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Real-time fluorescence polymerase chain reaction (PCR) detection kit for screening listeria monocytogenes and detection method thereof
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A detection kit and Listeria detection technology, applied in the direction of fluorescence/phosphorescence, measurement/inspection of microorganisms, biochemical equipment and methods, etc., to achieve the effects of saving time, improving accuracy, and improving sensitivity
Inactive Publication Date: 2013-04-10
ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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[0003] Among the current routine identification methods, only there are standard identification methods for Listeria monocytogenes, and they are all based on conventional culture methods. Identification is based on biochemical characteristics, pathogenicity, and synergistic hemolysis tests. The identification cycle needs 5 ~10 days, and there is no standard identification method for other pathogenic bacteria in the genus
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Embodiment 1
[0039] Example 1: Obtaining specific primers and standards
[0040] 1. Material:
[0041] Bacterial genomic DNA extraction reagents were purchased from Dalian Bao Biological Engineering Co., Ltd.; PCR reaction system and Taq DNA polymerase were purchased from Bao Biological (Dalian) Co., Ltd., pGEM-T-Easy cloning system and Eva Green dye were purchased from Shanghai Huirui Biotechnology Co., Ltd., type 377 sequencer, Bio-Rad icycler PCR instrument, and type 480 quantitative PCR instrument are the products of Roche, Switzerland.
[0042] 2. Primer synthesis:
[0043] Using Listeria monocytogenes ssrA gene sequence (registration number AF440341) as a template, primer 5.0 software was used to analyze the primers, and the best combination was selected. The PCR primer sequence for detection is as follows:
[0046] All are synthesized by Dalian Bao Biological Engineering Co., Ltd.
[0047] 3...
Embodiment 2
[0068] Example 2: Fluorescence quantitative PCR combined with high-resolution melting curve method to detect Listeria
[0069] 1. Sample testing:
[0070] For 7 actual specimens, genomic DNA extraction reagents were used to extract genomic DNA, and 1.0 μL of them were used as templates. The upstream and downstream primers for detection were used for PCR amplification on the Roche 480 quantitative PCR machine.
[0071] The composition of the PCR reaction solution is as follows:
[0072] 1×PCR buffer 2μL
[0073] 1×Eva Green 2μL
[0074] Upstream primer (10μM) 1μL
[0075] Downstream primer (10μM) 1μL
[0076] DNA polymerase (5U / μL) 0.2μL
[0077] dNTPs (each 250mM) 1.60μL
[0078] (The amount ratio of dATP, dTTP, dCTP, dGTP substances is 1:1:1:1)
[0079] Template DNA (50ng / μL) 1μL
[0080] Make up to 20 μL of water.
[0081] The PCR reaction conditions were: 95°C pre-denaturation for 2 minutes, 95°C for 15 seconds, 60°C for 45 seconds, 40 cycles of amplification, and 60°C to gradually increase ...
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Abstract
The invention provides a real-time fluorescence polymerase chain reaction (PCR) detection kit for screening listeria monocytogenes. The kit mainly comprises a specific primer, an Eva Green fluorescent dye, PCR buffer solution, a deoxynucleotide triphosphate mixture and (deoxyribonucleic acid) DNA polymerases. The kit is characterized in that: the specific primer and a fluorescence probe have the following sequences: a forward primer: 5'-CGTGCATCGCCCATGTGC-3' and a reverse primer: 5'-ATCTACGAGCGTAGTCAC-3'. The kit has the main advantages that: six kinds of listeria monocytogenes can be easily screened with high sensitivity and high specificity; rapid, precise and specific detection and analysis of the six kinds of listeria monocytogenes can be realized; and standard products can be added for quantitative detection.
Description
(1) Technical field [0001] The invention relates to a real-time fluorescent PCR detection kit for screening Listeria and a detection method. (2) Background technology [0002] Listeria is classified as an indeterminate genus of Gram-positive abacillus in the classification of microorganisms. There are 6 species of bacteria in this genus-Listeria monocytogenes, Listeria Ivanovii ( Listeria ivanovii), Listeria innocua (Listeria innocua), Listeria grayi (Listeria grayi), Listeria seeligeri (Listeria seeligeri), Listeria welshimeri (Listeria welshimeri). Previous reports pointed out that only Listeria monocytogenes of the genus Listeria is pathogenic to humans. Listeria monocytogenes is widely present in soil, water, plants, human and animal feces. The symptoms of poisoning are serious. In addition to the common gastrointestinal manifestations such as vomiting and diarrhea, it can also erode the human central nervous system (main manifestations) Meningitis, sepsis, miscarriage and ...
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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04G01N21/64
Inventor 金大智张政罗芸程苏云
Owner ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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