Rapid DNA extraction method from citrus leaves and its application in detection of citrus Huanglongbing

Abstract

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. The rapid DNA extraction method includes: mince the petiole and midrib of the citrus leaves from the citrus plants to be tested, add 40 μL of lysate to each 30 mg sample, vortex and shake for 10-15 seconds, bathe in 95°C water for 15 seconds, and then add 4 times the lysate Volume 0.1mol / L, Tris-HCL with pH=8, vortexed for 10-15s, centrifuged to get the supernatant to obtain the DNA solution; the formula of the lysate is: 0.5mol / L NaOH, 1% TritonX -100. The combination of this method and qPCR can realize high-throughput rapid molecular detection of citrus Huanglongbing, and provide a favorable tool for large-scale and accurate diagnosis of Huanglongbing.

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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