Method for detecting huanglongbing bacteria based on aPCR and DNA walker

Abstract

The invention discloses a method for detecting huanglongbing bacteria based on aPCR and DNA walker, and relates to the field of bacterial detection. The method comprises the following steps of selecting a to-be-detected target, reacting the to-be-detected target with a DNA walker probe, drawing a standard curve, reacting the to-be-detected target with a specific enzyme chain DNAzyme, and generating fluorescence by a system, wherein the DNA walker probe comprises nanoparticles, the nanoparticles are modified with the specific enzyme chain DNAzyme and a plurality of substrate chains with FAM fluorophores, and the specific enzyme chain DNAzyme can cut the FAM fluorophores on the substrate chains; extracting total DNA from the to-be-detected sample, carrying out aPCR amplification, and carrying out a mixed reaction on an amplification product and the DNA walker probe for 0.5-2 hours; and comparing a detection result with the standard curve. The method is lower in cost in high-throughput detection, and relates to secondary conversion of signals, so that the accuracy is higher.

Description

technical field [0001] The invention relates to the field of bacteria detection, in particular to a detection method for Huanglong bacteria based on aPCR and DNA walker. Background technique [0002] Huanglongbing (HLB) is caused by a Gram-negative bacterium limited to the phloem, which can infect a variety of Rutaceae plants, including Citrus, Citrus Citrus, Kumquat and Murata, and citrus is grown in my country. One of the larger fruits, it occupies an important position in the agricultural economy. Once citrus is infected with Huanglongbing, it will not only affect the yield and quality of the fruit, but even cause the death of a large area of ​​citrus plants in severe cases, resulting in a decline in the yield and quality of citrus , The huge loss is an important factor restricting the development of the citrus industry and increasing farmers' income. [0003] Due to the strong infectivity of Huanglongbing, once citrus infection occurs in the orchard, the infected plants s...

AI Technical Summary

Problems solved by technology

[0005] PCR techniques include conventional PCR, real-time quantitative PCR, and nested PCR. However, because the essence of PCR techniques is "length recognition" and has the risk of false positives, the reliability of the detection results is low; at the same time, when using PCR to detect In order to improve the reliability of the results, it is usually combined with Sanger sequencing (the first generation sequencing technology, the "gold standard" of nucleic acid detection) for "sequence recognition". Although this mode has high accuracy, it is applied to a large number of samples High-throughput detection will bring expensive detection costs (a Sanger sequencing reaction is generally 20 to 40 yuan, and 1000 reactions are 20,000 to 40,000 yuan)

In addition, the loop-mediated isothermal amplification technology involves 4 primers, which mainly solves the problem of temperature change and detection time in nested PCR, but its false positives are high, and it still does not solve the above-mentioned "sequence recognition" problem

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