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Detection primers, detection methods and applications of leaf spot pathogen phoma exigua

A technology for detecting primers and pathogenic bacteria, applied in the biological field, can solve the problems of short time consumption and achieve the effects of short time consumption, high precision and simple operation

Active Publication Date: 2021-05-14
湖北省农业科学院中药材研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The present invention collects the ITS (gene transcription spacer) region DNA sequence of all Phomaexigua in NCBI data, and then with the same genus Phoma glaucii, Phomatanaceti, Phoma herbarum, Phoma bellidis, Phoma senecionis, Phoma macrostoma, Phoma aliena, Phoma aloes, Phoma neerlandica, Phoma ITS (gene transcription spacer) region base of schachtii, Phomaodoratissimi, Phoma helianthi, and fungal microorganisms such as Magnaporthe oryzae, Rhizoctonia, Anthracnose, Fusarium, Alternaria, Sclerotinia, Aspergillus oryzae, and Pleurotus spp. Specific primers were designed based on differences in base composition, and nested PCR (Nested PCR) was used for molecular detection of Phoma exigua. The detection operation is simple, accurate, high-sensitivity, and time-consuming. This method has not been reported at home and abroad.

Method used

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  • Detection primers, detection methods and applications of leaf spot pathogen phoma exigua
  • Detection primers, detection methods and applications of leaf spot pathogen phoma exigua
  • Detection primers, detection methods and applications of leaf spot pathogen phoma exigua

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Construction of a specific PCR detection method for Phoma exigua, the pathogen of Panax ginseng leaf spot

[0035] 1. Extraction of pathogenic genomic DNA of Panax ginseng leaf spot disease

[0036] Phoma exigua, the pathogenic bacterium of Phoma exigua, is preserved by the Plant Protection Laboratory of the Institute of Chinese Materia Medica, Hubei Academy of Agricultural Sciences. After culturing on PDA plates containing cellophane for one week, the total genomic DNA of pathogenic bacteria was extracted by CTAB method.

[0037] 2. Design of specific molecular detection primers

[0038] Using ITS1 and ITS4 primers, the total genomic DNA of the obtained pathogen Phoma exigua was amplified and sequenced by PCR to obtain its ITS sequence. Phoma aliena, Phoma aloes, Phoma neerlandica, Phoma schachtii, Phoma odoratissimi, Phoma helianthi, and fungi such as Magnaporthe oryzae, Rhizoctonia, Anthracnose, Fusarium, Alternaria, Sclerotinia, Aspergillus oryzae, White...

Embodiment 2

[0055] Example 2 Specific detection for Phoma exigua genomic DNA

[0056] Phoma glaucii, Phoma tanaceti, Phoma herbarum, Phomabellidis, Phoma senecionis, Phoma macrostoma, Phoma aliena, Phoma aloes, Phomaneerlandica, Phoma schachtii, Phoma odoratissimi, Phoma helianthi, and blast fungus, Rhizoctonia anthracnose, Genomic DNA of fungal microorganisms such as Fusarium, Alternaria, Sclerotinia, Aspergillus oryzae, and Sclerotinia spp., and Panax ginseng plants. The genomic DNA of Phoma exigua was used as a positive control, and clear water was set as a negative control. Take the above pathogenic bacteria 1 μL of DNA was used as a template for PCR.

[0057] The reaction system was: 12.5 μL of 2×Taq Master Mix, 1 μL of 10 μM upstream and downstream primers ZJSF / ZJSR, 9.5 μL of double distilled water.

[0058] The amplification reaction program was: pre-denaturation at 94°C for 5 minutes, followed by 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, and fin...

Embodiment 3

[0061] Example 3 Detection of Phoma exigua Genomic DNA by Conventional PCR

[0062] The Phoma exigua genomic DNA was sequentially diluted to a total of 9 concentration gradients of 100ng / μL, 10ng / μL, 1ng / μL, 100pg / μL, 10pg / μL, 1pg / μL, 100fg / μL, 10fg / μL, 1fg / μL, and then Perform PCR.

[0063] The total volume of the PCR reaction system is 25 μL, and the reaction system is: 12.5 μL of 2×Taq Master Mix, 1 μL of 10 μM upstream and downstream primers ZJSF / ZJSR, 9.5 μL of double distilled water.

[0064] The amplification reaction program was: pre-denaturation at 94°C for 5 minutes, followed by 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, and finally extension at 72°C for 3 minutes.

[0065] Take 7 μL of the amplification product, use 1% (mass / volume) agarose gel, add 100 μL (mass volume ratio) of Goldview I nucleic acid stain to 100 mL of agarose gel, electrophoresis at a voltage of 120 V for 20 minutes, and image on the gel Take pictures under the ...

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Abstract

The invention discloses a detection primer, a detection method and an application of the leaf spot pathogen Phoma exigua, and the primers are shown in SEQ ID NO.1-2. By proposing the target genomic DNA, using the universal primers for the first round of amplification, using the above primers for the second round of amplification, and gel electrophoresis detection, if there is a 138bp DNA-specific band, then it is determined that there is leaf spot in the tested sample Pathogen Phoma exigua. The primer was used to detect the leaf spot disease of Panax ginseng caused by Phoma exigua, and proved that it has the characteristics of high efficiency, rapidity, precision and sensitivity, and effectively improved the convenience, timeliness and quality of detecting the leaf spot disease of Panax ginseng caused by Phoma exigua. Accuracy.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a detection primer, a detection method and an application of the leaf spot pathogen Phoma exigua. Background technique [0002] Bamboo ginseng (scientific name: Panax japonicus (T.Nees) C.A.Mey.) is a perennial medicinal plant of the Araliaceae genus Panax. Distributed in the alpine areas of Hubei, Sichuan, Hunan, Jiangxi, Guizhou, Shaanxi and other provinces. It is warm in nature, sweet in taste, slightly bitter, and returns to the liver meridian, spleen meridian, and lung meridian. It has anti-inflammatory, anti-aging, hypoglycemic effects, and adrenocortical hormone-like effects. This medicinal material has high benefits and is currently planted in many places across the country, but leaf spot disease caused by Phoma exigua (the applicant has submitted the ITS gene sequence of the pathogen to NCBI earlier, and the GenBank accession number is KP284148) causes leaves t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12Q1/04C12N15/11C12R1/645
CPCC12Q1/686C12Q1/6895C12Q2565/125
Inventor 游景茂段媛媛唐涛王帆帆郭杰郭晓亮
Owner 湖北省农业科学院中药材研究所
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