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Primers for detecting acanthamoeba protozoa and kit

A technology of Acanthamoeba and detection kit, which is applied in the field of primers and kits for detection of Acanthamoeba, can solve the problems of low DNA yield, difficulty in adapting to clinical needs of ordinary PCR, sample loss, etc., and achieve high Amplification efficiency, save template DNA extraction step, good effect of early diagnosis

Inactive Publication Date: 2013-04-17
SHANDONG EYE INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the common PCR method is highly efficient and specific in diagnosing pathogens, it needs to extract the template DNA from the sample first, and this process itself will cause the loss of samples; method, DNA yield is still very low
Therefore, considering the need for DNA extraction, ordinary PCR is difficult to adapt to clinical needs

Method used

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  • Primers for detecting acanthamoeba protozoa and kit
  • Primers for detecting acanthamoeba protozoa and kit
  • Primers for detecting acanthamoeba protozoa and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] The sensitivity detection of embodiment 1 kit of the present invention

[0059] Gradiently dilute the known concentration of Acanthamoeba suspension to 10 4 ,10 3 ,10 2 ,10 1 ,10 0 copies / μL, using the DNA amplification reagent and primer combination in the kit of the present invention to amplify, and evaluate the sensitivity of direct PCR to the detection of Acanthamoeba, the specific method is as follows:

[0060] 1) 10.5 μL of DNA amplification reagent, 1 μL of Acanthamoeba suspension in each gradient, 2 μL of forward and reverse primer combinations, and 20 μL of sterilized distilled water;

[0061] 2) 98°C for 5 minutes / 98°C for 30 seconds, 65°C-60°C for 30 seconds (the temperature decreases by 0.5°C for each cycle), 72°C for 30 seconds; (9 cycles) / 98°C for 30 seconds, 60°C for 30 seconds, 72°C for 30 seconds; (25 cycles) / 72°C for 10 minutes, hold at 4°C.

[0062] 3) After the PCR reaction is completed, add loading buffer solution to each concentration gradie...

Embodiment 2

[0064] Embodiment 2: the specific detection of kit of the present invention

[0065] Utilize other common pathogens in ophthalmology, such as Staphylococcus epidermidis in bacteria, Fusarium solani in fungi, herpes simplex virus type I in viruses, and corneal epithelial cells often mixed in corneal scrapings against Acanthamoeba Primers were tested for specificity.

[0066] The specific operation is the same as the specific method of Example 1, and the results show that the Acanthamoeba primer combination in the kit of the present invention only has specific amplification for Acanthamoeba, and does not have amplification for other pathogenic microorganisms, which illustrates the present invention There will be no false positives when the primers are tested.

Embodiment 3

[0067] Example 3: Detection of animal diseased tissue samples by the kit of the present invention

[0068] Use clinically highly suspected acanthamoeba keratitis lesions corneal tissue scrapings to verify the effectiveness of the kit of the present invention, the specific method is as follows:

[0069] (1) Add 5-10 μL of sterilized distilled water to the lesioned corneal scrapings and blow until evenly distributed.

[0070] (2) Depending on the sample volume, take 1-5 μL of the above sample suspension and add it to a small PCR reaction tube, add 10.5 μL of DNA amplification reagent, then add 2 μL of Acanthamoeba primer combination, add sterilized distilled water to make up 20 μL . At the same time, make a positive control with positive nucleic acid and a negative control without template.

[0071] (3) Put the above-mentioned reaction tube into the PCR instrument for TouchDown PCR reaction: 98°C for 5 minutes / 98°C for 30 seconds, 65°C-60°C for 30 seconds (each cycle temperat...

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Abstract

The invention relates to primers for detecting acanthamoeba protozoa and a kit. Sequences of the primers respectively are SEQ ID NO:1 to 2. The kit disclosed by the invention consists of a DNA (Deoxyribose Nucleic Acid) amplification reagent, a forward and reverse primer combination, positive control nucleic acid and sterilized distilled water. The primers adopted by the invention are sensitive and specific for the acanthamoeba protozoa, a direct PCR (Polymerase Chain Reaction) reaction system is simple to prepare and a reaction program has high efficiency; a step of extracting DNA of a template is omitted and the highest utilization degree of limited clinical samples is ensured; and meanwhile, detection time is also greatly shortened, a detection result can be obtained in 2 hours, and the primers and the kit can take a good auxiliary effect on early diagnosis of clinical pathology.

Description

technical field [0001] The invention belongs to the technical field of rapid detection of common pathogenic microorganisms of infectious eye diseases, and in particular relates to a 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; 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 often severe pa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 赵格孙士营袁青谢立信
Owner SHANDONG EYE INST
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