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Loop-mediated isothermal amplification primers and kit for detecting acanthamoeba

An Acanthamoeba, ring-mediated isothermal technology, which is applied to the determination/testing of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of restricting wide application, cumbersome cultivation process, and long time , to achieve the effect of safety for operators and the environment, high detection sensitivity and easy identification

Inactive Publication Date: 2012-10-24
SHANDONG EYE INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Microscopic observation of lesion scrapings has the advantages of being fast and simple, and does not require special instruments, but the positive rate is not high, and the detection of amoeba cysts requires a lot of experience
The isolation and culture of amoeba in diseased tissue or scraping is a very accurate test method, but the culture process is cumbersome and takes a long time, which also restricts the wide application of this method
Direct observation of amoeba cysts in the lesion by confocal microscope is a very direct detection method, because the low light level reflection of corneal tissue and the movement of eyeballs limit the generation of images, and confocal microscopes are not very popular testing equipment
The loop-mediated isothermal amplification method can make up for these deficiencies, but there is no report on the detection of Acanthamoeba using the loop-mediated isothermal amplification method

Method used

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  • Loop-mediated isothermal amplification primers and kit for detecting acanthamoeba
  • Loop-mediated isothermal amplification primers and kit for detecting acanthamoeba

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1: Primer of the present invention and test kit are to the detection of positive sample

[0051] The positive nucleic acid sample in this kit is a DNA plasmid containing Acanthamoeba nucleic acid. The specific operation of its preparation is to amplify Acanthamoeba genomic DNA by PCR, use the kit to recover the target fragment, connect it to the cloning plasmid, transfer it into Escherichia coli E. Sequencing verification of positive clones, extracting the plasmids of positive clones can be used as positive nucleic acids.

[0052] 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:

[0053] 1) 15 μl of LAMP reaction solution, 4 μl of LAMP primer mixture, 1 μl of Acanthamoeba positive nucleic acid in each gradient, 0.5 μl of UNG enzyme, 4 ...

Embodiment 2

[0058] Embodiment 2: Primer of the present invention and test kit are to the detection of negative sample

[0059] The primers and the kit of the present invention are used for specific detection of LAMP using common pathogenic microorganisms in ophthalmology, including bacteria Pseudomonas aeruginosa, fungus Fusarium solani, virus herpes simplex virus type 1, Acanthamoeba and human corneal epithelial cell genomic DNA.

[0060] 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 Acanthamoeba, but do not amplify other common bacteria, fungi, viruses and cornea itself 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.

Embodiment 3

[0061] Embodiment 3: Detection of the diseased tissue sample by primers and kits of the present invention

[0062] The effectiveness of the primers and kits of the present invention is tested by using the scraped corneal tissue of pathological changes that is highly suspected of acanthamoeba keratitis clinically, and the specific methods are as follows:

[0063] (1) Take the corneal lesion tissue scrapings and place them in a microcentrifuge tube, add 100 μL tissue lysate and 1 μL proteinase K, act at 60°C for 60 minutes, and repeatedly pipette several times;

[0064] (2) After adding an equal volume of nucleic acid extraction solution for extraction, centrifuge at 12,000 rpm / min for 5 minutes, absorb the upper layer and transfer it to a new microcentrifuge tube;

[0065] (3) Add 2 times the volume of nucleic acid extraction solution pre-cooled at -20°C, mix well, centrifuge at 12000r / min for 5, and discard the supernatant;

[0066] (4) Wash the above precipitate twice with n...

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Abstract

The invention discloses loop-mediated isothermal amplification primers and kit for detecting acanthamoeba. The sequences of the primers are represented by SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 respectively. The loop-mediated isothermal amplification primers designed according to the sequence of the conserved region of the gene of the acanthamoeba can flexibly, quickly and safely detect acanthamoeba; meanwhile, in the kit disclosed by the invention, false positive caused by nucleic acid pollution in a multiple detection process can be radically eliminated by using uracil-DNA glycosylase (UNG), so the problem of susceptibility to pollution and interference, which limits the wide application of a loop-mediated isothermal amplification technique, is solved. When the kit disclosed by the invention is used, the acanthamoeba causing amebic keratitis can be detected within 3 hours, and instead of expensive instrument and equipment, only a water bath pot or metal bath and a centrifuge are needed in a detection process; the detection result is very easy to determine; and compared with the conventional detection technique, the kit is low in cost, the kit is very safe for operators and the environment for the whole process is free from toxic reagents, and the detection sensitivity of the kit is very high.

Description

technical field [0001] The invention belongs to the technical field of rapid detection of common pathogenic microorganisms, and in particular relates to a loop-mediated isothermal amplification primer and a kit for detecting Acanthamoeba. Background technique [0002] Acanthamoeba is a self-living microorganism that widely exists in nature, and has two phases: trophozoite and cyst. Trophozoites generally inhabit fresh water, sewage, sea water or soil, and transform into cysts when the environment is unfavorable, and cysts can exist in the air. Under certain conditions, Acanthamoeba can enter the human body from skin wounds, penetrating corneal trauma, damaged conjunctiva, or through the respiratory tract, reproductive tract, etc., and most of them parasitize in the eyes, skin and other parts. Can further lead to encephalitis. Acanthamoeba keratitis is a persistent and progressive corneal ulcer. Patients have foreign body sensation, blurred vision, tearing, photophobia, and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 赵格谢立信孙士营陈豪
Owner SHANDONG EYE INST
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