Primer combination and kit for detecting mould in nuts and method for detecting types of mycotoxin-producing fungi in nuts
A combination of primers and mycotoxin technology, which is applied in the field of food safety biology, can solve the problems of false positives, poor detection result accuracy, and difficulty in distinguishing microbial types, etc., and achieve the effect of simple and fast operation and simple and fast detection
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Embodiment 1
[0057] Example 1 Design and verification of primers
[0058] (1) Specific primer design:
[0059] Bena (β-tube protein) region of the uchiline genome, AMY-1 (alpha-amylase) region of the pyrogenic genome, AMY-1 (alpha-amylase) region, Penicilli, a pyrochiosis (β-microtubule protein) and the RPB2 region of the Sickle genome is a conservative sequence, and the bacteria species, Oligo7 and Primer5 design primers, synthesis, Table 1 To design primer sequences and parameters:
[0060] Table 1 Nutsprint primer sequence
[0061]
[0062] (2) Extraction of the above-mentioned reagent strain genome DNA, the specific method is as follows:
[0063] Take the mycerene in the liquid medium, germ strain in liquefaction nitrogen; taking no more than 50 mg of fried fungi tissue, adding 1.5 ml of trace centrifugal tube; plus 400 μL Le Buffer and 4 μL 20 mg / mlRNase A, mixed uniform; at 65 ° C in an environment for 30 min, the centrifuge is vortexed every 10 min; adding 130 μl of DA buffer, and t...
Embodiment 2
[0070] Example 2 Specific test of each primer pair
[0071] (1) Extraction of DNA of Spirophosporospore genomic DNA
[0072] As described in Example 1 (2), the Mercu Mercusher, Huangchi, Colillter, Color Popular Millia, and Spiral Fusariosis. The resulting black craniosis genome concentration was 7.2 ng / μL, and A260 / A280 was 2.08; yellowula concentration was 7.2 ng / μL, and A260 / A280 was 2.03; the concentration of the pypermper genome was 6.9 ng / μL, A260 / A280 was 1.91; fresh The green alic genome concentration was 3.3 ng / μL, and A260 / A280 was 1.70; the spy psorospore genome concentration was 14 ng / μL, and A260 / A280 was 1.84.
[0073] (2) Establishment of the PCR system
[0074] The PCR reaction system is: 5 μL 10 × contains Mg 2+ PCR buffer, 4 μl of DNTP, 0.4 μl Taq DNA polymerase, upper and downstream primers 5 μl of DNA template, add sterile ultrapure water to 50 μl.
[0075] The amplification procedure was: 94 ° C Demographs were 5 min after entering the cycl...
Embodiment 3
[0081] Example 3 Natural pollution sample detection
[0082] (1) Sample processing:
[0083] Sample from Longgang Industrial Park, Lin'an City, Hangzhou, taking 25G sample nuts, and then cracked 225 ml of sterilized water, oscillated 10 min, and added 200 μl of oscillated solution to 100 ml of potato liquid medium, 28 ° C culture 40 h.
[0084] (2) Sample DNA extraction
[0085] Take the bacteria in the medium in the culture medium, and the sample genomic DNA is taken according to the method of the first step (2).
[0086] (3) Nut sample PCR system establishment
[0087] PCR reaction system: 5μL 10 × contains Mg 2+ PCR buffer, 4 μl of DNTP, 0.4 μl Taq DNA polymerase, upstream primer and downstream primer each 1 μl, step (2) extracted 5 μl, 33.6 μL of sterile ultrapure water.
[0088] The amplification program was: 94 ° C and then entered the cycle, 94 ° C 1 min, 57 ° C 45 s, 72 ° C 45s, a total of 30 cycles.
[0089] The PCR amplification was performed using the five groups of pri...
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