Primer for detecting trichothecene group B-class toxin of fusaria through PCR and application of primer

Abstract

The invention discloses a set of primer for detecting trichothecene group B-class toxin of fusaria through PCR (polymerase chain reaction). The primer comprises a share sense primer F shown in SEQID NO.1, 3-acetyl deoxygenation nivalenol reverse primer R1 shown in SEQID NO.2, 15-acetyl deoxygenation nivalenol reverse primer R2 shown in SEQID NO.3 and nivalenol reverse primer R3 shown in SEQID NO.4; the set of the primers can be applied to the PCR for directly detecting 3-acetyl deoxygenation nivalenol, 15-acetyl deoxygenation nivalenol and nivalenol. The primer is low in cost, simple in operation, short in consumption time, accurate in testing result and easy for large-scale popularization and application.

Description

technical field [0001] The invention belongs to the field of food safety detection, in particular to PCR detection primers for fusarium trichothecene family B toxoids and applications thereof. Background technique [0002] Fusarium head blight (or scab) caused by Fusarium is an important epidemic disease worldwide, which can harm many cereal crops including barley, wheat, corn, rice and so on. With global climate warming and abnormal regional climate and changes in farming systems, the frequency of disease occurrence has increased and the area of ​​occurrence has continued to expand. Fusarium scab not only leads to serious loss of grain yield, but also Fusarium can produce a variety of mycotoxins, reduce the quality of food and feed, seriously threaten the health of humans and animals, and bring major hidden dangers to food safety. [0003] Trichothecenes are the most important mycotoxins produced by Fusarium, which are mainly divided into two types: A and B. It can inhibi...

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Problems solved by technology

[0004] At present, the detection methods of mycotoxin content usually include biological detection methods, physical and chemical detection methods, immunochemical detection methods, etc. Due to the lack of uniform standardized standards and inaccurate detection and analysis of biological detection methods, they have not been used in the actual detection of fusarium toxins. It can be widely used; the chemical determination method is currently the main method used for the determination of Fusarium toxin content, which is characterized by accuracy and good repeatability, but the sample purification required by this method is cumbersome and requires high technical content, which cannot meet the requirements of rapid detection. Requirements; immunoassay, simple operation, low cost, but because the detected toxin often contains some other derivatives, so the result of the determination is often inaccurate, prone to false positives; these three detection methods are not suitable for rapid, Sensitive and Accurate Detection of Class B Trichothecenes Contamination

[0005] In recent years, with the publication of a variety of Fusarium genomes and the rapid development of molecular biology research, researchers at home and abroad have made important achievements in the synthesis of trichothecene toxins and the regulation of toxin synthesis gene expression, confirming that The important role of some genes in the pathway of toxin metabolism has been discovered. Based on these research results and the sequence information of related genes, establishing a simple, rapid and inexpensive toxin detection method has become a hot spot for researchers from different countries. Kim et al. used Tri5, Tri7 and Tri13 gene sequences, and detected the production of Fusarium graminearum DON and NIV toxins from multiple hosts (Kim et al.Polymorphism of trichothecene biosynthesis genes in deoxynivalenol-and nivalenol-producing Fusarium graminearum isolates.Mycol Res.2003 ,107:190-197), but the Tri5 identification method is cumbersome and expensive, and it is difficult to be widely used in practice; Chandler et al. used the gene sequences of Tri7 and Tri13 to design 10 pairs of primers to detect three kinds of Fusarium Toxins are produced, but there are inconsistent detection results between multiple pairs of primers (Chandler et al. Development of PCR assays to Tri7 and Tri13 trichothecene biosynthetic genes, and characterization of chemotypes of Fusarium graminearum, Fusarium culmorum and Fusarium cerealis. .Mol.Plant Pathol.2003,62:355-367); Li et al. (2005) designed primers to distinguish DON and NIV chemotypes by analyzing the sequence of the intergenic region of Fusarium graminearum Tri5~Tri6. The detection of bacterial strains proves that the stability of the primers is very good, and it is suitable for large-scale detection of Fusarium graminearum chemotypes, but this technique cannot distinguish between two derivatives of DON toxin (Li et al.Development of a generic PCR detection of deoxynivalenol-and nivalenol-chemotypes of Fusarium graminearum.FEMS Microbiol.Lett.2005,243:505-511); Wang et al. compared the Tri13 gene sequence, Specific primers were designed to simultaneously distinguish strains that synthesize NIV, 3ADON and 15ADON (Wang et al. .Mol.Sci.2008,9:2495-2504), but the latest research shows that the pair of primers also has the problem of inconsistent detection results (Wang et al.A multiplex PCR assay for genetic chemotyping of toxic Fusarium graminearum and wheat grains for 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol and nivalenol mycotoxin.J.Food Agric.Environ.2012,10:505-511); due to some of the above defects, the practical application of these methods in food and food safety toxins is restricted

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