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Weissella viridescens loop-mediated isothermal gene amplification rapid detection kit and detection method

A loop-mediated constant temperature, detection kit technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of difficult on-site detection, high cost, cumbersome operation, etc., and achieves low requirements for equipment and detection. Short, specific effects

Inactive Publication Date: 2016-11-09
HENAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Weissella viride has become an important spoilage bacteria that threatens the quality and safety of meat products, causing significant economic losses to the meat processing industry
[0004] However, current detection methods for Weissella viridans are limited
The traditional identification method needs to go through pre-enrichment, re-enrichment of selective medium, streak culture of the culture on a plate containing one or more agents that inhibit the growth of the tested bacteria, confirmation of characteristic colonies visible to the naked eye, A series of biochemical and serotype tests were carried out to identify the colony, which took a long time, complicated operations, and low reliability of the results; amplified ribosomal DNA restriction analysis (ARDRA) can be used in Differentiate Weissella at the species level, but the operation steps are cumbersome (PCR amplification, enzyme digestion and cluster analysis), require special instruments (PCR instrument) and a variety of restriction endonucleases, and the cost is high; real-time fluorescence Quantitative PCR technology has been successfully used in the molecular identification of Weissella viridans. Although this method has the advantages of good accuracy, high sensitivity, and strong specificity, the real-time fluorescent quantitative PCR instrument necessary for the experiment is expensive, and it is difficult to be widely used. On-site inspection

Method used

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  • Weissella viridescens loop-mediated isothermal gene amplification rapid detection kit and detection method

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Effect test

Embodiment 1

[0028] Establishment of rapid detection kit and detection method for Weissella viride ring-mediated constant temperature gene amplification

[0029] Step 1, design of primers and assembly of synthetic kits:

[0030] The primer sequences determined in this embodiment for detection are as follows:

[0031] Outer primer 1: CTTTGCTCAACGCAACAG,

[0032] Outer primer 2: ATTTTCGCGTCGCTCATG,

[0033] Internal primer 1: ACCTGCATTAACGCTTGGTG-GAAGTGGGCAATCATTTGG,

[0034] Inner primer 2: TAGATGAATACGCATCAAACGCT-CTAACTGTCGATACTCCGC.

[0035] On this basis, a rapid detection kit for Weissella viride ring-mediated constant temperature gene amplification is designed, which includes:

[0036] (1) Reaction solution 1: composed of 10mmol / L deoxynucleoside triphosphate (dNTP), 10×ThermoPol Buffer reaction buffer, 150mmol / L magnesium sulfate (MgSO 4 ), 5 mol / L betaine and sterilized double distilled water (ddH 2 O) Composition;

[0037] (2) Reaction solution 2: composed of 10 μmol / L outer ...

Embodiment 2

[0048] negative control

[0049] Step 1, design of primers and assembly of synthetic kits:

[0050] The sequence of primers used for detection determined in this embodiment is the same as that in Example 1.

[0051] On this basis, a rapid detection kit for constant temperature gene amplification of Weissella viride is designed, which includes:

[0052] (1) Reaction solution 1: composed of 10mmol / L deoxynucleoside triphosphate (dNTP), 10×ThermoPol Buffer reaction buffer, 150mmol / L magnesium sulfate (MgSO 4 ), 5mol / L betaine and sterilized double distilled water (ddH 2 O) Composition;

[0053] (2) Reaction solution 2: 10 μmol / L outer primer 1 (F3), 10 μmol / L outer primer 2 (B3), 40 μmol / L inner primer 1 (FIP) and 40 μmol / L inner primer 2 (BIP);

[0054] (3) 8U / μL Bst DNA polymerase;

[0055] (4) Chromogen: 10wt% SYBR Green I fluorescent dye;

[0056] The reaction solution 1 in the above-mentioned loop-mediated constant temperature gene amplification rapid detection kit is ...

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Abstract

The invention discloses a weissella viridescens loop-mediated isothermal gene amplification rapid detection kit and a detection method. The kit conducts large-scale genome analysis by means of a bioinformatic platform, four specific primers are designed according to a recN gene of weissella viridescens, an LAMP technology is combined, the rapid, sensitive and accurate detection method is established for the weissella viridescens, and the rapid detection kit used for the method is constructed. By means of the rapid detection kit and detection method, observing and identifying can be conducted with naked eyes after a reaction is conducted, other analysis steps of electrophoresis and the like are not needed, and the rapid detection kit and detection method have the advantages of being short in detection time, high in specificity, low in instrument and equipment requirement, easy and convenient to operate and the like and can be used for safe and rapid detection of samples of foods and environments, clinic samples and the like.

Description

technical field [0001] The invention relates to a biological detection reagent for rapid detection of bacterial samples using loop-mediated isothermalamplification of DNA (LAMP) technology, which belongs to the technical field of rapid gene molecular diagnostic reagents for Weissella viride, specifically The invention relates to a rapid detection kit and detection method for Weissella viride ring-mediated constant temperature gene amplification. Background technique [0002] Weissella viridans ( Weissella viridescens ) belonging to the Lactobacillus order ( Lactobacillales ) Leuconostocaceae ( Leuconostocaceae ) Weissella sp. ( Weissella ). The bacterium is similar to other representative strains of lactic acid bacteria in terms of microscopic and macroscopic morphology, especially it is easy to associate with Leuconostoc ( Leuconostoc ) and Lactobacillus ( Lactobacillus ) are confused. Weissella viride is a Gram-positive bacterium, which is irregularly short rod...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/6844C12Q1/689C12Q2531/119C12Q2563/107
Inventor 姜晓冰于涛田金河董斌周丹蕾任敏
Owner HENAN NORMAL UNIV
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