Method for detecting TCK (Tilletia controversa Kuhn) teliospores in soil through ddPCR (droplet digital polymerase chain reaction)

Abstract

The invention relates to a method for detecting TCK (Tilletia controversa Kuhn) teliospores in soil through ddPCR (droplet digital polymerase chain reaction). The method comprises steps as follows: (1), total DNA (deoxyribonucleic acid) of a to-be-detected soil sample is extracted; (2), the total DNA of the to-be-detected soil sample serves as a template for ddPCR, and a PCR product is obtained; (3), the PCR product is put in a droplet read instrument, a signal is read, experimental data are analyzed, and the absolute content of TCK in the soil sample is obtained. A total DNA extraction method comprises steps as follows: the taken-back soil sample is sieved by a mesh screen for impurity removal and then is frozen; total DNA of the soil sample is extracted by adopting FastDNA<TM> SPIN Kit for Soil. With the adoption of the method, higher-quality DNA can be extracted from the soil, ddPCR is adopted for detection, accordingly, the absolute quantification of TCK in the soil is realized, control measures are taken in time, and economic losses are reduced.

Description

technical field [0001] The invention relates to a method for detecting TCK teliospores in soil by microdroplet digital PCR (ddPCR), and belongs to the field of prevention and treatment of Ustilago dwarf. 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, ...

AI Technical Summary

Problems solved by technology

Therefore, fluorescent quantitative PCR technology is a relative nucleic acid quantitative method, and its sensitivity and accuracy are limited.

qRT-PCR must rely on the standard curve and genes with known copy numbers when analyzing the copy number of exogenous genes. It is only a relative quantitative method, and the quality of the standard curve is susceptible to DNA purity, concentration of primers and probes, and reaction inhibition. factor and many other factors (Cankare et al., 2006); in addition, the standard curve must be established based on standard materials, and the limited types and high prices of standard materials cannot be applied to all researches

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