High-throughput quantitative detection kit for food-borne pathogenic bacteria

A technology for food-borne pathogens and detection kits, applied in the determination/inspection of microorganisms, methods based on microorganisms, resistance to vector-borne diseases, etc., to achieve the effect of strong reliability, strong specificity and high sensitivity

Pending Publication Date: 2019-08-06
浙江正合谷生物科技有限公司
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

But these methods can only semi-qualitatively or the total number of bacteria

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  • High-throughput quantitative detection kit for food-borne pathogenic bacteria
  • High-throughput quantitative detection kit for food-borne pathogenic bacteria
  • High-throughput quantitative detection kit for food-borne pathogenic bacteria

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specific Embodiment 1

[0015] A high-throughput quantitative detection kit for food-borne pathogenic bacteria, including a LAMP detection primer set for nine kinds of food-borne pathogenic bacteria, the gene sequences of which are shown in Table 1 below:

[0016] Table 1

[0017] .

[0018] The design of the above-mentioned gene target site: choose inter-species specific and intra-species conservative gene design primers, because the external primer F3 also has the opportunity to combine with and extend the F3c on the template, and replace the complete complementary single strand connected by FIP. At this time, F1c on FIP can realize the complementarity with F1 on this single chain, and form a circular structure through self-base pairing. Similarly, the synthesis of downstream primers BIP and B3 is similar to that of primers FIP and F3, which provides the possibility of forming a dumbbell-shaped single-stranded structure. At this time, under the action of DNA polymerase with strand displacement ...

specific Embodiment 2

[0021] A high-throughput quantitative detection kit for food-borne pathogens, the LAMP reaction system and the final concentrations of the components are as follows: 1 μL of sample template DNA, 2.5 μL of 10× reaction buffer, 1.5 μL of MgSO with a concentration of 100 mM 4 , 3.5 μL of 10 mM dNTPs, 1 μL of 8 U / μl Bst DNA polymerase, 1 μL FIP / BIP primer set solution, 1 μL F3 / B3 primer set solution and 4 μL betaine, and then make up 25 μL with double-distilled water; the FIP / BIP primer set solution is listed in Table 1, respectively. Bacteria, Vibrio parahaemolyticus, Vibrio cholerae, Staphylococcus aureus, Bacillus cereus, Proteus mirabilis, Salmonella, Shigella and pathogenic Escherichia coli / BIP primer group solution, the final concentration of each primer is 40 μM, and the F3 / B3 primer group solution is Listeria monocytogenes, Vibrio parahaemolyticus, Vibrio cholerae, Staphylococcus aureus, wax sample shown in Table 1 respectively The F3 / B3 primer set solution of 9 kinds of...

specific Embodiment 3

[0024] A method for the high-throughput quantitative detection of food-borne pathogenic bacteria by using the detection kit of the second specific embodiment above, comprising the following steps:

[0025] Take the sample to be tested, extract the sample template DNA according to the commercial bacterial DNA extraction kit, take 1 μL of the sample template DNA, add 2.5 μL 10× reaction buffer according to the composition of the LAMP reaction system, 1.5 μL MgSO 4 , 3.5 μL dNTPs, 1 μL BstDNA polymerase, 1 μL FIP / BIP primer set solution, 1 μL F3 / B3 primer set solution and 4 μL betaine, then make up 25 μL with double distilled water and mix well, take 20 μL of the mixture and add it to each well of the sample detection chip (wherein the primer set solutions are Listeria monocytogenes, Vibrio parahaemolyticus, Vibrio cholerae, Staphylococcus aureus, Bacillus cereus, Proteus mirabilis, Salmonella, Shigella and pathogenic Escherichia coli The primer set solution of 9 kinds of food-b...

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Abstract

The invention discloses a high-throughput quantitative detection kit for food-borne pathogenic bacteria. The kit is characterized in that the kit includes a LAMP detection primer set, whose gene sequences are shown as SEQ ID NO.1-NO.36, of 9 kinds of food-borne pathogenic bacteria; and the kit also includes a sample template, 10*reaction buffer, MgSO4, dNTP, Bst DNA polymerase and double distilledwater. The kit has the advantages of being strong in specificity, high in sensitivity, high in throughput, strong in reliability and low in cost, and has no false negative results.

Description

technical field [0001] The invention relates to a detection kit for food-borne pathogenic bacteria, in particular to a high-throughput quantitative detection kit for food-borne pathogenic bacteria. Background technique [0002] Foodborne pathogens are pathogenic bacteria that can cause food poisoning or use food as a medium for transmission. Pathogenic bacteria directly or indirectly contaminate food and water sources, and human oral infection can lead to the occurrence of intestinal infectious diseases, food poisoning, and the prevalence of livestock and poultry infectious diseases. Foodborne pathogens are an important source of food safety problems. The pathogenic bacteria that can cause food poisoning or use food as the medium mainly include Shigella, pathogenic Escherichia coli, Salmonella, Vibrio cholerae, Staphylococcus aureus, Listeria monocytogenes, Vibrio parahaemolyticus, and Saccharomyces cereus Bacillus, Shigella. The traditional microbiological testing method...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12Q1/689C12Q1/04C12Q1/10C12Q1/14C12N15/11C12R1/01C12R1/63C12R1/445C12R1/42C12R1/19C12R1/085C12R1/37
CPCC12Q1/6851C12Q1/689C12Q2531/119Y02A50/30
Inventor 苏秀榕周君芦晨阳韩姣姣李晔
Owner 浙江正合谷生物科技有限公司
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