CN-121975966-A - AS-PCR primer and application thereof in detection of drug resistance of lychee anthracnose to azoxystrobin

Abstract

The invention discloses an AS-PCR primer and application thereof in detecting drug resistance of lychee anthracnose to azoxystrobin. The invention provides an AS-PCR primer and develops a method for detecting drug resistance of lychee anthracnose to azoxystrobin based on the primer. The method for detecting the drug resistance of the lychee anthracnose to the azoxystrobin is simple and convenient to operate, only the DNA of the lychee anthracnose is extracted and then is subjected to PCR amplification and electrophoresis analysis, a single target band can be observed in an electrophoresis pattern of the drug-resistant strain, and the sensitive strain has no amplified band, so that the method is suitable for large-scale sample screening. In addition, the primer designed by the invention has high specificity, can accurately distinguish drug resistance and sensitive strains, and has no cross amplification to other common plant pathogenic fungi. The invention provides reliable technical support for early and rapid diagnosis of the drug resistance of the lychee anthracnose to the azoxystrobin, and has high practical value and wide application prospect.

Inventors

• JI CHUNYAN
• Mo Xiumi
• PAN RUQIAN

Assignees

• 华南农业大学

Dates

Publication Date

20260505

Application Date

20250630

Claims (10)

1. 1. The AS-PCR primer is characterized by comprising a forward primer F2 and a reverse primer R1, wherein the sequence of the forward primer F2 is shown AS SEQ ID NO.2, and the sequence of the reverse primer R1 is shown AS SEQ ID NO. 4.
2. 2. A kit comprising the AS-PCR primer of claim 1.
3. 3. The kit of claim 2, further comprising a PCR reaction reagent.
4. 4. Use of the AS-PCR primer of claim 1 or the kit of claim 2 or 3 for detecting resistance of lychee anthracnose to azoxystrobin.
5. 5. Use of the AS-PCR primer of claim 1 or the kit of claim 2 or 3 in the preparation of a product for detecting resistance of lychee anthracnose to azoxystrobin.
6. 6. Use of the AS-PCR primer of claim 1 or the kit of claim 2 or 3 for detecting azoxystrobin-resistant colletotrichum gloeosporioides.
7. 7. Use of the AS-PCR primer of claim 1 or the kit of claim 2 or 3 for the preparation of a product for detecting azoxystrobin-resistant colletotrichum gloeosporioides.
8. 8. A method for detecting azoxystrobin-resistant lychee anthracnose, comprising the steps of: s1, extracting DNA of a strain to be detected; S2, carrying out PCR reaction by using the DNA obtained in the S1 AS a template and using the AS-PCR primer of claim 1 or the kit of claim 2 or 3 to obtain a PCR amplification product; s3, detecting the PCR amplification product obtained in the S2 by gel electrophoresis, wherein if a single band exists, the detected strain is the litchi anthracnose bacteria of the azoxystrobin.
9. 9. The method of claim 8, wherein in S2, the annealing temperature of the PCR reaction is 55-63 ℃.
10. 10. The method of claim 8, wherein in S3, the size of the band is 500 to 750bp.

Description

AS-PCR primer and application thereof in detection of drug resistance of lychee anthracnose to azoxystrobin Technical Field The invention belongs to the technical field of microorganism drug resistance detection. More specifically, relates to an AS-PCR primer and application thereof in detecting drug resistance of lychee anthracnose to azoxystrobin. Background The litchi anthracnose is an important litchi disease caused by Colletotrichum spp, and can infect leaves, branch tips, spikes and fruits of litchi, so that the leaves are brown and dead, a series of problems such as flower dropping, fruit dropping, brown stain and rot are further caused, and the healthy development of the litchi industry is seriously threatened. At present, chemical control is still a main means for controlling litchi anthracnose. Azoxystrobin is used as a broad-spectrum efficient bactericide, has both protection and treatment effects, and has been widely applied to the prevention and treatment of litchi anthracnose. However, the long-term single use of the agent can cause pathogenic bacteria to develop drug resistance, thereby reducing the control effect. Therefore, detecting the drug resistance level of pathogenic bacteria to azoxystrobin and monitoring the drug resistance is of great significance. Traditional drug resistance detection methods rely primarily on sensitive assays of pathogenic bacterial populations, such as hyphal growth rate or spore germination. Although the accuracy of the method is high, the method is complex in operation, takes a long time, usually takes 7-14 days, is large in workload, and is difficult to meet the rapid detection requirement of large-batch and large-scale samples. Therefore, a rapid and accurate molecular detection method is established, is used for identifying the drug resistance of the lychee anthracnose to azoxystrobin, and has important guiding significance and practical value for early monitoring, drug resistance management and scientific prevention and control of field drug-resistant groups. Disclosure of Invention Aiming at the defect of the existing molecular detection method for the drug resistance of the lychee anthracnose to the azoxystrobin, the invention provides an allele-specific PCR (AS-PCR) primer, and the primer can be used for rapidly detecting the drug resistance of the lychee anthracnose to the azoxystrobin. It is a first object of the present invention to provide an AS-PCR primer. It is a second object of the invention to provide a kit. The third object of the invention is to provide the application of the primer or the kit in detecting the drug resistance of the lychee anthracnose to azoxystrobin. The fourth object of the invention is to provide the application of the primer or the kit in preparing a product for detecting the drug resistance of lychee anthracnose bacteria to azoxystrobin. The fifth object of the invention is to provide the application of the primer or the kit in detecting the azoxystrobin-resistant lychee anthracnose. The sixth object of the invention is to provide the application of the primer or the kit in preparing products for detecting the azoxystrobin-resistant lychee anthracnose. The seventh object of the invention is to provide a method for detecting the anthracnose pathogen of litchi with azoxystrobin resistance. The above object of the present invention is achieved by the following technical scheme: The invention monitors the existence of a resistant strain of field lychee anthracnose to azoxystrobin by a research team, and develops a method capable of detecting the lychee anthracnose with azoxystrobin resistance by designing a primer, so the invention claims the following scheme: The invention provides an AS-PCR primer, which comprises a forward primer F2 and a reverse primer R1, wherein the sequence of the forward primer F2 is shown AS SEQ ID NO.2, and the sequence of the reverse primer R1 is shown AS SEQ ID NO. 4. The invention provides a kit comprising the AS-PCR primer. As an alternative embodiment, the kit further comprises PCR reaction reagents. As an alternative embodiment, the above kit further comprises a DNA extraction reagent. The invention provides application of the AS-PCR primer or the kit in detecting drug resistance of lychee anthracnose to azoxystrobin. The invention provides application of the AS-PCR primer or the kit in preparation of a product for detecting drug resistance of colletotrichum gloeosporioides to azoxystrobin. The invention provides application of the AS-PCR primer or the kit in detecting azoxystrobin-resistant litchi anthracnose bacteria. The invention provides application of the AS-PCR primer or the kit in preparation of products for detecting azoxystrobin-resistant litchi anthracnose bacteria. The invention provides a method for detecting azoxystrobin-resistant litchi anthracnose pathogen, which comprises the following steps: s1, extracting DNA of a strain to be detected; S2, carrying out PCR reaction by usin