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Corn and composite PCR detecting method of fumonisin toxigenic strain in corn products

A detection method and hummus technology are applied in the field of crop disease prevention and phytosanitary, which can solve the problems of not directly revealing the toxigenicity of the strain to be tested, and not completely guaranteeing the reliability of the results, saving detection time, improving accuracy, The effect of high detection accuracy

Inactive Publication Date: 2009-04-29
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the taxonomic genes are designed based on the homologous sequences of each species in the genus Fusarium, it directly reveals the type of the strain, but cannot directly reveal the toxicity of the strain to be tested, and it is likely to exclude unknown fumonisin-producing strains outer
At present, the primers for amplifying genes directly related to the mechanism of fumonin production are designed based on the FUM1 gene sequence of the known toxin-producing strain F. verticillioides. The synthesis of fumonin is a relatively complicated process, which is regulated by various genes. A single test for the FUM1 gene does not fully guarantee the reliability of the results

Method used

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  • Corn and composite PCR detecting method of fumonisin toxigenic strain in corn products
  • Corn and composite PCR detecting method of fumonisin toxigenic strain in corn products
  • Corn and composite PCR detecting method of fumonisin toxigenic strain in corn products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Isolation of bacterial strains on unprocessed corn kernels and detection of fumonisin toxin-producing strains

[0039] The unprocessed corn cobs and kernels used in this experiment were collected from all over the country, a total of 44 samples. Among them, 1 in North China (3 in Inner Mongolia, 1 in Hebei, 2 in Shanxi), 7 in Northwest China (Shaanxi), 1 in Northeast China (4 in Heilongjiang, 1 in Liaoning, 2 in Jilin), 15 in North and Central China (9 in Shandong, 6 in Henan), and 9 in Jiangnan (2 in Jiangsu, 2 in Zhejiang, and 5 in Shanghai).

[0040] Step 1: Primer Synthesis

[0041] The designed primer sequences of the present invention (see Table 1)

[0042] Table 1 PCR primers designed in the present invention

[0043]

[0044] ** Bluhm, 2002.

[0045] Submit to Shanghai Sangon Bioengineering Technology Service Company for synthesis. 1 OD U260 per tube, stock solution concentration 25 μmol / L, prepared with sterile double distilled water. Store ...

Embodiment 2

[0078] Example 2 Direct detection of fumonisin-producing strains on unprocessed corn kernels

[0079] The materials used in this experiment are the same as in Example 1.

[0080] Step 1: Primer Synthesis

[0081] The operation steps are the same as the first step of embodiment 1.

[0082] Step 2: Template DNA Extraction

[0083] Grind the corn kernels into powder, and put about 0.2g into a 50ml test tube. Add 4ml of DNA extraction solution and vortex to mix well. Add 2ml of phenol and 1.5ml of chloroform, vortex, and centrifuge at 2500g for 15min. Transfer the supernatant to 2ml centrifuge tubes, add 900 μl of supernatant and 900 μl of chloroform to each tube, and centrifuge at 13200 rpm for 5 minutes. Transfer the upper layer solution to a new 2ml test tube, and add RNase I (final concentration C≥100μg / ml). Then incubate at 37°C for 2-3h. Add an equal volume of cold 5M lithium chloride solution, shake gently to mix well, and place on ice for another 15 min. Centrifuge...

Embodiment 3

[0089] Example 3 Detection of processed corn products

[0090] The materials used in this experiment are corn grits, corn flour, corn cakes, quick-frozen corn cobs, etc., a total of 14 parts.

[0091] Step 1: Primer Synthesis

[0092] The operation steps are the same as the first step of embodiment 1.

[0093] Step 2: Template DNA Extraction

[0094] After the sample was ground, the template DNA was extracted according to the operation steps of the second step of Example 2.

[0095] Step 3: Multiplex PCR amplification

[0096] With the fourth step of embodiment 1.

[0097] Step 4: Judgment of test results

[0098] Same as the fifth step in Example 1.

[0099] Implementation results:

[0100] No fumonisin-producing bacteria or other strains were detected on the corn products collected in the experiment. Perhaps it is related to the source of these products. Natural products such as corn cobs and corn kernels are mostly collected from rural farmers. The storage condition...

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Abstract

The invention relates to a composite PCR detection method for fumonisin toxicogenic strains in corn and corn products, wherein a plurality of pairs of primers are designed by application of DNAman sequence analysis software respectively on the basis of polyketide synthetase genes FUM1, serine-hexadecanoyl transferase genes FUM8 and longevity guarantee factor genes FUM17 required for biosynthesis of fumonisin, and primer matched groups with optimized specificity are obtained through experimental comparison of the primers and fusarium specific primers IstF / IstR; the fumonisin toxicogenic strains are simultaneously detected in the same reaction system; and the plurality of pairs of primers are subjected to amplification simultaneously in the same reaction system by the composite PCR technology. The composite PCR detection method comprises the following detection steps: template DNA is directly extracted from a sample to be detected by the improved SDS method; and the template DNA is subjected to composite PCR reaction, electrophoresis, dyeing and rinsing, a gel imaging device is used for observing the result and taking a picture, and finally the detection result is obtained after spectrogram analysis. The composite PCR detection method improves the detection accuracy, saves the detection time and can be massively promoted by means of a reagent kit.

Description

technical field [0001] The "complex PCR detection method for fumonisin-producing strains in corn and its products" of the invention belongs to the technical field of crop disease prevention and plant quarantine. Background technique [0002] Fumonisins are a group of mycotoxins mainly produced by fungi of the genus Fusarium, such as Fusarium verticillioides (Fusarium moniliforme) and Fusarium proliferatum. It not only has acute toxicity and potential carcinogenicity to some livestock, such as causing equine encephalitis, pig pulmonary edema or liver cancer in rats, but also can cause esophageal cancer and neural tube defects in humans, seriously threatening food safety and human and animal health. healthy. Due to its wide distribution and strong toxicity, fumonisin has become another new hotspot of mycotoxin research after aflatoxin. In recent years, elevated levels of fumarin in corn have attracted the attention not only of corn growers, but also of livestock farmers, agr...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
Inventor 汪俏梅魏佳王建升周莹杜良成
Owner ZHEJIANG UNIV
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