Method for manufacturing multiple amplification internal mark for four-bacteria PCR test

A technology of multiple amplification and amplification of internal standards, applied in biochemical equipment and methods, DNA preparation, recombinant DNA technology, etc., can solve problems such as unavailability, and achieve the effect of improving accuracy

Inactive Publication Date: 2008-07-16
SHANGHAI JIAO TONG UNIV
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Problems solved by technology

However, it cannot be used for ordinary PCR detection of Staphylococcus aureus, Salmonella, Listeria monocytogenes and Vibrio parahaemolyticus

Method used

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  • Method for manufacturing multiple amplification internal mark for four-bacteria PCR test

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Embodiment

[0030] 1. Design of specific detection primers

[0031] The known specific genes of Staphylococcus aureus, Salmonella, Listeria monocytogenes and Vibrio parahaemolyticus were analyzed by bioinformatics, and the target genes vicK, invA, hlyA and toxR were selected for detection. The sequences of vicK, invA, hlyA, and toxR genes were respectively compared with other microorganisms through the public BLAST software in Genbank (an existing technology, shared free of charge), and sequence segments with higher specificity were selected. Then use the software Primer 5.0 (commercially available, Premier, Canada) to design a pair of internal standard primers in this specific sequence. The primer sequences are as follows:

[0032] 1. Primers for detection of Staphylococcus aureus (vicKF / vicKR)

[0033] vicKF: 5'- CGCAGGCTAATACTGAAAG -3'

[0034] vic KR: 5'- TTCTGTTTCTTCACGGGTA -3'

[0035] (a) Detect the sequence characteristics of the target gene:

[0036] * Length: 512 bp

[...

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Abstract

The invention relates to a preparation method of a multiple amplification internal standard used for the PCR detection of four bacteria of the technical field of food safety, the steps are that: a specific detection primer is designed according to a staphylococcus aureus vicK gene, a salmonella invA gene, a listeria monocytogene hlyA gene and vibrio parahaemolyticus toxR gene; the invention adopts an overlap PCR technology for assembling the sequence of the specific detection primer of the staphylococcus aureus vicK gene, the salmonella invA gene, the listeria monocytogene hlyA gene and the vibrio parahaemolyticus toxR gene into a nucleotide segment so as to construct the multiple amplification internal standard; the artificial sequence is cloned to a plasmid vector pMD-18T to constitute a plasmid pMD-mIAC which comprises the sequence of the multiple amplification internal standard; the multiple amplification internal standard is respectively verified in the detections of staphylococcus aureus, salmonella, listeria monocytogenes and vibrio parahaemolyticus. The invention can greatly reduce the detection cost and ensure the accuracy and the high efficiency of the detection at the same time.

Description

technical field [0001] The invention relates to a method in the technical field of food safety, in particular to a method for preparing a multiple amplification internal standard for PCR detection of four types of bacteria. Background technique [0002] Staphylococcus aureus, Salmonella, Listeria monocytogenes and Vibrioparahaemolyticus are four important food-borne pathogenic bacteria that are well known and published , which are widely distributed in nature, they mainly spread and infect the human body through food (especially animal food), directly causing damage to human health. PCR (polymerase chain reaction) technology has the characteristics of simple operation, rapidity, high sensitivity and strong specificity, and has been widely used in the detection of Staphylococcus aureus, Salmonella, Listeria monocytogenes and Vibrio parahaemolyticus . However, there are certain differences in the target genes and operating procedures detected by PCR methods in different labo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12N15/10C12N15/63
CPCY02A50/30
Inventor 史贤明龙飞施春雷马瑜丹张忠明
Owner SHANGHAI JIAO TONG UNIV
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