LAMP (loop-mediated isothermal amplification) detection primer, kit and detection method for fusarium solani

A technology of Fusarium solani and a detection kit, which is applied in the biological field to achieve the effects of strong technicality, accurate and reliable test results, and high sensitivity

Pending Publication Date: 2021-06-22
SHANXI AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no reports at home and abroad on the LAMP detection of Fusarium solani which causes Astragalus root rot

Method used

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  • LAMP (loop-mediated isothermal amplification) detection primer, kit and detection method for fusarium solani
  • LAMP (loop-mediated isothermal amplification) detection primer, kit and detection method for fusarium solani
  • LAMP (loop-mediated isothermal amplification) detection primer, kit and detection method for fusarium solani

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1: the design of Fusarium solani LAMP primer composition

[0040] The EF-1alpha gene sequence of Fusarium solani (Fusarium solani) was determined, and compared with the EF-1alpha gene sequences of other Fusarium spp. in the GenBank database, using the online LAMP primer design software Primer Explorer V5 software (http: / / primerexplorer.jp / lampv5e), design specific LAMP primers of Fusarium solani with the nucleotide sequence shown in SEQID NO1-6, the primers include specifically including 1 outer primer F3 / B3, 1 pair of inner Primer FIP / BIP and 1 pair of loop primers Loop F / Loop B. The specific primer sequences are as follows:

[0041] F3: 5'-GATCGCGCCTTGCTATTC-3', SEQ ID NO: 1;

[0042] B3: 5'-TTAGCGTCTGTTGTGAACC-3', SEQ ID NO: 2;

[0043] FIP: 5'-AATTACGGTCGGACCGCAAAACATCGAATTCCCTCCCCT-3', SEQ ID NO: 3;

[0044] BIP: 5'-CGGGCGACGTTGGACAAAACCAAGAGGGTTTGGTGTTT-3', SEQ ID NO: 4;

[0045] Loop F: 5'-ATTTTGGGACTCGGGAGAAG-3', SEQ ID NO: 5;

[0046] Loop B: 5...

Embodiment 2

[0047] Embodiment 2: the extraction of pathogenic bacteria genome DNA

[0048] Genomic DNA of pathogenic bacteria was extracted using Biospin Fungal Genomic DNA Extraction Kit, and extracted according to its operating instructions:

[0049] 1) Pick a small amount of fresh mycelium of the activated pathogenic bacteria, add liquid nitrogen to grind it into powder, put it into a 1.5mL centrifuge tube, add 500μL LE Buffer, and mix well.

[0050] 2) Incubate at 65°C for 60 minutes (take out every 15 minutes and mix well);

[0051] 3) Add 130 μL DA Buffer, mix well and put in a water bath at 65°C for 5 minutes;

[0052] 4) Centrifuge at 14,000×g for 3 minutes;

[0053] 5) Aspirate the supernatant, transfer it to a new 1.5mL centrifuge tube, add 500μL E Binding Buffer, and mix well;

[0054] 6) Transfer the mixture to the Spin Column and centrifuge at 6,000×g for 1 min;

[0055] 7) Add 500μL G Binding Buffer to the Spin Column, centrifuge at 10,000×g for 30s, and discard the liqu...

Embodiment 3

[0061] Embodiment 3: the establishment of the LAMP detection reaction system of Fusarium solani

[0062] Using the DNA extracted in Example 2 as a template, and using the LAMP primer composition designed in Example 1, a LAMP detection reaction system for Fusarium solani was established: each of the outer primers F3 and B3 was 0.2 μM, and the inner primers FIP and BIP were each 0.8 μM. Loop primers Loop F and Loop B each 0.4 μM, 2×LAMP PCR isothermal amplification reaction mixture (general-purpose, Sangon Bioengineering (Shanghai) Co., Ltd.) 12.5 μL, 8U Bst polymerase 0.5 μL, DNA template 1.0 μL , make up to 25 μL with sterilized ultrapure water. Before amplification, add 1.0 μL of SYBR green I chromogen to the cap of the centrifuge tube. After amplification, centrifuge at 5000r / min for 30s, and mix well by inverting up and down. After the reaction, 2.0 μL of the LAMP amplification product was taken for 2.0% agarose gel electrophoresis detection, and the electrophoresis detec...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to an LAMP (loop-mediated isothermal amplification) detection primer, a kit and a detection method for fusarium solani. The primers comprise a forward outer primer F3, a reverse outer primer B3, a forward inner primer FIP, a reverse inner primer BIP, a forward loop primer LF and a reverse loop primer LB, and nucleotide sequences of the forward outer primer F3, the reverse outer primer B3, the forward inner primer FIP, the reverse inner primer BIP, the forward loop primer LF and the reverse loop primer LB are respectively shown as SEQ ID NO: 1-6; the LAMP detection primer composition can be used for detecting fusarium solani. The designed fusarium solani LAMP detection primer composition is high in specificity, high in sensitivity and accurate and reliable in result, the detection method is easy and convenient to operate, pathogenic bacteria do not need to be separated and cultured, the detection time is short, the detection process can be completed within 2 h, the detection result is visual, and the detection cost is low. The method can be used for detection of fusarium solani in astragalus membranaceus and early diagnosis of astragalus membranaceus root rot.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a LAMP detection primer, a kit and a detection method of Fusarium solani. Background technique [0002] Astragalus membranaceus (Astragalus membranaceus) is a perennial herbaceous plant of the Leguminosae sp. Astragalus Linn. It has a thick main root and can be used as medicine. Crop rotation in soil. In recent years, due to the increase in demand and the continuous expansion of cultivation area, the rotation cycle of Astragalus membranaceus has been shortened and the area of ​​continuous cropping has continued to increase, which has led to a significant increase in the incidence of Astragalus root rot. The incidence rate is 10% to 30%, and the severe case is as high as 60%. , more than 3 years of plant disease is more serious, has become one of the main factors limiting the production of Astragalus membranaceus, seriously restricting the sustainable development of Astra...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04C12N15/11C12R1/77
CPCC12Q1/6895C12Q1/6844
Inventor 王春伟党海燕王燕张仙红
Owner SHANXI AGRI UNIV
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