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Quick molecular detection method for simultaneously detecting three kinds of fusarium toxins and application thereof

A Fusarium and deoxynivalenol technology, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve the problem that the two derivatives of DON toxin cannot be detected, and cannot be truly Meet the problems of rapid detection and achieve the effect of low price, low cost and strong practicability

Inactive Publication Date: 2012-11-14
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Li He et al. designed a pair of primers using the sequence of the Tri5-Tri6 intergenic region, which can stably detect the NIV and DON toxins produced by Fusarium graminearum (Li et al.2005.Development of a generic PCR detection of deoxynivalenol-and nivalenol-chemotypes ofFusarium graminearum.FEMS Microbiol.Lett.243:505-511.), can be applied to the detection of wheat and corn grains, but still can not detect the two derivatives of DON toxin, and only applicable to Fusarium graminearum, for Detection of other Fusarium species that produce this toxin is not indicated
Moreover, due to the limitations of the DNA extraction method, the above methods cannot really meet the needs of rapid detection.

Method used

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  • Quick molecular detection method for simultaneously detecting three kinds of fusarium toxins and application thereof
  • Quick molecular detection method for simultaneously detecting three kinds of fusarium toxins and application thereof
  • Quick molecular detection method for simultaneously detecting three kinds of fusarium toxins and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Implementation example 1: detection and identification of toxins produced by NRRL international standard strains

[0045] 1) Rapid extraction of strain DNA

[0046] NRRL strains were provided by Dr. O'Donnell, USDA (Table 1), and their toxin types are known. To activate the NRRL strain, first pick mycelia on PDA medium (200g of peeled potatoes, 20g of glucose, 20g of agar, 1000ml of distilled water) at 28°C for 3 days, pick a small amount of mycelia with a pipette tip, and put them into PCR tube, add 50μl BufferA solution (100mM NaOH, Buffer A with 10M NaOH and Now prepared), incubate at 95°C for 10 minutes. Then add 50 μl of Buffer B solution (100 mM Tris-HCl, 2 mM EDTA), vortex and mix, centrifuge at 12000 rpm for 15 seconds, take 1 μl of the supernatant as a template for PCR amplification, and directly add it to the PCR reaction system.

[0047] Table 1 Fusarium strains of toxin production in different countries and regions adopted by the present invention

[...

Embodiment 2

[0063] Implementation Example 2: Detection and identification of toxins produced by strains collected in China.

[0064] The strains were collected from 8 provinces in China (Table 2), and the type of toxin was determined by GC / MS method. The rapid extraction of bacterial strain DNA, the synthesis of specific primers, the PCR amplification reaction, the PCR amplification procedure and product identification are the same as in Example 1.

[0065] Table 2 The Chinese collection Fusarium bacterial strain that the present invention adopts

[0066]

[0067] Implementation results

[0068] Utilize the compound primer of the present invention to use the hyphae of the bacterial strain collected in China as a template for PCR amplification. The result is as image 3, 643bp of the NIV toxin-specific band was amplified in lanes 1-3; 424bp of the 15ADON toxin-specific band was amplified in lanes 4-8; It is consistent with the determination method (Table 1).

Embodiment 3

[0069] Implementation example 3: Toxin detection of wheat scab grains inoculated in the field

[0070] 1) Preparation of conidia suspension

[0071] The purified and identified strains 13033 (NIV), 7076 (15ADON) and 13082 (3ADON) were inoculated in sterilized mung bean soup medium, cultured at 28° C. with shaking at 150 rpm for 4 days. Count with a hemocytometer and adjust the spore concentration to 5 x 10 5 pieces / ml or so.

[0072] 2) Field flowering inoculation

[0073] The wheat at the flowering stage was divided into 5 groups, a little of the top of the spikelets in the middle of the wheat was cut off, and 20 μl of the conidia suspension were injected with a micro-syringe. The first group was injected with sterile water as a control, the second group was injected with strain 13033, the third group was injected with 7076, the fourth group was injected with 13082, and the fifth group was injected with spore suspension mixed with equal amounts of the three strains. Bag t...

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Abstract

The invention discloses a quick molecular detection method for simultaneously detecting three kinds of fusarium toxins and application thereof, is special for detecting three kinds of common fusarium toxins, and belongs to the technical field of plant quarantine, in particular to the technical field of molecular detection of the fusarium toxins. A plurality of composite primers are designed by the invention according to the closely related Trill gene which is synthesized by the toxins in the fusarium genome; the specific segments of the fusarium toxins nivalenol (NIV), 15 acetyldeoxynivalenol(ADON) and 3ADON are obtained from the fusarium collected in China and foreign countries through detection and separation; and the overall lengths of the specific segments of the NIV, 15ADON and 3ADON are 643bp, 424bp, and 342bp respectively. Only one group of specificity primers are used for performing polymerase chain reaction (PCR) amplification reaction, the PCR product is detected by 1.5 percent agarose gel electrophoresis, and the types of the three kinds of B type toxins produced by fusarium graminearum schw can be directly detected according to the length of the product segments; and the method can be applied to the detection of the deoxynivalenol (DON) and NIV toxin pollution in grain, feed and food safety.

Description

(1) Technical field [0001] The invention "a rapid molecular detection method and application for simultaneously detecting three types of Fusarium toxins" is specially used for detecting three types of Fusarium toxins, belongs to the technical field of plant quarantine, and is related to molecular detection technology. (2) Background technology [0002] Fusarium head blight (or scab) caused by Fusarium is an important disease that harms wheat, barley, oats, corn, rice, rye and other cereal crops, and is widely distributed in warm and humid regions of the world. In the 1890s, barley and wheat scab broke out in North America, Europe, Asia and other places; in recent years, wheat scab has become more and more serious in my country, and it has spread to almost all wheat-growing areas in the country, causing serious Yield loss and substantial drop in quality. In addition, the more important thing is that the toxin produced by Fusarium can poison humans and animals, seriously threa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 张昊冯洁徐进许景升潘哲超张力勍田茜
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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