Real-time fluorescence PCR kit used for detecting salmonella and detection method thereof

A Salmonella, kit technology, applied in fluorescence/phosphorescence, biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as poor stability and repeatability, complex process, application limitations, etc. The effect of good specificity and improved detection sensitivity

Inactive Publication Date: 2010-05-12
ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Currently commonly used methods for the detection of Salmonella are complex, long-term, and require a large number of reagents
The Salmonella PCR detection technology developed since the 1980s has provided the possibility for the rapid diagnosis of Salmonella, but the PCR method is prone to false positives, and its stability and repeatability are also poor, which limits its application.

Method used

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  • Real-time fluorescence PCR kit used for detecting salmonella and detection method thereof
  • Real-time fluorescence PCR kit used for detecting salmonella and detection method thereof
  • Real-time fluorescence PCR kit used for detecting salmonella and detection method thereof

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Experimental program
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Effect test

Embodiment 1

[0042] Embodiment 1: Obtaining of specific primers and fluorescent probes

[0043] 1. Materials:

[0044] Premix Ex Taq TM (2×) was purchased from TaKaRa Company (TaKaRa, DRR039S);

[0045] MX3000P quantitative PCR instrument is a product of STRATAGNE, USA.

[0046] 2. Primer and probe design and synthesis:

[0047] Using the Salmonella invE gene sequence (registration number U43253) as a template, use PrimerExpress TM (V2.0, American ABI Company) software analyzes TaqMan-MGB primers and probe sites, and selects the best combination:

[0048] Upstream primer: 5′-TTGTCCAGTCRACRGAYGAA-3′

[0049] Downstream primer: 5′-TCGCGACGGTTACGRAATTC-3′

[0050] Fluorescent probe: 5′-FAM-TGTCAGCGGCGCTG-MGB-3′

[0051] Wherein FAM is a fluorescent reporter group, and MGB is a fluorescent quencher group.

[0052] Both were synthesized by Dalian Bao Biological Engineering Co., Ltd.

Embodiment 2

[0053] Embodiment 2: Salmonella fluorescent quantitative PCR method specificity evaluation

[0054] 1. Strain detection:

[0055] Salmonella typhi (ATCC 50079), Salmonella typhimurium (ATCC 50013), Salmonella paratyphi A (ATCC 50002), Salmonella duck (ATCC 50083), Shigella dysenteriae (ATCC51570), Shigella sonnei (ATCC 51334), Shigella flexneri (ATCC 51573), Vibrio parahaemolyticus (ATCC 33847), Escherichia coli (ATCC 25922), Listeria monocytogenes (CMCC 54001), Staphylococcus aureus (ATCC 25923) , Bacillus cereus (CMCC63301), Pseudomonas aeruginosa (ATCC 27853), Proteus mirabilis (ATCC 43071) were purchased from China Institute for the Control of Biological Products and China Microorganism Culture Collection Center; beta-hemolytic streptococcus (ATCC 33152 ) was purchased from Shanghai Hanni Company; Salmonella Jiwei, Salmonella Brendenloop, Salmonella Corvallis, Salmonella Heidelberg, Salmonella Manchester, and Salmonella Mbanda long were tested by the World Health Organiza...

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Abstract

The invention provides a real-time fluorescence PCR kit used for detecting salmonella and detection method thereof. The kit mainly comprises a specific primer, a mixture of fluorescence probe, PCR buffer solution and deoxynucleotide triphosphate, and DNA polymerases, wherein the sequences of the specific primer and the fluorescence probe are as follows: the sequence of the upstream primer is 5'-TTG TCC AGT CRA CRGAYG AA-3', the sequence of the downstream primer is 5'-TCG CGA CGG TTA CGR AAT TC-3', and the sequence of the fluorescence probe is 5'-FAM-TGT CAG CGG CGC TG-MGB-3', wherein the FAM is fluorescence report gene and the MGB is fluorescence quenching gene. The beneficial effects of the invention are mainly as follows: the detection sensitivity of the target bacteria is high, the specificity is good, the false positive rate of conventional PCR amplification is reduced; and the rapid, accurate and specific detection of salmonella can be realized.

Description

(1) Technical field [0001] The invention relates to a real-time fluorescent PCR kit for detecting Salmonella and a detection method thereof. (2) Background technology [0002] Salmonella widely exists in nature, and more than 2,500 serotypes have been discovered so far, and 216 serotypes have been found in my country. The serotypes related to human diseases are mainly concentrated in groups A to E, including: Salmonella typhi, Salmonella paratyphi A, B, C, Salmonella typhimurium, Salmonella choleraesuis, Salmonella enteritidis, Salmonella duck, Salmonella newport, etc., among which Salmonella typhimurium, Salmonella enteritidis, and Salmonella choleraesuis are the most common. Food poisoning caused by Salmonella ranks first in Japan, Europe and the United States, and it is also a common bacteria in bacterial food poisoning in my country. In my country's inland areas, 70% to 80% of bacterial food poisoning is caused by Salmonella, which is a type of food-borne pathogen that...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/10C12Q1/06G01N21/64
CPCY02A50/30
Inventor 程苏云梅玲玲朱敏龚璞
Owner ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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