Quintuple fluorescent PCR detection kit for foodborne pathogenic bacteria

A food-borne pathogenic bacteria and detection kit technology, applied in the biological field, can solve the problems of dye redistribution, limited melting curve, poor stability, etc., and achieve the effect of sensitive automation and low detection cost

Active Publication Date: 2015-06-24
嘉兴市疾病预防控制中心
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Because the fluorescent dye SYBR Green I used in the early stage is an unsaturated dye, its stability is p

Method used

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  • Quintuple fluorescent PCR detection kit for foodborne pathogenic bacteria
  • Quintuple fluorescent PCR detection kit for foodborne pathogenic bacteria
  • Quintuple fluorescent PCR detection kit for foodborne pathogenic bacteria

Examples

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Effect test

Embodiment 1

[0037] Example 1: Establishment of real-time fluorescent PCR detection method for five kinds of food-borne pathogens

[0038]Extract Staphylococcus aureus, Listeria monocytogenes, Salmonella, Vibrio parahaemolyticus and Shigella standard strain DNA as a template, and use five pairs of specific primers to perform amplification reaction on a real-time fluorescent PCR instrument, 20 μL amplification reaction The system is: template DNA 1 μL, Bio-Rad SsoFast EvaGreen master mix 10 μL, 10 μmol / L nuc upstream and downstream primers 1.4 μL each, 10 μmol / L hlyA upstream and downstream primers 1.2 μL, 10 μmol / L invA upstream and downstream primers 1 μL each, 10 μmol / L 0.8 μL of tlh upstream and downstream primers, 0.8 μL of 10 μmol / L ipaH upstream and downstream primers, and make up to 20 μL with ultrapure water. The amplification reaction conditions are: pre-denaturation at 95°C for 2 minutes, 30 cycles at 95°C for 5s and 62.3°C for 30s, collecting fluorescence during the annealing st...

Embodiment 2

[0039] Embodiment 2: specificity experiment

[0040] Enterobacter sakazakii (ATCC 51329), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Yersinia enterocolitica (ATCC 23715), Bacillus cereus (ATCC 11778 ), Vibrio mimicus (ATCC 33653), Vibrio alginolyticus (ATCC 17749) as samples to be tested, Staphylococcus aureus (ATCC 25923), Salmonella typhi (ATCC 50097), Shigella flexneri (ATCC 12022) , Vibrio parahaemolyticus (ATCC 33847) and Listeria monocytogenes (CMCC 54006) were used as positive controls, and ultrapure water was used as negative controls. Enterobacter sakazakii (ATCC 51329), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Yersinia enterocolitica (ATCC 23715), Bacillus cereus (ATCC 11778) , Vibrio mimeticum (ATCC 33653), Vibrio alginolyticus (ATCC 17749), Staphylococcus aureus (ATCC 25923), Salmonella typhi (ATCC 50097), Shigella flexneri (ATCC 12022) and Vibrio parahaemolyticus Bacteria (ATCC 33847) was obtained from the Amer...

Embodiment 3

[0041] Embodiment 3: Sensitivity experiment

[0042] Using standard strain DNA extracts of Staphylococcus aureus, Listeria monocytogenes, Salmonella, Vibrio parahaemolyticus, and Shigella as templates, dilute with ultrapure water to form a series of dilutions, and take 1 μL template for each dilution The DNA was detected according to the method described above, and three replicate wells were made for each dilution, and the detection was carried out according to the method described in Embodiment 1. The results show that the concentration of each standard strain template DNA has a good linear relationship with the Cq value, and the higher the concentration, the lower the Cq value. For the test results, see Figure 6-10 .

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Abstract

The invention provides a quintuple fluorescent PCR detection kit for foodborne pathogenic bacteria. The quintuple fluorescent PCR detection kit for foodborne pathogenic bacteria mainly comprises five pairs of specific primers, an EvaGreen PCR premixed solution, ultrapure water and a positive control, wherein the specific primers comprise the nuc genes of staphylococcus aureus, the hlyA genes of listeria monocytogenes, the invA genes of salmonella, the tlh genes of vibrio parahaemolyticus and the ipaH genes of Shigella. The kit provided by the invention is short in detection period, low in detection cost, and reliable in detection result.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a five-fold fluorescent PCR detection kit and a detection method for food-borne pathogenic bacteria. technical background [0002] Food is food to safety first. Food safety is closely related to people's lives. Without food safety guarantee, it will not only affect human health and survival, but also seriously affect social and economic development. In 2000, the World Health Assembly passed the "Food Safety Resolution", formulated a global food safety strategy, and listed food safety as a priority area of ​​public health. However, food safety incidents occur from time to time, especially in recent years, the incidence of food poisoning incidents caused by foodborne pathogens has increased significantly in most countries around the world, and has become an important public health problem. Pathogenic bacteria such as Staphylococcus aureus, Listeria monocytogenes, Salmonella, Vibrio par...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/14C12Q1/10C12Q1/04C12R1/445C12R1/42C12R1/63C12R1/01
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143C12Q2561/101C12Q2531/113
Inventor 何培彦陈中文罗建勇王恒辉燕勇
Owner 嘉兴市疾病预防控制中心
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