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LAMP primer for detecting Asperillus Parasiticus, and kit including primer

A technology of Aspergillus parasiticus and kit, which is applied to LAMP primers and kits containing the primers, can rapidly detect Aspergillus parasiticus, and can solve problems such as poor timeliness, false positives, and long identification period.

Inactive Publication Date: 2016-03-02
黄耀江 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the detection of toxin-producing fungi in my country is based on the classification and identification of bacterial morphology and characteristics under the microscope. However, this method has a long identification cycle and poor timeliness. It requires experienced experts or experimenters to accurately distinguish, and is prone to false positives. Or false negative results, which are far from meeting the rapid development of food and feed import and export testing needs

Method used

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  • LAMP primer for detecting Asperillus Parasiticus, and kit including primer
  • LAMP primer for detecting Asperillus Parasiticus, and kit including primer
  • LAMP primer for detecting Asperillus Parasiticus, and kit including primer

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Effect test

Embodiment 1

[0035] Example 1 is used to detect the design and synthesis of Aspergillus parasitica LAMP primers

[0036] The histidine kinase mRNA gene (AY430047.1) of Aspergillus parasitica was selected, and the specific sequence region was selected to design primers according to the NCBI comparison results. Primers were designed using PrimerExplorer, a primer design support software for the LAMP method, and the online design website is: http: / / primerexplorer.jp / e / . A set of primers that meet the design requirements of LAMP primers was selected by screening. The primers include 2 outer primers (F3, B3), 2 inner primers (FIP, BIP) and 2 loop primers (LF and LB), synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The histidine kinase mRNA gene can be used as a specific gene for specific identification of Aspergillus parasitica.

Embodiment 2

[0037] The preparation of embodiment 2 Aspergillus parasitica genome DNA template

[0038] Genomic DNA of Aspergillus parasitica was extracted using a modified CTAB method. The standard strain of Aspergillus parasitica (40365) was provided by Liaoning Entry-Exit Inspection and Quarantine Bureau. The detailed steps are: (1) Take a certain amount of mycelium in a mortar, grind it into powder with liquid nitrogen and transfer it to a 1.5mL centrifuge tube; (2) Add 600 μL of preheated CTAB solution (2% CTAB, 200mmol / LTris-HCl, 20mmol / LEDTApH=8.0, 1.4mmol / LNaCl, 1% PVP-40, 0.2% β-mercaptoethanol), quickly close the tube cap, fully shake, 65 ° C water bath for 30min, during which the shake is constantly; (3 ) After cooling at room temperature, add an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1), repeatedly invert and mix for 1 min, and centrifuge at 13000r / min for 10 min; (4) draw the supernatant into another centrifuge tube, add volume of chloroform-isoamyl alcoho...

Embodiment 3

[0039] Embodiment 3 utilizes LAMP primer to detect the specificity analysis of Aspergillus parasitica

[0040] Aspergillus flavus, Aspergillus glider, Aspergillus fumigatus, Aspergillus versicolor within the genus Aspergillus and Fusarium graminearum, Fusarium solani, Penicillium insulae, Penicillium aeruginosa, Staphylococcus aureus and Escherichia coli Bacillus, etc. were used as samples for specificity testing, and amplification was performed according to the LAMP reaction system to verify the specificity of the Aspergillus parasitica primer combination. Analyze the peak time and peak value of the LA-320C program, such as figure 1 and figure 2 shown. From figure 1 and figure 2 It can be seen that the primers only positively reacted with Aspergillus parasitica, and with Aspergillus flavus, Aspergillus spp., Aspergillus fumigatus, Aspergillus versicolor within the genus; ; and Staphylococcus aureus and Escherichia coli have no amplification reaction, which proves that ...

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Abstract

The invention relates to an LAMP primer for detecting Asperillus Parasiticus, and a kit including the primer. The LAMP primer comprises inner primers FIP and BIP, outer primers F3 and B3, and loop primers LF and LB; and the kit comprises the LAMP primer, a Bst DNA polymerase, a reaction buffer solution containing dNTPs, and deionized water. A loop-mediated isothermal amplification technology is used to rapidly detect Asperillus Parasiticus in the invention, so the Asperillus Parasiticus can be rapidly, accurately and conveniently detected, and the LAMP primer and the kit are of great significance to prevent and detect Asperillus Parasiticus pollution. A detection method also disclosed in the invention has the advantages of strong specificity, high sensitivity, lower detection limit reaching 1*10<-12>g / [mu]L, short reaction time, strong operability, no need of expensive apparatuses, convenient product detection, realization through macroscopic observation, and very high practical values.

Description

technical field [0001] The invention relates to a LAMP primer for detecting Aspergillus parasitica and a kit containing the primer, and also relates to a method for rapidly detecting Aspergillus parasitica using the kit containing the primer. The invention belongs to the technical field of food safety detection and microbial detection. Background technique [0002] Aspergillus parasitica is a common saprophytic fungus in the Aspergillus genus. It is more common in moldy grain, grain products and other moldy organic matter. Aflatoxins are toxic secondary metabolites produced by fungi, mainly produced by the metabolism of Aspergillus flavus, Aspergillus parasiticus and Aspergillus luteus, especially Aspergillus flavus and Aspergillus parasitica have the most research reports. [0003] Aflatoxin was discovered in 1961, when hundreds of thousands of turkeys died suddenly in the UK. It was finally determined that this toxic substance was a secondary metabolite produced by Asper...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 黄耀江靳卫林蒋丹孙雷闫强
Owner 黄耀江
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