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CN-116377119-B - PCR detection method for fusarium graminearum and application thereof

CN116377119BCN 116377119 BCN116377119 BCN 116377119BCN-116377119-B

Abstract

The invention discloses a PCR detection method of fusarium brachypus and application thereof, belonging to the technical field of biological detection and identification. The primer set used in the PCR detection method comprises an upstream primer and a downstream primer, wherein the sequence of the upstream primer is shown as SEQ ID NO.1, and the sequence of the downstream primer is shown as SEQ ID NO. 2. The PCR detection method has good specificity, can amplify fusarium brachypus from 23 test strains, and can detect pathogenic bacteria genome DNA as low as 10 pg/mu L. Therefore, the primer and the detection method designed by the invention can rapidly, specifically and sensitively identify the fusarium graminearum.

Inventors

  • WANG HUA
  • LIU JUN
  • CHANG WEI
  • DENG SIYI

Assignees

  • 湖北省农业科学院植保土肥研究所

Dates

Publication Date
20260512
Application Date
20230529

Claims (8)

  1. 1. The primer set for PCR detection of fusarium graminearum is characterized by comprising an upstream primer and a downstream primer, wherein the sequence of the upstream primer is shown as SEQ ID NO.1, and the sequence of the downstream primer is shown as SEQ ID NO. 2.
  2. 2. A reagent for detecting fusarium graminearum, comprising the primer set according to claim 1.
  3. 3. A kit for detecting fusarium graminearum, comprising the primer set of claim 1 or the reagent of claim 2.
  4. 4. A PCR identification method of fusarium graminearum is characterized in that the identification method uses total DNA of a sample as a template, and uses the primer group of claim 1 to carry out PCR amplification, and the result is judged according to agarose gel electrophoresis after the reaction is finished.
  5. 5. The method according to claim 4, wherein the PCR is performed by a reaction sequence of 94℃pre-denaturation for 5min, 94℃denaturation for 30s,60℃annealing for 30s,72℃extension for 30s,35 cycles, and 72℃extension for 10min.
  6. 6. The method according to claim 5, wherein the total volume of the PCR reaction system is 25. Mu.L, which comprises 2.5. Mu.L of 10 XPCR buffer, 1.5. Mu.L of 25mM Mg 2+ , 2. Mu.L of 10mM dNTP, 0.125. Mu.L of 5U/. Mu.L Taq DNA polymerase, 10. Mu.M of upstream primer SEQ ID NO. 11. Mu.L, 10. Mu.M of downstream primer SEQ ID NO. 21. Mu.L, 1. Mu.L of DNA template, and ddH 2 O15.875. Mu.L.
  7. 7. The PCR identification method as claimed in claim 6, wherein the DNA template is extracted from tomato.
  8. 8. Use of the primer set of claim 1 or the reagent of claim 2 or the kit of claim 3 for identifying fusarium graminearum.

Description

PCR detection method for fusarium graminearum and application thereof Technical Field The invention belongs to the technical field of biological detection and identification, and particularly relates to a PCR detection method of fusarium graminearum and application thereof. Background Tomatoes are widely planted in various areas of China, and as the planting area is gradually enlarged, the reseeding index is improved year by year, and the occurrence and hazard of soil-borne diseases are also in a trend of increasing year by year. Common tomato soil-borne diseases comprise bacterial wilt, fusarium wilt, root rot, verticillium wilt, brown root rot, damping off and the like, and dominant pathogenic bacteria of the tomato soil-borne diseases are Laurella solanaceae (Ralstonia solanacearum), fusarium oxysporum (Fusarium oxysporum), fusarium solani (Fusarium solani), verticillium dahliae (Verticillium dahliae), tomato acanthosporium (Pyrenochaeta lycopersici) and rhizoctonia solani (Rhizoctonia solani) respectively, so that serious economic losses are caused to the producing areas of solanaceae crops in various places in China. Recently, in investigation of tomato soil-borne diseases, it was reported （Jun Liu, Yi Si Deng, Wei Chang, and Hua Wang. 2023. First report of tomato wilt caused by Fusarium brachygibbosum in China. https://doi.org/10.1094/PDIS-01-23-0076-PDN). for the first time that fusarium graminearum causes tomato wilt symptom diseases in China, and the bacteria may pose serious threat to tomato planting and may cause huge economic loss to tomato industry. Therefore, the rapid identification of the pathogenic bacteria can help to research an effective disease management strategy and prevent the serious yield reduction of tomatoes. However, studies on rapid detection of PCR molecules of Fusarium breve have not been reported. With the rapid development of molecular biology techniques, many molecular biology-based methods for detecting pathogenic bacteria molecules have been developed successively. Compared with the traditional detection method based on separation culture and morphological observation, the molecular identification method of the pathogenic bacteria is accurate and efficient. Disclosure of Invention The invention aims to provide a primer set for PCR detection of fusarium graminearum, which comprises an upstream primer and a downstream primer, wherein the sequence of the upstream primer is shown as SEQ ID NO.1, and the sequence of the downstream primer is shown as SEQ ID NO. 2. The second object of the invention is to provide a reagent for detecting fusarium graminearum, which contains the primer groups SEQ ID NO.1 and SEQ ID NO.2. The invention also aims to provide a kit for detecting fusarium graminearum, which contains the primer groups SEQ ID NO.1 and SEQ ID NO.2 or the reagents. The fourth object of the invention is to provide a PCR identification method of fusarium graminearum, which uses total DNA of a sample as a template, uses the primer groups SEQ ID NO.1 and SEQ ID NO.2 to carry out PCR amplification, and judges the result according to agarose gel electrophoresis after the reaction is finished. Preferably, the PCR is performed by a reaction sequence of 94℃for 5min, 94℃for 30s,60℃for 30s,72℃for 30s,35 cycles, and 72℃for 10min. More preferably, the total volume of the PCR reaction system is 25. Mu.L, which comprises 2.5. Mu.L of 10 XPCR buffer, 1.5. Mu.L of 25mM Mg 2+, 2. Mu.L of 10mM dNTP, 0.125. Mu.L of 5U/. Mu.L Taq DNA polymerase, 10. Mu.M upstream primer SEQ ID NO. 11. Mu.L, 10. Mu.M downstream primer SEQ ID NO.2 1. Mu.L, 1. Mu.L of DNA template, and ddH 2 O15.875. Mu.L. More preferably, the DNA template is extracted from tomato. The fifth object of the present invention is to provide the use of the above primer set SEQ ID NO.1 and SEQ ID NO.2 or the above reagent or the above kit for identifying Fusarium graminearum. Compared with the prior art, the invention has the following beneficial effects: The invention has the advantage that the designed primer combination can rapidly detect the existence of fusarium brachypus in tomato samples. The PCR detection has good specificity, can only amplify fusarium brachypus from 23 test strains, and can detect pathogenic bacteria genome DNA as low as 10 pg/mu L. Therefore, the primer and the detection method designed by the invention can rapidly, specifically and sensitively identify the fusarium graminearum. Drawings FIG. 1 shows agarose gel electrophoresis of PCR primer pair-specific detection of Fusarium graminearum in example 2, wherein M is 2000bp DNA Marker;NC is sterile water, negative control, PC is Fusarium graminearum DNA, positive control, lane 1 is Fusarium oxysporum DNA, lane 2 is Fusarium verticillatum DNA, lane 3 is Fusarium roseum DNA, lane 4 is Fusarium graminearum DNA, lane 5 is Fusarium pseudograminearum DNA, lane 6 is Fusarium putrescens DNA, lane 7 is Fusarium rubrum DNA, lane 8 is Fusarium equisetum DNA, lane 9 is Fu