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Primers and kit for detecting fusarium on basis of loop-mediated isothermal amplification technology

A loop-mediated isothermal and amplified primer technology, applied in the direction of microorganism-based methods, recombinant DNA technology, microorganisms, etc., can solve the problems of low positive rate, cumbersome process, and restrictions on popularization and application, and achieve the safety of operators and the environment , High detection sensitivity and good detection specificity

Inactive Publication Date: 2011-11-23
SHANDONG EYE INST
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Among them, the fungal isolation and culture period of lesion specimens is long and the process is cumbersome, which is not conducive to rapid early diagnosis; direct microscopic examination of lesion specimens is fast and simple, but the positive rate is not high; serological methods are less effective in detecting superficial and local infections , and it is not conducive to the detection of other organisms; it is a very direct detection method to directly observe whether there are Fusarium hyphae or spores in the lesion with a confocal microscope, but the confocal microscope is not a very popular detection instrument, which limits its popularization and application
Although the common PCR detection method is sensitive and specific, it is expensive and requires a special PCR instrument
The loop-mediated isothermal amplification (loop-mediated isothermal amplification, LAMP) technology not only has the sensitivity and specificity of ordinary PCR detection methods, but also can rapidly, efficiently and specifically amplify target sequences under isothermal conditions; Expensive instruments and reagents, low cost, have shown broad development prospects in the field of pathogenic microorganism detection, but there is no report on the detection of Fusarium by loop-mediated isothermal amplification method

Method used

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  • Primers and kit for detecting fusarium on basis of loop-mediated isothermal amplification technology
  • Primers and kit for detecting fusarium on basis of loop-mediated isothermal amplification technology

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preparation example Construction

[0029] Preparation of Fusarium nucleic acid isothermal amplification detection kit:

[0030] (1) LAMP reaction solution:

[0031] A total of 22 μL / part, each containing 2.5 μL 10× Thermopol reaction buffer, 3.5 μL dNTP (dATP, dGTP, and dCTP each 10 mM, dTTP, dUTP each 5 mM), 1 μL 40 μM upstream internal primer, 1 μL 40 μM downstream internal primer, 1 μL 5 μM upstream outer primer, 1 μL 5 μM downstream outer primer, 3 μL 100 mM MgSO4, 5 μL 5M betaine (betaine), and 4 μL sterile double distilled water (ddH2O).

[0032] The primers described herein are forward and reverse inner primers and forward and reverse outer primers, and the sequences are SEQ ID NO: 1-4 respectively.

[0033] (2) UNG enzyme: 1U / μL;

[0034] (3) Bst DNA polymerase: 8U / μL;

[0035] (4) Chromogen: 10 times diluted fluorescent dye GeneFinder TM ;

[0036] (5) Positive control nucleic acid: a plasmid containing Fusarium DNA.

[0037] The detection method is carried out according to the following procedur...

Embodiment 1

[0047] Embodiment 1: Sensitivity detection of the kit of the present invention

[0048] The positive nucleic acid sample in this kit is a DNA plasmid containing Fusarium nucleic acid. The specific operation of its preparation is to amplify the genomic DNA in the conserved region of Fusarium by PCR, use the kit to recover the target fragment, connect it to the cloning plasmid, transfer it to Escherichia coli E. Sequencing verification of positive clones, extracting the plasmids of positive clones can be used as positive nucleic acids.

[0049] Dilute the positive nucleic acid gradient to 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , 10 0 copies / uL, directly use the LAMP primers of the present invention and the LAMP reaction solution in the kit for LAMP sensitivity detection, the specific method is as follows:

[0050] 1) 22 μL of LAMP reaction solution, 2 μL of Acanthamoeba positive nucleic acid in each gradient, add 0.5 μL of UNG enzyme, react at 50°C for 3 minutes...

Embodiment 2

[0055] Embodiment 2: the specific detection of kit of the present invention

[0056] The primers and kits of the present invention are paired with the genome DNA of common pathogenic microorganisms in ophthalmology including fungi Fusarium solani, Fusarium oxysporum, Fusarium moniliforme, bacteria Pseudomonas aeruginosa, virus herpes simplex virus type 1 and Acanthamoeba A LAMP-specific assay was performed.

[0057] The specific operation is the same as the specific method in Example 1. The results show that the primers and LAMP reaction system of the present invention only amplify the DNA of three kinds of Fusarium, and do not amplify other common bacteria, viruses and amoebas in ophthalmology. , that is, the above-mentioned amplification reaction tube will not turn green, indicating that the primers of the present invention will not cause false positives during detection.

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Abstract

The invention relates to a loop-mediated isothermal amplification kit and method for detecting fusarium. The kit comprises loop-mediated isothermal amplification reaction liquid, UNG (uracil-DNA-glycosylase), Bst DNA polymerase, developer and fusarium positive DNA, wherein the reaction liquid contains forward / reverse inner and outer primers, of which the sequences are respectively SEQ ID NO: 1-4. The detection method comprises the steps of fungus DNA extraction, loop-mediated isothermal amplification and development detection. The loop-mediated isothermal amplification primers are designed according to the fusarium gene conserved region sequences, and the fusarium in the sample is detected by loop-mediated isothermal amplification technology. The invention has the advantages of high speed, high sensitivity, high specificity, low cost and the like; and the whole process is not related to toxic reagents, thereby ensuring the safety of operating personnel and environment. Meanwhile, the UNG adopted in the invention can thoroughly eliminate false positive caused by nucleic acid pollution in multiple detection processes.

Description

technical field [0001] The invention belongs to the technical field of rapid detection of common pathogenic fungi, and in particular relates to a primer and a kit for detecting Fusarium based on a loop-mediated isothermal amplification technology. Background technique [0002] Fusarium (Fusarium) is widely distributed in soil, water and organic matter in nature. It is an important plant pathogen that can infect a variety of plants (food crops, economic crops, medicinal plants and ornamental plants). Known hosts There are more than 100 species of plants. Fusarium infects the vascular system of the host plant, causing various diseases such as root rot, stem rot, stem base rot, flower rot, ear rot, etc., and produces toxins in the process of growth, development and metabolism to harm crops, causing crops to wilt and die , seriously affecting the yield and quality of crops, and is one of the most difficult diseases to control in production. In addition, Fusarium can also infec...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12R1/77
Inventor 赵格孙士营谢立信
Owner SHANDONG EYE INST
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