Primers for detecting acanthamoeba protozoa and kit
A technology for Acanthamoeba and reagents, which is applied in the field of primers and kits for detecting Acanthamoeba, can solve the problems of low DNA yield, difficulty in adapting to clinical needs by ordinary PCR, loss of samples, and the like, and achieves high amplification Efficiency, omitting template DNA extraction step, good effect of early diagnosis
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Embodiment 1
[0058] Example 1 Sensitivity detection of the kit of the present invention
[0059] Serial dilution of Acanthamoeba suspension of known concentration to 10 4 ,10 3 ,10 2 ,10 1 ,10 0 copies / μL, adopt 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, and the specific method is as follows:
[0060] 1) 10.5 μL of DNA amplification reagent, 1 μL of each gradient Acanthamoeba suspension, 2 μL of forward and reverse primer combinations, and sterilized distilled water to 20 μL;
[0061] 2) 98℃ for 5 minutes / 98℃ for 30 seconds, 65℃-60℃ for 30 seconds (temperature decrease by 0.5℃ for each cycle), 72℃ for 30 seconds; (9 cycles) / 98℃ for 30 seconds, 60℃ for 30 seconds, 72°C for 30 seconds; (25 cycles) / 72°C for 10 minutes, 4°C hold.
[0062] 3) After the PCR reaction is completed, add the loading buffer to each concentration gradient reaction tube and p...
Embodiment 2
[0064] Example 2: Specific detection of the kit of the present invention
[0065] The use of other common ophthalmic pathogens such as Staphylococcus epidermidis in bacteria, Fusarium solanacearum in fungi, herpes simplex virus type I in viruses and corneal epithelial cells often mixed in corneal scrapings to Acanthamoeba Primers were tested for specificity.
[0066] The specific operation is the same as the specific method of Example 1. The results show that the Acanthamoeba primer combination in the kit of the present invention only has specific amplification for Acanthamoeba, and does not amplify other pathogenic microorganisms, indicating the present invention The primers will not produce false positives during detection.
Embodiment 3
[0067] Example 3: Detection of animal diseased tissue samples by the kit of the present invention
[0068] Utilize the clinically highly suspected acanthamoeba keratitis lesion corneal tissue scraping material to carry out validity verification to the kit of the present invention, and the concrete method is as follows:
[0069] (1) Add 5-10 μL of sterilized distilled water to the scraped cornea of the lesion to blow and disperse until it is uniform.
[0070] (2) Depending on the amount of the sample, 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, and add sterilized distilled water to make up 20 μL . At the same time, a positive control with positive nucleic acid and a negative control without template were made.
[0071] (3) Put the above reaction tube into the PCR machine for TouchDown PCR reaction: 98°C for 5 minutes / 98°C for 30 seconds, 65°C...
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