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CN-121137253-B - LAMP primer group, kit and detection method for detecting oat smut pathogen

CN121137253BCN 121137253 BCN121137253 BCN 121137253BCN-121137253-B

Abstract

The invention belongs to the technical field of molecular biology detection of plant fungi, and discloses an LAMP primer group, a kit and a detection method for detecting oat smut pathogenic bacteria. 1 group of LAMP specific primers are designed according to the specific sequence on the whole genome of the oat smut pathogen Ustilago hordei, and the oat smut pathogen carried by oat seeds is detected through a real-time fluorescent quantitative method, an agarose gel electrophoresis method and a SYBR GreenI fluorescent dye chromogenic method judgment result. The LAMP detection method for the oat smut pathogenic bacteria has the advantages of strong specificity, high sensitivity, high speed and low cost, provides a new technical means for detecting oat smut pathogenic bacteria carried by oat seeds, and has higher practical application value.

Inventors

  • DANG JINJIN
  • WU BIN
  • LI JIA

Assignees

  • 中国农业科学院作物科学研究所
  • 盐碱地综合利用技术创新中心

Dates

Publication Date
20260505
Application Date
20251117

Claims (6)

  1. 1. The detection method of the oat smut pathogen barley black powder fungus (Ustilago hordei) is characterized by comprising the following steps: 1) Extracting DNA in a sample to be detected; 2) Performing LAMP amplification reaction by using the DNA extracted in the step 1) as a template and using an LAMP specific primer of the oat smut pathogenic bacteria; 3) Judging an amplification result; The LAMP specific primers comprise an outer forward primer F3, an outer reverse primer B3, an inner forward primer FIP, an inner reverse primer BIP and loop primers LF and LB; F3:5′-GCAGAGGAGGAAGCTCGA-3′; B3:5′-TGGTCCGTCGCATGGTTA-3′; FIP:5′-GGATGCAGCAAAAGTTGCGGTCACCTGCTTTGGCTCCGAT-3′; BIP:5′-AACCCAACTTGCCGCTGCAGGACGATCCAGCAATGACACT-3′; LF:5′-AACAGGGCCGAAATGGGC-3′; LB:5′-CGCATGCTTCTCTGGCATCTG-3′。
  2. 2. the method according to claim 1, wherein the reaction system used in step 2) is 100 fg/. Mu.L-10 ng/. Mu.L of DNA template 2.0. Mu.L, 10mM dNTP 3.5. Mu.L, 10. Mu.M primers F3 and B3 each 0.2. Mu.L, 10. Mu.M primers FIP and BIP each 1.6. Mu.L, 10. Mu.M primers LF and LB each 0.4. Mu.L, 0.8M betaine 4.0. Mu.L, 8U/. Mu.L of Bst DNA polymerase 0.8. Mu.L, 10 Xisothermal amplification reaction buffer 2.5. Mu.L, 100 mM MgSO 4 1.5.5. Mu.L, and sterile distilled water 6.3. Mu.L.
  3. 3. The process according to claim 1, wherein the reaction conditions used in step 2) are 60-65℃for 40-60min.
  4. 4. The method according to any one of claims 1 to 3, wherein the amplification result is judged by a fluorescent dye color development method in step 3), wherein a dye SYBR GreenI is added into the LAMP amplification product solution to carry out a color development reaction, and if the amplification product solution turns from orange to green, the amplification product solution shows that the sample to be detected contains barley smut (Ustilago hordei) which is a pathogenic bacterium of oat smut.
  5. 5. The method according to any one of claims 1 to 3, wherein the amplification result is determined by agarose gel electrophoresis in step 3), wherein if the amplification product shows a characteristic ladder-like band on agarose gel, it indicates that the sample to be tested contains barley smut which is a pathogenic organism of oat smut (Ustilago hordei).
  6. 6. The method according to any one of claims 1 to 3, wherein the step 3) comprises the steps of determining the amplification product by a real-time fluorescent quantitative amplification curve method, wherein the fluorescent detection interval is 1min, determining whether the amplification is normal or not according to the change of the fluorescent signal, indicating that the amplification is normal if the amplification curve shows an exponential change, and generating detectable fluorescence after the SYBR GREEN I fluorescent dye is added, indicating that the sample to be detected contains barley smut powder (Ustilago hordei) which is a pathogenic bacterium of oat smut.

Description

LAMP primer group, kit and detection method for detecting oat smut pathogen Technical Field The invention belongs to the technical field of molecular biology detection of plant fungi, and particularly relates to an LAMP primer group, a kit and a detection method for detecting oat smut pathogenic bacteria. Background Loop-mediated isothermal amplification (LAMP) is a novel isothermal nucleic acid amplification technique developed by Notomi et al in 2000. The technology designs inner and outer 2 pairs of specific primers aiming at 6 specific areas of a target gene, and utilizes DNA polymerase with a strand displacement function to specifically amplify a target sequence under a constant temperature condition, thereby having the characteristics of strong specificity, high sensitivity, economy and simplicity. The isothermal nature of LAMP allows it to adapt to complex scenarios (e.g. field, substrate) with high sensitivity and fast response capability. At present, the LAMP technology is widely applied to a plurality of fields such as clinical pathogen detection, food safety, medicine quality control, customs quarantine, animal and plant disease diagnosis, agriculture and forestry environmental protection monitoring and the like. In Fukuta, 2003, a study of plant disease detection was carried out by combining the LAMP technique with the reverse transcription technique for the first time, and yam mosaic virus (JYMV) was successfully detected. Ma Jun and the like screen out specific molecular markers of fruit anthracnose (Colletotrichum fructicola) based on comparative genomics, and successfully establish an LAMP rapid detection system for hickory anthracnose. The highland barley sheath rot is a spike disease caused by the dextrorotatory fungus (Dactylobotrys gramincola) of the grass, and seriously affects the yield and quality of the highland barley. Zhang Haiqing and the like based on ITS sequences, design and screen out specific primers, and establish a method which has high sensitivity and can rapidly detect the staphylococcus epidermidis of highland barley. These studies indicate that the LAMP technique has wide applicability and reliability in pathogen detection. In recent years, the LAMP technology is combined with the microfluidic technology, the CRISPR technology and the like, so that the detection equipment is more portable and intelligent, the detection cost and the technical requirements on detection personnel are reduced, the detection result is obtained more simply and efficiently, but no research report on the application of the related technology in oat pathogen detection exists so far. The oat smut pathogen (Ustilago hordei) is also called oat smut, and is mainly transmitted through seeds, so that whether the seeds carry pathogen or not is detected before sowing, and whether prevention and control measures are needed or not is determined, and the method is the most economical and effective method for controlling diseases. However, since the spore of the smut pathogen is small (about 5-9 μm in diameter), it is difficult to observe by naked eyes, and a rapid detection method for the oat smut pathogen is not available at present. Disclosure of Invention The invention develops the LAMP specific primer capable of detecting the oat smut pathogen and establishes the detection method of the pathogen based on the LAMP technology, so that the oat smut pathogen can be rapidly detected from oat seeds, and the establishment of the method is favorable for controlling the occurrence of the oat smut. In order to achieve the purpose of the invention, in a first aspect, the invention provides a group of oat smut pathogen (barley smut Ustilago hordei) LAMP specific primers, according to the LAMP primer design principle, multiple sequence comparison is carried out according to oat smut pathogen specific genome sequences in Genbank databases, a section of highly conserved region is searched as an amplification object, then 1 group of specific primers comprising an outer forward primer F3 and an outer reverse primer B3 (SEQ ID NO: 1-2), an inner forward primer FIP and an inner reverse primer BIP (SEQ ID NO: 3-4), and loop primers LF and LB (SEQ ID NO: 5-6) are designed through online software Primer ExploreV (http:// primexduplex. Jp/lampv e/index. Html); F3:5′-GCAGAGGAGGAAGCTCGA-3′; B3:5′-TGGTCCGTCGCATGGTTA-3′; FIP:5′-GGATGCAGCAAAAGTTGCGGTCACCTGCTTTGGCTCCGAT-3′; BIP:5′-AACCCAACTTGCCGCTGCAGGACGATCCAGCAATGACACT-3′; LF:5′-AACAGGGCCGAAATGGGC-3′; LB:5′-CGCATGCTTCTCTGGCATCTG-3′。 in a second aspect, the invention provides a detection reagent or kit comprising said primer. In a third aspect, the present invention provides a kit for detecting oat smut pathogen (Ustilago hordei), the kit comprising the primer and further comprising at least one of dNTP, bst DNA polymerase, betaine, reaction buffer, etc. In a fourth aspect, the invention provides the use of said primer, said detection reagent or kit, or said detection kit, for de