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LAMP (loop-mediated isothermal amplification) universal primer for detecting aspergillus toxin-producing fungi and kit containing primer

A universal primer, Aspergillus technology, applied in the direction of microbe-based methods, microbiological determination/testing, microbiology, etc., can solve problems such as false negative results, poor timeliness, and false positives

Inactive Publication Date: 2016-06-08
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 (loop-mediated isothermal amplification) universal primer for detecting aspergillus toxin-producing fungi and kit containing primer
  • LAMP (loop-mediated isothermal amplification) universal primer for detecting aspergillus toxin-producing fungi and kit containing primer
  • LAMP (loop-mediated isothermal amplification) universal primer for detecting aspergillus toxin-producing fungi and kit containing primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Design and synthesis of LAMP universal primers for detecting Aspergillus toxin-producing fungi

[0037] The ATP citrate lyase genes shared by 5 species of Aspergillus toxigenic fungi were selected, and the consensus sequence region was selected to design primers based on the NCBI alignment results. Use the primer design support software PrimerExplorer of LAMP method to design the primers. The online design website is: http: / / primerexplorer.jp / e / . Select a set of primers that meet the requirements of LAMP primer design through screening. The primers include 2 outer primers (F3, B3), 2 inner primers (FIP, BIP) and 2 loop primers (LF and LB), synthesized by Shenggong Bioengineering (Shanghai) Co., Ltd. The ATP citrate lyase gene can be used as a universal gene to identify toxigenic fungi of the genus Aspergillus.

Embodiment 2

[0038] Example 2 Preparation of genomic DNA template of Aspergillus toxigenic fungi

[0039] A modified CTAB method was used to extract genomic DNA. The standard strains of Aspergillus flavus (40536), Aspergillus parasitica (40365), Aspergillus slip (30585), Aspergillus fumigatus (3.5835) and Aspergillus versicolor (2474) were provided by Liaoning Entry-Exit Inspection and Quarantine Bureau. The detailed steps are as follows: (1) Take a certain amount of mycelium in a mortar, grind it into powder with liquid nitrogen, and quickly transfer it to a 1.5mL centrifuge tube; (2) Add 600μL 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 cap of the tube, shake it thoroughly, bath at 65°C for 30 minutes, keep shaking during the period; (3 ) After cooling at room temperature, add an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1), invert and mix for 1 min, centrifuge at 13000r / min...

Embodiment 3

[0040] Example 3 Using LAMP universal primers to detect Aspergillus toxigenic fungi

[0041] Using Aspergillus flavus, Aspergillus parasiticus, Aspergillus slip, Aspergillus fumigatus, and Aspergillus versicolor as samples, the amplification was carried out according to the LAMP reaction system to verify the time and temperature of the LAMP universal primers to detect Aspergillus toxin-producing fungi. Analyze the peak time of LA-320C program, such as figure 1 Shown. From figure 1 It can be seen that the LAMP universal primers all start to undergo amplification reactions within 10-11 minutes, and the reaction temperature is appropriate.

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Abstract

The invention relates to an LAMP (loop-mediated isothermal amplification) universal primer for detecting aspergillus toxin-producing fungi and a kit containing the primer. The LAMP primer includes inner primers FIP and BIP, outer primers F3 and B3 and loop primers LF and LB; the kit consists of the LAMP primer, Bst DNA polymerase, a reaction buffer containing dNTPs and de-ionized water. According to the LAMP primer disclosed by the invention, by applying the loop-mediated isothermal amplification technology to the rapid detection of the aspergillus toxin-producing fungi, the aspergillus toxin-producing fungi can be rapidly, accurately and conveniently detected; therefore, the LAMP primer is of great significance to the prevention and detection of contamination caused by the aspergillus toxin-producing fungi. The detection method is strong in specificity and relatively high in sensitivity, short in reaction time and strong in operability, a detection lower limit can reach 1*10<-12>g / [mu]L or below and the detection method can be achieved without an expensive instrument; in addition, the detection of a finished product is convenient and the detection can be achieved by virtue of a naked eye observation method; therefore, the detection method is quite high in practical value.

Description

Technical field [0001] The invention relates to a LAMP universal primer for detecting toxin-producing fungi of the genus Aspergillus and a kit containing the primer, and also relates to a method for rapidly detecting the toxin-producing fungus of the genus Aspergillus using the kit containing the primer. It belongs to the technical field of food safety testing and microbial testing. Background technique [0002] Toxigenic fungi of the genus Aspergillus are common saprophytic fungi, which are more common on moldy grains, food products and other moldy organic matter. The colony grows faster, the structure is loose, and the bacteria are composed of many complex branched hyphae. Aspergillus is a genus in the order Arbuscularis and Arbuscularaceae. The main mycotoxin-producing strains are: Aspergillus flavus (A.flavus), Aspergillus parasiticus (A.parasiticus), Aspergillus versicolor (A.versicolor) , Aspergillus fumigatus (A.fumigatus), Aspergillus ochraceus (Aspergillus ochraceus), A...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/66
Inventor 黄耀江蒋丹靳卫林孙雷闫强
Owner 黄耀江
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