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Method for detecting fumonisin toxigenic strain in asparagus using composite PCR technique

A detection method and technical detection technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of not directly revealing the toxicity of the strains to be tested, and cannot fully guarantee the reliability of the results, so as to meet the requirements of plant quarantine And food safety testing, strong practicability and simple process

Inactive Publication Date: 2009-04-29
ZHEJIANG UNIV
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  • Abstract
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  • Claims
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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 sequence of the FUM1 gene of the known toxin-producing strain F. verticillioides, but the synthesis of fumonin is a relatively complicated process. Participation and regulation, a single test for the FUM1 gene does not fully guarantee the reliability of the results

Method used

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  • Method for detecting fumonisin toxigenic strain in asparagus using composite PCR technique
  • Method for detecting fumonisin toxigenic strain in asparagus using composite PCR technique
  • Method for detecting fumonisin toxigenic strain in asparagus using composite PCR technique

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Isolation of strains in asparagus in Zhejiang area to detect fumonin-producing strains

[0043] The asparagus materials used in this experiment are edible stems, collected from various planting bases in Zhejiang Province, a total of 16 parts, including 5 parts in Ningbo, 5 parts in Fuyang, and 6 parts in Xinchang.

[0044] Step 1: Isolation and purification of strains

[0045] Wash the asparagus samples and cut them into 2cm long sections, disinfect them with 70% alcohol for 3 minutes, 10% sodium hypochlorite for 7 minutes, and then rinse them with sterile water for 3 times. Under sterile conditions, spread the samples on PDA (potato-dextrose-agar) medium and seal with parafilm. Placed in an incubator at 25°C in the dark.

[0046] Count the colonies of each sample on the PDA medium, observe the growth of the fungi, observe the characteristics of the fungi under a microscope after 4-5 days, separate out different fungi, pick them out from the colonies, and in...

Embodiment 2

[0081] Example 2: Sensitivity and minimum detection limit of primers designed in the present invention to detect fumonisin-producing strains

[0082] Step 1: Primer Synthesis

[0083] The operation steps are the same as the third step of Example 1.

[0084] Step 2: Template DNA Dilution

[0085] Dilute Fusarium moniliforme wild-type chromosomal DNA concentration with sterile water to 10ng, 1ng, 100pg, 10pg, 1pg,

[0086] Step 3: Multiplex PCR amplification

[0087] The template DNA diluted in the second step was directly added to the PCR reaction system in the fourth step of Example 1 for multiplex PCR amplification.

[0088] Step 4: Judgment of test results

[0089] Same as the fifth step in Example 1.

[0090] Implementation results:

[0091] Measured primer pair rp32 / rp33, the detection limit of rp679 / rp680 is 10pg ( figure 2 ), the water control result without adding DNA was negative, and the detection limit of composite PCR was 100pg ( image 3 ).

Embodiment 3

[0092] Example 3: Detection of virulence of isolated bacterial strains under in vitro conditions

[0093] The strain materials used in this experiment were all the strains isolated from asparagus samples collected from various places in previous experiments.

[0094] Step 1: Preparation of strain spore suspension

[0095] Take 15ml of GYP liquid medium (glucose-peptone-yeast extract), add it to a 50ml Erlenmeyer flask, and use an inoculation loop to pick out a small amount from the preserved wild-type Fusarium moniliforme (F.verticillioides) or the test tube of the test strain The bacteria were put into a conical flask, placed in a constant temperature shaking incubator at 200rpm / min, 27°C, and cultivated for 40-48h. Take 2ml of culture solution and add it to a 2ml centrifuge tube, centrifuge at 12000rpm / min for 10min, discard the upper layer carefully, add 1ml of sterile water, centrifuge at 12000rpm / min for 10min, discard the supernatant, then add 1ml of sterile water, 1200...

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Abstract

The invention relates to a method for detecting fumonisin toxicogenic strains in asparaguses by the composite PCR technology, which uses primer pairs rp32 / rp33 and primer pairs rp679 / rp680 which are designed according to polyketide synthetase genes FUM1 and serine-hexadecanoyl transferase genes FUM8 required for biosynthesis of fumonisin, and fusarium specific primers IstF / IstR respectively. The method simultaneously detects the fumonisin toxicogenic strains in the same reaction system, and not only can detect unknown fumonisin toxicogenic strains but also can identify whether the detected strains belong to fusarium. Moreover, a plurality of pairs of primers are subjected to amplification in the same reaction system simultaneously by the composite PCR technology, so that the method improves the detection accuracy, saves the detection time, is a simple, quick and effective composite PCR detection method for the fumonisin toxicogenic strains in the asparaguses, and can be massively promoted by means of a kit.

Description

technical field [0001] The "method for detecting fumonisin toxin-producing strains in asparagus by using composite PCR technology" of the invention belongs to the technical field of crop disease prevention and plant quarantine. Background technique [0002] Asparagus is a high-grade vegetable that can be used as both food and medicine. It not only tastes delicious, but also has the best anti-cancer and anti-cancer effects found and recognized in the world today. It is deeply loved by consumers at home and abroad. my country is the largest asparagus producer and exporter in the world. The main asparagus growing areas are located in Shandong, Jiangsu, Zhejiang, Shanxi, Fujian, Hebei and other provinces. Most of the asparagus produced are for export. [0003] Fumonisin is a group of mycotoxins mainly produced by Fusarium fungi such as Fusarium verticillioides and Fusarium proliferatum. It has the characteristics of wide distribution and strong toxicity. It has become another n...

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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