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LAMP primer for detecting enoki mushroom surface lactococcus and detection method

A technology for Lactococcus and Flammulina velutipes, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of not finding and detecting Lactococcus on the surface of Flammulina velutipes, achieve fast detection speed, easy observation, and method simple effect

Inactive Publication Date: 2017-05-31
INST OF AGRI RESOURCES & REGIONAL PLANNING CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] After searching, there is no report of using LAMP method to detect Lactococcus on the surface of Flammulina velutipes

Method used

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  • LAMP primer for detecting enoki mushroom surface lactococcus and detection method
  • LAMP primer for detecting enoki mushroom surface lactococcus and detection method
  • LAMP primer for detecting enoki mushroom surface lactococcus and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Bacteria Isolation and Identification Test on the Surface of Fresh Flammulina velutipes

[0057] (1) Test materials

[0058] Flammulina velutipes sample source: Fresh Flammulina velutipes were purchased from the supermarket Fa Supermarket at the South Gate of the Chinese Academy of Agricultural Sciences, packed in plastic wrap on trays.

[0059] (2) Preparation of beef extract-peptone medium: the composition and proportion of the medium are: 3g beef extract, 10g peptone, 5g NaCl, 20g agar powder, 20g glucose, 1000ml distilled water, pH 7.0-7.2, sterilized at 121°C 20min.

[0060] (3) Test method

[0061] (1) Sampling: Aseptically cut the surface tissue of Flammulina velutipes into pieces, mix them evenly, take 10g and put them into a sterile bag.

[0062] (2) Preparation of bacterial suspension: add 90ml of sterile saline to the sterile bag, and vibrate for 30s with a pulse homogenizer (Guangdong Huankai Microbial Technology Co., Ltd.: ShockMixer-1) to obta...

Embodiment 2

[0084] Example 2 Screening test of primers for three dominant Lactococcus strains on the surface of Flammulina velutipes

[0085] (1) Strain material

[0086] Among the 22 bacterial strains (see Table 1) isolated from the surface of Flammulina velutipes in Example 1, Nos. 1 to 3 are bacteria of the genus Lactococcus, and other bacterial strains are used as specificity test controls.

[0087] (2) Test method

[0088] (1) Determine candidate genes: take Lactococcus lactis subsp. The target gene, wherein the gene sequence of rpoA is shown in SEQ ID No.14.

[0089] (2) Design primers: according to the LAMP primer design software (URL: http: / / primerexplorer.jp / e / ), the DNA to be amplified is divided into six independent regions, and the primers required for the LAMP reaction (inner primers FIP and BIP, outer primers F3 and B3, loop primers LB and LF) are designed according to these six regions, each Five sets of LAMP primers were designed for each target gene, and a total of ...

Embodiment 3

[0106] Example 3 rpo289 primer set sensitivity detection test

[0107] (1) Test materials

[0108] Bacterial strain: Lactococcus lactis subsp. lactis (Lactococcus lactis subsp. lactis) FVL2 strain isolated from the surface of Flammulina velutipes.

[0109] (2) Reagent preparation:

[0110] The kit for LAMP nucleic acid amplification was purchased from Beijing Lanpu Biotechnology Co., Ltd. The kit mainly includes: 20mM Tris-HCl (pH 8.8), 10mM KCl, 10mM (NH 4 ) 2 SO 4 , 0.1% Tween20, 0.8M Betaine, 8mM MgSO 4 , 1.4mM dNTPs and 8U Bst DNA polymerase. Fluorescent visual detection reagents for detecting LAMP reactions were purchased from Beijing Lanpu Biotechnology Co., Ltd. Primers were synthesized by Sangon Biotech Co., Ltd. Takara ExTaq Mix was purchased from Bao Biological Engineering Co., Ltd. TIANGEN bacterial genome extraction kit.

[0111] (3) Test method

[0112] (1) Genome concentration sensitivity experiment

[0113] (a) Template preparation: Lactococcus lactis...

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Abstract

The invention discloses an LAMP primer for detecting needle mushroom surface Lactococcus. The LAMP primer consists of nucleotide sequences shown by SEQ ID No. 1 to SEQ ID No. 6. The needle mushroom Lactococcus mainly refers to Lactococcus lactis subsp. hordniae, Lactococcus lactis subsp. Lactis or Lactococcus garvieae strains. The invention also discloses a detection kit containing the LAMP primer and a corresponding detection method. The LAMP primer provided by the invention has strong specificity and high sensitivity, and has the advantages of simple method, high detection speed, easy observation, and no need for expensive equipment and professionals.

Description

technical field [0001] The invention belongs to the field of preservation of edible fungi, and in particular relates to a LAMP primer for detecting Lactococcus surface of Flammulina velutipes and a method for detecting Lactococcus surface of Flammulina velutipes using the primer. Background technique [0002] Flammulina velutipes fresh products have high water content and strong metabolism, and are fresh agricultural products. Therefore, freshness preservation is very important for Flammulina velutipes. Fresh Flammulina velutipes has a sour phenomenon after opening the package, but the reason for the sourness is still unclear. Bacteria grow and reproduce by decomposing sugar, protein, lipid and other substances in food, and play a key role in the process of food spoilage (Li Xuepeng et al. Journal of Food and Fermentation Industry, 2015, 41(8):244-250), such as Pseudomonas, Erwinia, etc. decompose pectin and protein in fruits and vegetables, resulting in spoilage; Lactococc...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6844C12Q1/6895C12Q2531/119Y02A50/30
Inventor 王琼英黄晨阳陈强高巍赵梦然曲积彬
Owner INST OF AGRI RESOURCES & REGIONAL PLANNING CHINESE ACADEMY OF AGRI SCI
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