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Triple real-time fluorescent PCR detection primers, probes, detection kits and detection methods for three bacteria

A technology for real-time fluorescence and detection probes, applied in biochemical equipment and methods, microbial-based methods, microbial determination/inspection, etc.

Active Publication Date: 2014-10-01
许龙岩 +5
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the specific gene detection of Salmonella Enteritidis and Salmonella typhimurium, Wood et al. used the 2Kb Pst I / BgIII DNA fragment of the Salmonella Enteritidis serotype-specific plasmid as the target sequence to establish a PCR detection method, but many Salmonella Enteritidis isolates have no specific serotype plasmid , this method can only detect specific serotypes of Salmonella enteritidis; Edel O'Regan et al. used flic, sefa, sdf, and acek as target genes to detect Salmonella with fluorescent PCR, but this method could not accurately distinguish Salmonella typhimurium, Salmonella kentucky, Salmonella Dublin and Salmonella chicken

Method used

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  • Triple real-time fluorescent PCR detection primers, probes, detection kits and detection methods for three bacteria
  • Triple real-time fluorescent PCR detection primers, probes, detection kits and detection methods for three bacteria
  • Triple real-time fluorescent PCR detection primers, probes, detection kits and detection methods for three bacteria

Examples

Experimental program
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Effect test

Embodiment 1

[0069] A total of 15 strains of Salmonella enteritidis were selected, of which 1 strain was the standard strain ATCC13076, 13 strains were isolated from human sources, and 1 strain was isolated from frozen chicken samples; a total of 11 strains of Salmonella typhimurium were selected, of which 1 strain was the standard strain ATCC14028, 10 The strains were food isolates (as shown in Table 1); a total of 21 strains of different serotypes of non-Salmonella enteritidis and non-typhimurium isolated from food were selected (as shown in Table 2); 1 strain of Salmonella Dublin CMCC 50042 was selected; There were 11 negative control strains, including Listeria monocytogenes ATCC19115, Staphylococcus aureus ATCC6538, Enterobacter sakazakii ATCC29544, Escherichia coli ATCC25922, Shigella baumannii CMCC51582, Shigella sonnei CMCC51334, dysentery Shigella NICPBP51252, Yersinia enterocolitica CMCC52221, Yersinia pseudotuberculosis CMCC53510, Klebsiella pneumoniae CMCC46102, Vibrio parahaemo...

Embodiment 2

[0096]The 21 strains of non-Salmonella enteritidis non-typhimurium different serotype Salmonella strains (shown in Table 2), 15 strains of Salmonella enteritidis, 11 strains of Salmonella typhimurium, 1 strain of Salmonella Dublin CMCC 50042 and 11 negative control strains were used Buffered peptone water (BP) was cultured at 37°C for 10 hours, then 1ml of the cultured bacteria was inoculated into the TTB enrichment solution, cultured at 44.5°C for 18 hours, and then 1ml of the cultured bacteria suspension was transferred into a centrifuge tube, 12000r / min Centrifuge for 8 minutes to remove the supernatant, float the sediment with 1ml of deionized water, then centrifuge at 12000r / min for 5 minutes to remove the supernatant, repeat twice, finally add 200μl of deionized water, extract DNA on a nucleic acid extractor for real-time fluorescent PCR amplification.

[0097] Embodiment 3: detection primer and detection probe specificity test

Embodiment 3

[0098] Single-plex real-time fluorescent PCR reaction system 30μl, including: template DNA 1μl, 10×TaqMan buffer 4μl, 5mmol / L MgCl 2 2μl, 2.5mmol / L dNTPs 3μl, 20μmol / l TaqMan detection probe 1μl, 20μmol / l detection primer 1μl each (total 2μl), 0.55U UNG enzyme 0.2μl, 2.5U / μl Taq polymerase 3μl, deionized water 13.8 μl. The parameters of the fluorescent PCR reaction were 95°C for 30s, 95°C for 5s, 60°C for 34s, and 40 cycles.

[0099] 1. Specificity test 1: Specificity test of detection primers and detection probes for Salmonella

[0100] According to the method described in Example 2, 21 different serotype Salmonella strains of non-Salmonella enteritidis non-Typhimurium, Salmonella typhimurium standard strain ATCC14028, Salmonella enteritidis standard strain ATCC 13076, Salmonella Dublin CMCC 50042 and 11 negative control strains were extracted. DNA is amplified by real-time fluorescent PCR according to the above-mentioned single-plex real-time fluorescent PCR reaction syste...

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Abstract

The invention discloses a triple real-time fluorescent PCR detection primer, probe, detection kit and detection method for Salmonella, Salmonella enteritidis and Salmonella typhimurium. The present invention utilizes a triple real-time fluorescent PCR method to use the aceA gene of Salmonella to detect Salmonella of different serotypes, the specific sequence of Salmonella enteritidis to detect Salmonella enteritidis, and the STM4599 sequence of Salmonella typhimurium to specifically detect Salmonella typhimurium, and optimize the reaction Conditions, through a real-time fluorescent PCR amplification to determine whether the sample is contaminated by Salmonella, Salmonella enteritidis and Salmonella typhimurium, the detection is fast, and the process from preparing the sample to issuing the test result can be completed within 30 hours, free from false positives and cross-contamination and other interference, the results are reliable, and the sensitivity and specificity are strong, which provides a favorable tool for the epidemiological investigation of Salmonella.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to primers, probes and detection reagents for triple real-time fluorescent PCR detection of important food-borne pathogenic bacteria Salmonella, Salmonella Enteritidis and Salmonella Typhimurium Boxes and assay methods. Background technique: [0002] Salmonella (Salmonella) is one of the important food-borne pathogens, in the cases of bacterial food poisoning in various countries in the world, food poisoning cases caused by Salmonella often rank first or second. There are more than 2,500 serotypes of Salmonella, all of which are pathogenic, but the scope of host infection can be divided into host-adapted serotypes and non-host-adapted serotypes. It is pathogenic only in the adapted host, but Salmonella enteritidis, Salmonella typhimurium, and Salmonella duck can make a variety of hosts pathogenic. A British survey showed that Salmonella Enteritidis and Salmonella Typhimuri...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/42
Inventor 许龙岩袁慕云曹际娟柯碧霞相大鹏邹志飞谢力
Owner 许龙岩
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