Sugarcane smut bacteria nest type polymerase chain reaction (PCR) quick detection method

Abstract

The invention relates to the field of sugarcane pathogenic bacteria detection, and provides a sugarcane smut bacteria nest type polymerase chain reaction (PCR) quick detection method. The method comprises the steps of DNA extraction, primary PCR amplification, secondary PCR amplification and PCR amplification product detection, wherein universal primers ITS4 and ITS5 of a fungal ribosome genetic transcription spacer ITS are adopted in the primary PCR amplification; and specific primers Smut-L1 and Smut-R2 of a sugarcane smut ribosome genetic transcription spacer ITS are adopted in the secondary PCR amplification. The method has the substantial characteristics that: 1, the detection sensitivity is extremely high, and the smut bacteria can be accurately detected even if the sugarcane has nosmut symptom or is infected little by the smut bacteria; 2, the method is strong in specificity, low in interference of other bacteria and high in accuracy; and 3, the method has short time consumption and high detection speed, and is suitable for sugarcane variety introduction quarantine, toxin-free seedling quality detection, sugarcane smut resistance identification, early symptom-free leaf detection and the like.

Description

technical field [0001] The invention relates to the field of detection of sugarcane pathogenic bacteria, in particular to a method for detecting sugarcane smut. Background technique [0002] by Saccharomyces sativa ( Ustilago scitaminea ) caused by sugarcane smut is a worldwide important sugarcane disease, and it is also the most economically harmful sugarcane disease in my country. Powdery mildew, sugarcane smut spreads infection during storage and planting. Therefore, the detection method of sugarcane smut during the sugarcane introduction and quarantine process is an important way to prevent the widespread spread of sugarcane smut. [0003] The routine PCR detection method of sugarcane smut, such as the patented "Sugarcane smut rapid detection method" (application number: 201010230534), this conventional PCR detection method mainly includes the following steps: Step 1, using CTAB method to extract sugarcane smut genomic DNA ; Step 2, using the sugarcane smut genomic DNA...

AI Technical Summary

Problems solved by technology

Although this method can also detect whether sugarcane is infected with smut, but because it uses fungal ITS general primers instead of sugarcane smut specific primers, its detection specificity is extremely poor, and it is easy to be affected during the detection process. Interference from other pathogens; the sensitivity is limited, and it cannot be accurately detected when the infection amount of smut is small

Based on the shortcomings of the above method, the present invention designs specific primers according to the difference in base composition between the sugarcane smut and the ITS region (gene transcription spacer) of other species of the same genus, and uses nested PCR to detect the sugarcane smut, and the detection is accurate High sensitivity and high sensitivity, no reports at home and abroad

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