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High-throughput quantitative detection kit for diarrhea pathogenic bacteria

A detection kit, quantitative detection technology, 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 low cost

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

AI Technical Summary

Problems solved by technology

They generally require large-scale detection equipment, expensive detection reagents, and the detection time exceeds 24 hours, and they are all qualitative tests

Method used

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  • High-throughput quantitative detection kit for diarrhea pathogenic bacteria
  • High-throughput quantitative detection kit for diarrhea pathogenic bacteria
  • High-throughput quantitative detection kit for diarrhea pathogenic bacteria

Examples

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specific Embodiment 1

[0016] A high-throughput quantitative detection kit for diarrheal pathogens, including 9 kinds of LAMP detection primer sets for diarrheal pathogens, and its gene sequence is shown in Table 1 below:

[0017] Table 1

[0018] .

[0019] The above-mentioned gene target site design: select gene design primers that are specific between species and conserved within species because the external primer F3 also has the opportunity to bind and extend with F3c on the template to replace a complete FIP-linked complementary single strand. At this time, the F1c on the FIP can be complementary to the F1 on the single strand, forming a circular structure through self-base pairing. Similarly, the downstream primers BIP and B3 are similar to the synthesis of primers FIP and F3, which makes it possible to form a dumbbell-shaped single-stranded structure. At this time, under the action of DNA polymerase capable of strand displacement, the F1 segment at the 3'end will use itself as a template for DNA...

specific Embodiment 2

[0022] A high-throughput quantitative detection kit for diarrheal pathogens. The LAMP reaction system and the final concentration of each component are as follows: sample template DNA 1μL, 2.5μL 10× reaction buffer, 1.5μL 100mM MgSO 4 , 3.5μL of 10mM dNTP, 1μL of 8 U / μl Bst DNA polymerase, 1μL FIP / BIP primer set solution, 1μL F3 / B3 primer set solution and 4μL betaine, and then make up to 25μL with double distilled water; the FIP / BIP primer set solutions are listed in Table 1 respectively. FIP / BIP primer set solution of 9 bacteria, Vibrio cholerae, Staphylococcus aureus, Salmonella, Shigella, pathogenic Escherichia coli, Bacillus cereus, Proteus mirabilis and Vibrio parahaemolyticus, The final concentration of each primer is 40 μM, and the F3 / B3 primer set solutions are Listeria monocytogenes, Vibrio cholerae, Staphylococcus aureus, Salmonella, Shigella, and pathogenicity as shown in Table 1 above. F3 / B3 primer set solution of 9 bacteria of Escherichia coli, Bacillus cereus, Prot...

specific Embodiment 3

[0025] A method for high-throughput quantitative detection of diarrheal pathogens by using the detection kit of the second embodiment described above, including the following steps:

[0026] Take the sample to be tested, extract the sample template DNA according to the commercially available bacterial DNA extraction kit, take 1μL of the sample template DNA, add 2.5μL of 10× reaction buffer, 1.5μL of MgSO according to the composition of the LAMP reaction system 4 , 3.5μL dNTP, 1μL Bst DNA polymerase, 1μL FIP / BIP primer set solution, 1μL F3 / B3 primer set solution and 4μL betaine, then make up 25μL with double distilled water and mix well. Take 20μL of the mixed solution and add it to each loading hole of the sample detection chip (The primer set solutions are Listeria monocytogenes, Vibrio cholerae, Staphylococcus aureus, Salmonella, Shigella, pathogenic Escherichia coli, Bacillus cereus, Proteus mirabilis and Vibrio parahaemolyticus The primer set solution of 9 bacteria, the primer...

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Abstract

The invention discloses a high-throughput quantitative detection kit for diarrhea 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 diarrhea pathogenic bacteria; and the kit also includes a sample template, 10*reaction buffer, MgSO4, dNTP, Bst DNA polymerase and double distilled water. 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 rapid detection kit for diarrheal pathogenic bacteria, in particular to a high-throughput quantitative detection kit for diarrheal pathogenic bacteria. Background technique [0002] Diarrhea is a common clinical symptom that can be caused by a variety of diseases. Generally refers to the increase in the number of bowel movements per day or frequent bowel movements, thin stools or containing mucus, pus and blood, or indigestible food and other pathological contents. The main reasons are bacterial infections, viral infections, food poisoning, cold food, and food Retention, intestinal infection and small intestinal malabsorption, etc. Bacterial infection means that people may develop enteritis or bacillary dysentery after eating foods contaminated with bacteria such as E. coli, salmonella, Shigella, or drinking beverages contaminated with bacteria, and have different degrees of abdominal pain, diarrhea, vomiting, tenesmus , Fever a...

Claims

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Application Information

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