LAMP (Loop-Mediated Isothermal Amplification) primer group and method for rapidly detecting ditylenchus destructor from complex samples

Abstract

The invention discloses an LAMP (Loop-Mediated Isothermal Amplification) primer group and method for rapidly detecting ditylenchus destructor from complex samples. The LAMP primer group comprises a pair of outer primers F3/B3, a pair of inner primers FIP/BIP and a loop primer LF, and sequences of the primers are sequentially shown as SEQ ID NO. 1-5. The LAMP primer group and the detection method thereof disclosed by the invention have excellent specificity and sensitivity for detecting the ditylenchus destructor, individuals of the ditylenchus destructor in different insect stages (such as eggs, larva, female and male) can be detected, the ditylenchus destructor can be directly detected from multiple nematode mixed samples, plant tissue samples and soil samples, and the detection sensitivity can reach 1/1000 individual DNA. A novel detection method is provided for rapid detection and identification and early diagnosis of the ditylenchus destructor, and the method has the advantages ofbeing accurate, sensitive, stable and intuitive in result judgment. The LAMP primer group disclosed by the invention is simple and convenient to operate and high in practicality, and has important significances and application value on quarantine on ports and transfer and origin monitoring operations.

Description

technical field [0001] The invention belongs to the technical field of pathogen detection. More specifically, it relates to a LAMP primer set and method for rapid detection of D. destructor from complex samples. Background technique [0002] Demia destructor ( Ditylenchus destructor Thorne, 1945) is a migratory endoparasitic nematode with more than 120 known hosts, mainly damaging sweet potatoes and potatoes, and is an important pathogenic nematode in agriculture. The loss of potato yield due to this nematode damage averages 10% per year in the United States. In China, stem nematode rot is one of the three major diseases restricting sweet potato production. In China, the nematode also harms various medicinal plants such as angelica, ginseng and notoginseng, and the incidence rate is as high as 84.9% when the damage to angelica is serious. In order to control the spread of D. destructor and cause greater damage and loss, China and other countries and regions have listed ...

AI Technical Summary

Problems solved by technology

[0003] At present, for the molecular biological identification of D. destructor, both Wendt and Wan Fei designed specific primers based on the rDNA-ITS region of D. destructor, and used conventional PCR techniques for molecular identification (Wendt, 1993; Wan Fei et al., 2008). Although this method is accurate and sensitive, it still has certain limitations: (1) For physical samples such as mixed samples of various nematodes, plant tissue samples, and soil samples, existing PCR detection methods need to be separated from the samples first. The target nematode can only be detected before it can be detected, which is a cumbersome process and a large workload; (2) the detection method requires several hours, and the detection sensitivity is low. When the content of D. destructor in the sample is low, the purpose cannot be achieved; ( 3) A sophisticated and expensive RCR instrument is required, the detection process is complicated, and the results can only be judged by agarose gel electrophoresis detection, which cannot meet the needs of port and transportation quarantine and field monitoring

Invention patent 201110228196.6 discloses a kit for detection of D. destructor in sweet potato based on loop-mediated isothermal amplification. Three sets of primers are designed for the rDNA-ITS region of D. destructor in sweet potato, which can realize the detection of D. The identification of type B solves the problem of high cost, low efficiency and slow speed of conventional molecular detection, which cannot meet the current needs of plant quarantine and plant protection.

This has caused a lot of trouble for the direct and specific detection of a certain nematode. In addition, there is an interaction between a large number of microorganisms in the soil and the environment. During the DNA extraction process, some humic substances are often extracted, which will seriously Interference detection process

In addition, there are still many unknown factors in the soil that seriously affect the detection effect of the target nematodes, which has been an insurmountable problem in this field.

[0005] In summary, the biggest problem and obstacle in the actual detection of plant nematodes, including D. destructor, is the inability to directly detect samples mixed with various nematodes, plant tissue samples, especially soil samples, etc. The target nematodes need to be isolated from the sample before testing. The process is cumbersome and the workload is heavy, so it is not suitable for grassroots detection and mass screening.

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