Quintuple PCR primers for detecting pathogenic bacteria in fresh agricultural products, probe and detection kit

A technology for pathogenic bacteria and agricultural products is applied in the field of five-fold PCR primers, probes and kits for detecting pathogenic bacteria in fresh and live agricultural products, which can solve the problem of increased non-specific amplification reactions, decreased sensitivity, and restricted development of multiplex PCR technology. To achieve the effect of strong primer specificity, high accuracy, and ensuring sensitivity and specificity

Active Publication Date: 2016-02-17
DALIAN NATIONALITIES UNIVERSITY +1
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  • Abstract
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  • Claims
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Problems solved by technology

However, in the traditional multiplex PCR system, due to the existence of multiple pairs of primers, the interference between primers and the mismatch between primers and templates lead to problems such as decreased sensitivity and increased non-specific amplification reactions, which restrict the development of multiplex PCR technology.

Method used

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  • Quintuple PCR primers for detecting pathogenic bacteria in fresh agricultural products, probe and detection kit
  • Quintuple PCR primers for detecting pathogenic bacteria in fresh agricultural products, probe and detection kit
  • Quintuple PCR primers for detecting pathogenic bacteria in fresh agricultural products, probe and detection kit

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Experimental program
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Embodiment

[0075] 1. Primer design

[0076] The genomes of 132 strains of five pathogenic bacteria were downloaded from the NCBI database, and one strain genome sequence was selected from each strain, and the selected genome sequences were compared with BLAT software to find the difference between any two genomes. For each bacterial species genome, according to the obtained sequence intervals with similarity comparison results, perform a complementary inversion operation to obtain sequence intervals specific to the bacterial species. The purpose of this step is preliminary screening and narrowing the scope of screening. These screened segments were manually blasted on the NCBI database to screen out species-specific gene segments. The species-specific gene segments obtained by screening are then aligned within the species, and finally the gene sequence segments with intra-species homology > 95% and inter-species homology < 75% in the gene sequences of the pathogenic bacteria to be teste...

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Abstract

The invention discloses quintuple PCR primers for detecting pathogenic bacteria in fresh agricultural products, a probe and a detection kit. The quintuple PCR primers comprises five primer pairs, and the five primer pairs are salmonella forward primer and salmonella reverse primer, staphylococcus aureus forward primer and staphylococcus aureus reverse primer, escherichia coli O-157 forward primer amd escherichia coli O-157 reverse primer, vibrio parahaemolyticus forward primer and vibrio parahaemolyticus reverse primer, and listeria monocytogenes forward primer and listeria monocytogenes reverse primer. The sequence information is shown as SEQ ID No.1-10. According to the technical scheme, five pathogenic bacteria can be simultaneously detected, interference among the primers is small, non-specific amplification reaction is less, detection sensitivity and specificity are guaranteed, detection speed is improved and also detection cost is reduced.

Description

technical field [0001] The invention relates to the technical field of detection of pathogenic bacteria, in particular to a five-fold PCR primer, a probe and a kit for detecting pathogenic bacteria in fresh and live agricultural products. Background technique [0002] Fresh and live agricultural products are the most important source of food nutrition for Chinese consumers besides food, and occupy a pivotal position in daily life. Fresh and live agricultural products are susceptible to infection by various pathogenic microorganisms (mainly bacteria and fungi) during raw materials, processing, storage, and transportation. The cross-infection after the occurrence of diseases caused by microbial infection will cause a large loss of fruits and vegetables, lose their commodity value, and greatly limit the development of the fruit and vegetable industry. At the same time, once the fresh and live agricultural products contaminated by various pathogenic microorganisms are purchased...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
CPCY02A50/30
Inventor 胡文忠何煜波姜爱丽冯可刘星伯郎秋蕾方超梁洪钱刚
Owner DALIAN NATIONALITIES UNIVERSITY
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