A pair of specific molecular detection primers and detection method for endophytic fungi of locoweed

Abstract

The invention discloses a pair of specific molecular detection primers for undifilum oxytropis and a method for detecting whether locoweed is infected by undifilum oxytropis with the pair of specific molecular detection primers for undifilum oxytropis. The pair of specific molecular detection primers include a base sequence equipped upstream primer OmtssuF and a downstream primer OmtssuR, the upstream primer OmtssuF and the downstream primer OmtssuR are 164bp products specifically amplified from undifilum oxytropis infected locoweed. According to the mtSSU coding sequence of undifilum oxytropis, the pair of specific molecular detection primers are designed. The detection primers have strong specificity and good sensitivity. The detection method provided by the invention carries out DNA amplification on the to-be-detected locoweed to obtain a 164bp strip, cannot amplify any product from other fungal DNA and locoweed DNA itself, can be used for fast, accurately and conveniently detecting whether a locoweed sample is infected by undifilum oxytropis, has strong specificity and good practicability, and has important significance to locoweed control.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a pair of specific molecular detection primers and a detection method of locoweed endophytic fungi. Background technique [0002] Locoweed (locoweed) is a leguminous species of Astragalus ( Astragalus ) and Oxytropis ( Oxytropis ) is a general term for poisonous plants, and it is one of the most serious poisonous weeds that endanger the development of grassland animal husbandry. Worldwide locoweeds are mainly distributed in the United States, Canada, Mexico, Russia, Spain and other countries; in my country, locoweeds are mainly distributed in western provinces, including Gansu, Inner Mongolia, Ningxia, Shaanxi, Sichuan, Qinghai, Tibet and Xinjiang, etc. Grasslands, deserts and mountains in provinces and regions. Locoweeds not only reduce the utilization rate of pastures and cause locoweed poisoning of livestock, but also accelerate the degradation of pastures and b...

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Problems solved by technology

However, the traditional method of detecting whether locoweed is infected by locoweed endophytes through separation and identification has the disadvantages of long time and cumbersome operation. Molecular biological methods for fungal infection, Cook et al. designed a pair of primers ITS5 and OR1 using the ITS sequence in 2009 to detect locoweed endophytes (Daniel Cook, Dale R. Gardner, Kevin D. Welch, et al. Quantitative PCR Method To Measure the Fungal Endophyte in Locoweeds[J]. Journal of Agricultural and Food Chemistry, 2009,57(14):6050–6054.), but the primers have poor specificity and cannot isolate locoweed endophyte ( Undifilum oxytropis ) and its closely related Alternaria ( Alternaria ) to distinguish multiple species, and at the same time, the product after PCR amplification needs restriction endonuclease Ava II enzyme digestion, cumbersome, troublesome, high cost

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