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Aspergillus flavus LAMP (loop-mediated isothermal amplification) detection primer and visualized detection method thereof

A technology of Aspergillus flavus and detection method, applied in biochemical equipment and methods, microorganism-based methods, microorganisms, etc., can solve the problems of long cycle, poor specificity and low sensitivity of detection methods, and achieve reliable results and strong specificity. , the effect of high sensitivity

Inactive Publication Date: 2015-05-20
INST OF PLANT PROTECTION FAAS
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to provide a kind of LAMP detection primer of Aspergillus flavus and reliable result, easy operation, Visual detection method of Aspergillus flavus with strong specificity and high sensitivity

Method used

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  • Aspergillus flavus LAMP (loop-mediated isothermal amplification) detection primer and visualized detection method thereof
  • Aspergillus flavus LAMP (loop-mediated isothermal amplification) detection primer and visualized detection method thereof

Examples

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Effect test

Embodiment 1

[0024] Example 1: Specific amplification of LAMP primers to Aspergillus flavus

[0025] 1. Design of LAMP primers

[0026] According to the Aspergillus flavus ribosomal transcriptional spacer (ITS) sequence, PrimerExplorer V4 software was used to design a LAMP detection primer, including 1 outer primer (F3 and B3) and 1 pair of inner primers (FIP and BIP). The primer sequences were:

[0027] F3: 5'-GTGAATTGCAGAATTCCGTGAA-3'

[0028] B3: 5'-CCTACAGAGCGGGTGACAA-3'

[0029] FIP: 5'-ATGACGCTCGGACAGGCATG-ATCGAGTCTTTGAACGCACA-3'

[0030] BIP: 5'-TTGGGTCGTCGTCCCTCTCTC-CCCCATACGCTCGAGGAT-3'

[0031] 2. Genomic DNA extraction

[0032] Genomic DNA of 5 species of Aspergillus including Aspergillus flavus and 17 species of different fungi and bacteria were extracted by CTAB method.

[0033]The specific method is as follows: take 50 mg of freeze-dried mycelium powder in a 1.5 ml centrifuge tube, add 900 μl of 2% CTAB (cetyltrimethylammonium bromide) extract (the formula of the e...

Embodiment 2

[0040] Embodiment 2: Sensitivity detection of LAMP primers to Aspergillus flavus

[0041] 1. LAMP Sensitivity Detection of Aspergillus flavus

[0042] The extracted Aspergillus flavus DNA was diluted into 7 different concentration gradients of 1ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg by 10-fold concentration serial dilution method.

[0043] ①LAMP reaction system 25 μL: including 0.25 μL each of outer primers F3 (5 μM) and B3 (5 μM), 0.25 μL each of inner primers FIP (40 μM) and BIP (40 μM), 12.5 μL reaction mixture [40 mM Tris-HCL, 20 mM ( NH 4 ) 2 SO 4 , 20 mM KCl, 16 mM MgSO 4 , 0.2% Triton X-100, 1.6M Betaine, 2.8 mM dNTPs], 1 μL 50 μM Calcein-500 μM MnCl 2 , 1 μL (8U) Bst DNA polymerase, 25ng DNA template, make up to 25 μL with sterilized double distilled water. The LAMP reaction conditions were incubation at 65°C for 60 min and incubation at 82°C for 10 min.

[0044] ② Add 1 μL of chromogen to the LAMP reaction solution, the chromogen is 50 μM Calcein-50...

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Abstract

The invention discloses an aspergillus flavus LAMP (loop-mediated isothermal amplification) detection primer and a visualized detection method of the aspergillus flavus LAMP detection primer, which are specially used for specific detection of aspergillus flavus. The invention mainly designs one aspergillus flavus LAMP detection primer (comprising a pair of outer side primers and one pair of inner side primers); green fluorescence or a trapezoidal zone with an LAMP characteristic can be observed by carrying out isothermal amplification and adding a 50micronM Calcein-500micronM MnCl2 developing agent to develop or carrying out agarose gel electrophoresis detection. The aspergillus flavus LAMP detection primer and the visualized detection method of the aspergillus flavus LAMP detection primer can be used for visualized detection of aspergillus flavus in crops including peanuts, corns and the like which are infected by the aspergillus flavus in production practices, and can also be used for early diagnosis, monitoring and identification of bacteria in tissues of the peanuts or the corns which are naturally attacked.

Description

technical field [0001] The invention relates to a LAMP detection primer for Aspergillus flavus and a visual detection method thereof, which is specially used for rapid visual detection of Aspergillus flavus, and can be used for early diagnosis and monitoring and identification of pathogens in naturally occurring peanut or corn tissues, and belongs to the detection of harmful organisms , Identification field. Background technique [0002] Aflatoxin is a fungal secondary metabolite produced during the growth and reproduction of Aspergillus flavus and Aspergillus parasiticus. It is the most serious environmental pollution and the most harmful toxin to humans and animals among all mycotoxins. It has carcinogenic and teratogenic properties. , mutagenic effect, mainly infecting peanuts, corn, soybeans, wheat, nuts and other crops and their products, most of the food and feeds infected by them lose their nutritional value and economic value. Therefore, in order to protect the natio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/67
Inventor 陈庆河李本金尹容美刘裴清翁启勇
Owner INST OF PLANT PROTECTION FAAS
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