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Real time fluorescent PCR reagent kit for discriminating active Listeria monocytogenes and detection method

A Listeria and real-time fluorescence technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of inability to distinguish the dead and alive state of pathogenic bacteria, incomplete data, etc., and achieves improved detection sensitivity and high Sensitivity, the effect of reducing false positive rate

Inactive Publication Date: 2009-06-24
ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the methods used for the diagnosis of pathogenic bacteria in food safety are all aimed at the genomic DNA of pathogenic bacteria. Incomplete data lead to unnecessary preventive and therapeutic measures

Method used

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  • Real time fluorescent PCR reagent kit for discriminating active Listeria monocytogenes and detection method
  • Real time fluorescent PCR reagent kit for discriminating active Listeria monocytogenes and detection method
  • Real time fluorescent PCR reagent kit for discriminating active Listeria monocytogenes and detection method

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Experimental program
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Effect test

Embodiment 1

[0044] Example 1: Specific primers, fluorescent probes

[0045] 1. Materials:

[0046] Bacterial RNA extraction reagents were purchased from Promega Company (SV Total RNA IsolationZ3100); Taq DNA polymerase was purchased from Promega Company in the United States, and the 377 sequencer and the 7000 quantitative PCR instrument were all products of the American ABI Company.

[0047] 2. Primer and probe design and synthesis:

[0048] Using the Listeria monocytogenes A gene sequence (registration number EU372055) as a template, use Primer Express TM (V2.0, American ABI Company) software analyzes TaqMan primer and probe site, selects the optimal combination therefrom.

[0049] Upstream primer: 5′-CGGAGGTTCCGCAAAAGATG-3′

[0050] Downstream primer: 5′-CCTCCAGAGTGATCGATGTT-3′

[0051] Fluorescent probe: 5′-FAM-AAATCATCGACGGCAACCTC-TAMRA-3′

[0052] Among them, FAM is a fluorescent reporter group, and TAMRA is a fluorescent quencher group.

[0053] Both were synthesized by Dalian...

Embodiment 2

[0054] Example 2: Detection of simulated food contamination Listeria monocytogenes by fluorescent quantitative PCR method

[0055] 1. Sample testing:

[0056] Prepare 20 cases of double-blind food samples with a standard strain (CMCC 54001), wash with normal saline and centrifuge, use bacterial RNA extraction reagent to extract RNA, take 2.5 μg as a template for reverse transcription reaction, and then use upstream and downstream primers for detection and The probes were amplified by PCR on a 7000 quantitative PCR instrument from ABI Company.

[0057] (1) Reverse transcription reaction

[0058] The reverse transcription reaction is as follows: ① Mix 2.5 μg total RNA with 1.0 μL of 10 mM deoxynucleoside triphosphate mixture and 1.0 μL of random primers (0.5 μg / μL) (Bao Biology Dalian Co., Ltd., D3801) and mix the reaction volume Make up to 10 μL with DEPC (diethylpyrocarbonate) water. After incubating at 65°C for 5 minutes, place it in an ice bath for 3 minutes. ② Add 2 μL ...

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Abstract

The invention provides a real-time fluorescent PCR reagent kit for screening active Listeria monocytogenes. The real-time fluorescent PCR reagent kit mainly comprises specific primers, a fluorescent probe, PCR buffer solution, deoxidant nucleoside triphosphate mixture and DNA polymerase. The sequence of the specific primers and the fluorescent probe is as follows: an upstream primer: 5'- CGGAGGTTCCGCAAAAGATG-3', a downstream primer: 5' -CCTCCAGAGTGATCGATGTT-3' and the fluorescent probe: 5' FAM-AAATCATCGACGGCAACCTC-TAMRA-3', wherein, the FAM is a fluorescent reporter group, and the TAMRA is a fluorescent quenching group. The real-time fluorescent PCR reagent kit has the beneficial effects that the real-time fluorescent PCR reagent kit can effectively screen the life and death state of bacteria and has the high sensitivity and the good specificity of the detection of bacteria, thereby reducing the false positive rate of regular PCR amplification; and the real-time fluorescent PCR reagent kit can realize fast, exact and specific detection towards the active Listeria monocytogenes.

Description

(1) Technical field [0001] The invention relates to a real-time fluorescent PCR kit and a detection method for screening active Listeria monocytogenes (Listeria monocytogenes, referred to as Listeria monocytogenes). (2) Background technology [0002] Listeria belongs to the indeterminate genus of Gram-positive non-bacillus in the microbial classification, and there are 7 species of bacteria in this genus (Listeria monocytogenes L.monocytogenes, Listeria ovis L.iuanuii, Listeria innoculus L.innocua, L.welshimeri, L.seeligeri, L.grayi, L.murray), of which only monocytogenes Listeria is pathogenic to humans. Listeria monocytogenes widely exists in soil, water, plants, human and animal feces, and the symptoms of poisoning are severe. In addition to common gastrointestinal manifestations such as vomiting and diarrhea, it can also erode the human central nervous system (main manifestations meningitis, septicemia, miscarriage and other diseases), causing great damage to the body,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/06
Inventor 金大智张政梅玲玲程苏云
Owner ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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