Method for detecting TCK (Tilletia controversa Kuhn) in soil through QPCR (quantitative polymerase chain reaction)

Abstract

The invention relates to a method for detecting TCK (Tilletia controversa Kuhn) in soil through QPCR (quantitative polymerase chain reaction). The method comprises steps as follows: (1), a taken-back soil sample is sieved by a mesh screen, impurities are removed, and the soil sample is frozen overnight; (2), a FastDNA<TM>SPIN Kit for Soil is adopted to extract total DNA (deoxyribonucleic acid) of the soil sample; (3), the total DNA of the to-be-detected soil sample and positive control templates obtained by gradient dilution have the QPCR simultaneously, and amplification curves and Ct values of the soil sample and the positive control templates are obtained; (4), whether the soil sample contains TCK is judged according to the amplification curve and the Ct value of the positive control templates obtained by the gradient dilution and the amplification curve and the Ct value of the to-be-detected soil sample. According to the method, the soil sample is subjected to freezing treatment, a DNA extraction method is improved, so that DNA with better quality can be extracted, the problem that DNA is extracted from the soil difficultly is solved, and the detection of TCK in the soil is realized.

Description

technical field [0001] The invention relates to a method for QPCR detection of Tilletia tritici in soil, and belongs to the field of prevention and control of Tilletia tritici. Background technique [0002] Tilletia controversa Kühn (TCK) is an important international quarantine disease caused by Tilletia controversa Kühn (TCK), and it is also a kind of dangerous plant disease in foreign quarantine in my country. The disease is very harmful and difficult to control. Once introduced, it will cause devastating damage to wheat production, and the incidence rate is usually approximately equal to the loss rate of wheat yield. According to Hoffmann's report, in popular years, the loss rate caused by TCK is generally 20% to 50%, and the highest can reach 75% to 90%. In addition to yield loss, pollination of infested wheat decreases with infection severity and quality is greatly affected by the presence of TCK teliospores. TCK has a wide range of hosts, harming more than 70 kinds ...

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Problems solved by technology

[0003] T. tritici teliospores hide in the soil in winter. Qualitative and quantitative detection of the content of T. tritici teliospores in soil is of great significance for early warning and disease prevention. However, T. tritici teliospores The volume and quality of spores are very small, and there are many types of microorganisms in the soil. Although the molecular biology detection method has high sensitivity, the proportion of TCK DNA in the total DNA of soil samples is extremely low, and the DNA extraction rate of TCK spores is extremely low. The method extracts the total DNA of the soil sample as a template, which can easily lead to false negative results

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