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Kit and detection method for detecting pathogenic microorganisms of infectious eye disease

A technology of pathogenic microorganisms and infectivity, which is applied in the field of kits for detecting pathogenic microorganisms of infectious eye diseases, can solve the problems that ordinary PCR is difficult to adapt to clinical needs, sample loss, etc., to avoid non-specific amplification and shorten the detection time , the effect of high amplification efficiency

Active 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 ordinary PCR method is highly efficient and specific in diagnosing pathogens, the template DNA must be extracted from the sample first, which will cause the loss of the sample; in addition, the sample size of clinical ophthalmic lesions is very small, even if the efficient and convenient kit is used to extract method, DNA extraction of different pathogenic microorganisms also requires different kits
Therefore, considering the need for DNA extraction, ordinary PCR is difficult to adapt to clinical needs

Method used

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  • Kit and detection method for detecting pathogenic microorganisms of infectious eye disease
  • Kit and detection method for detecting pathogenic microorganisms of infectious eye disease
  • Kit and detection method for detecting pathogenic microorganisms of infectious eye disease

Examples

Experimental program
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Effect test

Embodiment 1

[0069] The sensitivity detection of embodiment 1 kit of the present invention

[0070] The bacteria (Pseudomonas aeruginosa), fungi (Candida albicans), type I herpes simplex virus and Acanthamoeba suspensions with known concentrations or titers determined by counting and other methods were respectively diluted to 10 4 ,10 3 ,10 2 ,10 1 ,10 0 copies / μL, using the DNA amplification reagent in the kit of the present invention and the corresponding primer combination to amplify, evaluate the sensitivity of direct PCR to the detection of various pathogenic microorganisms, the specific methods are as follows:

[0071] 1) 10.5 μL of DNA amplification reagent, 1 μL of each gradient pathogenic microorganism suspension, 2 μL of forward and reverse primer combinations, and sterilized distilled water to 20 μL;

[0072] 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 se...

Embodiment 2

[0075] Embodiment 2: the universality in the kind and the specificity detection outside the kind of the kit of the present invention

[0076] Common pathogenic bacteria in ophthalmology such as Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus pneumoniae, Serratia marcescens, Bacillus subtilis and Pseudomonas aeruginosa were used for universal detection of bacterial universal primers, and common pathogenic fungi, Bacterial universal primers were specifically detected for herpes simplex virus and acanthamoeba as well as corneal epithelial cells. Common pathogenic fungi in ophthalmology such as Fusarium solani, Aspergillus fumigatus, Candida albicans and Alternaria were also used for universal detection of fungal universal primers, as well as other bacteria, viruses and Acanthamoeba and cornea Epithelial cells were tested specifically for fungal universal primers. In addition, the primers for herpes simplex virus type I and Acanthamoeba were also tested specifica...

Embodiment 3

[0078] Example 3: Detection of animal diseased tissue samples by the kit of the present invention

[0079] The effectiveness of the kit of the present invention was verified by utilizing three scrapings of diseased corneal tissues that were clinically highly suspected of fungal keratitis, viral keratitis, and acanthamoeba keratitis. The specific methods are as follows:

[0080] (1) Add 20 μL of sterilized distilled water to the lesioned corneal scrapings and beat until evenly distributed.

[0081] (2) Depending on the sample size, take 1-5 μL of the above sample suspension and add them to 4 small PCR reaction tubes, add 10.5 μL of DNA amplification reagent, and then add bacteria, fungi, type I herpes simplex virus and Acanthamoeba Primer combination 2μL, add sterilized distilled water to make up 20μL. At the same time, a positive control with positive nucleic acid and a negative control without template were made for the four primer combinations.

[0082] (3) Put the above-m...

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Abstract

The invention relates to a kit and a detection method for detecting pathogenic microorganisms of the infectious eye disease. The kit consists of a DNA (Deoxyribose Nucleic Acid) amplification reagent, a universal primer combination, positive control nucleic acid and sterilized distilled water, wherein the DNA amplification reagent is formed by a premixed reagent which directly carries out amplification without extracting DNA of a sample and corresponding DNA polymerase mixed solution; and the universal primer combination is mixed solution of equal quantities of universal forward and reverse primers, which is designed and synthesized for respective conserved sequences of a plurality of types of common pathogenic microorganism bacteria, fungi, I-type herpes simplex virus and acanthamoeba protozoa of the infectious eye disease. The primers adopted by the invention are universal in various pathogenic agent types, but are specific for other pathogenies; a direct PCR (Polymerase Chain Reaction) reaction system and a program also have 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 and the kit and the detection method 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 pathogenic microorganisms of infectious eye diseases, and specifically relates to a kit and a detection method for detecting pathogenic microorganisms of infectious eye diseases, that is, detecting common pathogenic bacteria and fungi of infectious eye diseases by direct PCR method , A kit and a detection method for the specific DNA of type I herpes simplex virus and Acanthamoeba. Background technique [0002] Infectious eye diseases are the most common frequently-occurring diseases of the eye, including keratitis, conjunctivitis, and endophthalmitis. The microorganisms that cause eye lesions mainly include bacteria, fungi, viruses, and Acanthamoeba. If patients with keratitis and endophthalmitis are not diagnosed and treated in time, they will be at risk of blindness. Only by early and timely diagnosis of which pathogenic microorganism infection is the cause can better guide the ration...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12Q1/04
Inventor 赵格孙士营翟华蕾袁青谢立信
Owner SHANDONG EYE INST
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