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Primer probe composition capable of detecting five pathogenic bacteria at same time and multiplex real-time fluorescence PCR method

A primer probe, pathogenic bacteria technology, applied in the field of molecular biology, to achieve the effect of compensating for complicated steps, improving detection efficiency, and improving sensitivity

Active Publication Date: 2017-06-09
中谱安信(青岛)检测科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

As a result, the use of real-time fluorescent quantitative PCR detection at this stage is mostly single-plex real-time fluorescent quantitative PCR detection method, and there are few detection systems that can detect multiple target genes at the same time. Therefore, it is necessary to establish a method based on multiple real-time fluorescent quantitative PCR. , Vibrio vulnificus, Shigella, Staphylococcus aureus, Listeria monocytogenes rapid simultaneous detection method is of practical significance

Method used

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  • Primer probe composition capable of detecting five pathogenic bacteria at same time and multiplex real-time fluorescence PCR method
  • Primer probe composition capable of detecting five pathogenic bacteria at same time and multiplex real-time fluorescence PCR method
  • Primer probe composition capable of detecting five pathogenic bacteria at same time and multiplex real-time fluorescence PCR method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The design of embodiment 1 primer and probe

[0031] Based on Salmonella invA gene sequence (geneID: 1254419), Vibrio vulnificus vvhA gene sequence (geneID: 2827959), Shigella ipaH gene sequence (gene ID: 3776622), Staphylococcus aureus nuc gene sequence (gene ID: 767621) , Listeria monocytogenes prfA gene sequence (gene ID: 2744465), using the primer design software PrimerExpress3.0, we designed the specific invA gene of Salmonella, the specific vvhA gene of Vibrio vulnificus, and the specificity of Shigella The ipaH gene, the specific nuc gene of Staphylococcus aureus, the upstream and downstream primers of the specific prfA gene of Listeria monocytogenes, and Taqman probes labeled with different fluorescein for each gene.

[0032] The designed primers were analyzed as follows: interference between primers, hairpin structure and dimer. And through the BLAST function of NCBI to preliminarily identify its specificity. After screening, the primers with complementary se...

Embodiment 2

[0038] Determination of the optimal reaction conditions of embodiment 2 multiplex fluorescent quantitative PCR

[0039] The method steps for detecting Salmonella, Vibrio vulnificus, Shigella, Staphylococcus aureus, and Listeria monocytogenes using primers, probes and multiple real-time fluorescent quantitative PCR detection methods in Table 1 are as follows:

[0040] (1) DNA extraction:

[0041] Bacterial DNA was extracted by boiling water bath method. The specific operation is to pick a single colony purely cultured on agar and add it to 100 μL sterilized double-distilled water, shake and mix well, boil in a boiling water bath for 10 minutes, let it stand at room temperature, and centrifuge at 12000 r / min for 2 minutes after it drops to room temperature, take the above Store at -20°C for later use.

[0042] (2) Establishment of single-plex real-time fluorescent quantitative PCR system and condition optimization

[0043] Using TAKARA's Premix Ex Taq reagent as the reaction ...

Embodiment 3

[0063] Embodiment 3 is detected to the specificity of five kinds of pathogenic bacteria

[0064] (1) In order to verify the performance of probes and primers, carry out specificity tests:

[0065] After rapidly amplifying 40 known bacterial strains, the target pathogenic bacteria were detected using single-plex and multiple real-time fluorescent quantitative PCR methods (using the PCR system and reaction conditions determined in Example 2). Use specific primers and probes to perform single-plex real-time fluorescent quantitative PCR reactions on the corresponding target pathogenic bacteria. The Ct values ​​of the detected genes are all less than 36. Use mixed primer probes to perform multiple real-time fluorescent quantitative PCR reactions on these 40 known strains Detection, the detection results are completely consistent with the design: the five target pathogenic bacteria all have the amplification of the corresponding reporter group, and no amplification occurs for other kn...

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Abstract

The invention relates to a primer probe composition capable of detecting five pathogenic bacteria at the same time. The primer probe composition comprises a sequence primer sequence or a probe sequence shown as SEQ ID No.1-15 as shown in a chart 1; a report radical group is modified at the 5' end of each probe sequence, and a quenching radial group is modified at the 3' end of each probe sequence; the report radial group has FAM, FITC, HEX, JOE and CY3, and the quenching radial group has TAMRA, BHQ and Eclipse. According to a detection method of five-fold real-time fluorescence quantification PCR, the five pathogenic bacteria such as salmonella, vibrio vulnificus, Shigella, staphylococcus aureus and Listeria monocytogenes can be detected at the same time in the same reaction system. Moreover, the five pathogenic bacteria are detected by using the five-fold real-time fluorescence quantification PCR method, the sensitivity and specificity of detection can be improved, the detection efficiency can be greatly improved, step complication and long detection period existing in conventional detection are remedied, and the primer probe composition cannot be applicable to the defects of large-scale quick detection.

Description

technical field [0001] The invention relates to a primer probe composition for rapid and simultaneous detection of Salmonella, Vibrio vulnificus, Shigella, Staphylococcus aureus, Listeria monocytogenes and a detection method for multiple real-time fluorescent PCR, belonging to molecular biology technology field. Background technique [0002] Salmonella, Vibrio Vulnificus, Shigella Castellani, Staphylococcus aureus, and Listeria monocytogenes are five common foodborne pathogens. It is also an important indicator of pathogenic bacteria detection in my country's national food hygiene standards. Among them, Salmonella is one of the zoonotic diseases of great significance in public health. The pathogenic Salmonella belongs to the Enterobacteriaceae family, including those bacteria that cause food poisoning, gastroenteritis, typhoid fever and paratyphoid fever. In addition to infecting humans, they can also infect many animals including mammals, birds, reptiles, fish, amphibians ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/14C12Q1/10C12Q1/04C12R1/42C12R1/63C12R1/445C12R1/01
CPCC12Q1/6851C12Q1/689C12Q2600/16C12Q2531/113C12Q2537/143C12Q2561/101Y02A50/30
Inventor 张鹏宋智斌王萌竹官宁张加美
Owner 中谱安信(青岛)检测科技有限公司
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