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Amplification primer for detecting food-borne pathogenic microorganisms and liquid chip kit

A technology of pathogenic microorganisms and amplification primers, applied in the field of molecular biology, can solve the problems of increased sensitivity and complexity of detection methods, and achieve the effects of avoiding uncertain factors, precise and deterministic analysis of characteristics, and improving sensitivity

Inactive Publication Date: 2016-02-10
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method is still relatively complicated, and due to the complexity of foodborne pathogenic microorganism detection, the sensitivity of the detection method needs to be improved

Method used

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  • Amplification primer for detecting food-borne pathogenic microorganisms and liquid chip kit
  • Amplification primer for detecting food-borne pathogenic microorganisms and liquid chip kit
  • Amplification primer for detecting food-borne pathogenic microorganisms and liquid chip kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The liquid-phase chip kit for detecting food-borne pathogenic microorganisms described in this embodiment mainly includes:

[0028] 1. PCR primers

[0029] For the rpsU-dnaG gene of Enterobacter sakazakii, the prfA gene of Listeria monocytogenes, the flic gene of Escherichia coli O157, the Sa442 gene of Staphylococcus aureus, the ipaH gene of Shigella, The fim gene of Salmonella, the toxR gene of Vibrio parahaemolyticus, and the bacterial internal control sequence 16S-rRNA were respectively designed to amplify primer pairs.

[0030] Among them, the amplification primer F1 is composed of three parts, from the 5' end to the 3' end are: tag sequence, spacer arm, and the forward amplification primer of the corresponding gene. At the same time, the amplification primer R1 is the 5' end with biotin Labeled and corresponding reverse primers of microorganisms to be detected, the primer sequences are shown in the table below:

[0031] Table 1 Genes and amplification primers of...

Embodiment 3

[0100] The detection of the target gene by the liquid phase chip of the amplification primer of embodiment 3 different tag sequences

[0101] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0102] Taking the rpsU-dnaG gene of Enterobacter sakazakii and the prfA gene detection liquid chip of Listeria monocytogenes as examples, the amplification primers were respectively designed for the rpsU-dnaG and prfA genes, and the amplification primers consisted of three Partial composition, the sequence from 5' end to 3' end is: tag sequence + spacer arm + forward amplification primer of the corresponding gene, meanwhile, the 5' end of the reverse amplification primer of amplification primer R1 is labeled with biotin. Wherein, the tag sequences in the amplification primers are respectively selected from SEQ ID NO.17-SEQ ID NO.28, and correspondingly, the anti-tag sequences coated on the magnetic beads that are complementary to the correspond...

Embodiment 4

[0117] Example 4 Detection of Target Genes by Liquid Chips with Different Spacer Arms

[0118] 1. Design of liquid phase chip preparation (selection of spacer arm)

[0119] The amplification primer F1 of the present invention consists of three parts, from the 5' end to the 3' end: a tag sequence, a spacer arm, and a forward amplification primer of the corresponding gene. In the present invention, a section of spacer arm is inserted between the Tag sequence and the forward amplification primer, and the spacer arm can connect two sections of base sequences (tag sequence and amplification primer), but cannot be paired with deoxyribonucleotide bases. During the amplification process, the spacer can prevent the DNA polymerization reaction from proceeding forward, so that the PCR product does not have an anti-tag sequence complementary to the tag sequence, greatly improving the hybridization efficiency and the intensity of the detection signal.

[0120] In this example, taking the ...

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Abstract

The invention discloses an amplification primer for detecting food-borne pathogenic microorganisms and a liquid chip kit. The kit comprises an amplification primer pair designed for microorganism genes to be detected; and a forward primer and a reverse primer of microorganisms to be detected comprise at least one pair of SEQ ID NO. 1 and SEQ ID NO. 2 for enterobacter sakazakii, SEQ ID NO. 3 and SEQ ID NO. 4 for monocyte listeria monocytogenes, SEQ ID NO. 5 and SEQ ID NO. 6 for escherichia coli O157, SEQ ID NO. 7 and SEQ ID NO. 8 for staphylococcus aureus, SEQ ID NO. 9 and SEQ ID NO. 10 for shigella, SEQ ID NO. 11 and SEQ ID NO. 12 for salmonella, and SEQ ID NO. 13 and SEQ ID NO. 14 for vibrio parahaemolyticus, and magnetic beads coated with Anti-tag sequences. The amplification primer designed by the invention has excellent specificity, and can accurately distinguish and specifically amplify gene segments corresponding to various target detection pathogenic microorganisms. In addition, the sensibility of the detecting kit designed by the invention is greatly improved. An experiment result shows that sensibility of detection of seven food-borne pathogenic microorganisms can reach 10 CFU / mL.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to an amplification primer and a liquid phase chip kit for detecting food-borne pathogenic microorganisms. Background technique [0002] Pathogenic microbial contamination is one of the main sources of food safety risks during the processing and production of catering services. From food raw materials, processing processes, and tableware, it is possible to introduce pathogenic microorganisms to contaminate catering food and increase food safety risks. Common pathogens include: Enterobacter sakazakii, Listeria monocytogenes, Escherichia coli O157, Staphylococcus aureus, Shigella, Salmonella, Vibrio parahaemolyticus, etc. [0003] The detection technology of foodborne pathogenic microorganisms is a key technical link in the prevention and control of foodborne diseases. At present, the methods for detecting foodborne pathogenic microorganisms in food include traditional me...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/14C12Q1/04C12N15/11C12R1/185C12R1/01C12R1/445C12R1/42C12R1/19C12R1/63
Inventor 陈昌华陈颖吴诗扬
Owner SUREXAM BIO TECH
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