritis. The incubation period is generally four to forty-eight hours, several hours in the short term, and two to three days in the long term. The early symptoms include nausea, headache, malaise and chills, etc. The main symptoms are: Vomiting, diarrhea, abdominal pain, yellow-green watery stool, sometimes with pus, blood and mucus, the general fever temperature is 38 degrees Celsius to 40 degrees Celsius, severe patients have symptoms of chills, convulsions, convulsions and coma. Th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/10C12N15/11C12R1/42
CPCC12Q1/689C12Q1/6844C12Q2531/119C12Q2565/125Y02A50/30
Inventor 刘洪涛叶诚曹亚楠郑军平胡海明杨化冰张志刚殷明珠
Owner 武汉海关技术中心
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