Rapid DNA extraction method of citrus leaves, and application thereof in detection of citrus yellow shoot diseases

Abstract

The present invention relates to detection of citrus diseases and particularly relates to a rapid DNA extraction method of citrus leaves, and an application thereof in a high-throughput molecular detection of citrus yellow shoot diseases. The rapid DNA extraction method comprises the following steps: petioles and leaf midribs of the citrus leaves from citrus plants to be tested are cut and crushed, 40 [mu]L of lysate is added into each 30 mg of a sample, vortexing vibrating is conducted for 10-15 s, water bath at 95 DEG C is conducted for 15 s, then 0.1 mol/L of Tris-HCL with a pH of 8 and a volume 4 times of the lysate is added, the vortexing vibrating is conducted for 10-15 s, and centrifuging is conducted to obtain a supernatant, a DNA solution; and a formula of the lysate is as follows: 0.5 mol/L NaOH and 1% TritonX-100. The method is combined with qPCR to achieve the high-throughput rapid molecular detection of the citrus yellow shoot diseases, and provides a favorable tool for large-scale and accurate diagnosis of the citrus yellow shoot diseases.

Description

technical field [0001] The invention relates to citrus disease detection, in particular to a method for rapidly extracting DNA from citrus leaves and its application in high-throughput molecular detection of citrus Huanglongbing. Background technique [0002] Citrus Huanglongbing is a devastating disease of citrus, which occurs in major citrus producing areas in the world (Zhong et al., 2015). The pathogenic bacteria of citrus greening disease are Candidatus Liberibacterasiaticus, Candidatus Liberibacter africanus and Candidatus Liberibacter americanus. At present, the Asian species (Las) is the most widely damaged area in the world (Gottwald et al, 2010). A diseased citrus tree cannot produce fruit after 5 to 8 years. There is no effective treatment for citrus huanglongbing. At present, the methods of removing diseased trees, killing vector insects and planting non-toxic seedlings are generally implemented to prevent and control the disease. Therefore, rapid and accurate...

AI Technical Summary

Problems solved by technology

The CTAB and SDS methods need to place the citrus leaves or leaf midribs in a mortar, add liquid nitrogen to grind, and then perform steps such as protein extraction, DNA precipitation, and ethanol washing. The entire extraction process is cumbersome and takes a long time. It takes more than 5 hours to obtain DNA, and the subsequent cleaning of the mortar is also very labor-intensive and time-consuming, and the efficiency is not high when processing a large number of samples

In addition to the high cost of commercial kits, there are also problems such as unstable effects between different batches, cumbersome steps, and low throughput.

The alkaline lysis method has limitations in the molecular detection of citrus Huanglongbing. The citrus samples were directly used in the LAMP reaction after being treated with NaOH, and the results showed no amplification, and the inhibition could be eliminated only after being diluted 50 times (Qian Wenjuan, 2018). This reduces the detection accuracy, and the LAMP method cannot quantitatively analyze the pathogenic bacteria content in the sample

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