Primer set for detecting I-type herpes simplex virus and kit

A herpes simplex virus, detection kit technology, applied in recombinant DNA technology, microbial determination/inspection, biochemical equipment and methods, etc., can solve the problems of ordinary PCR difficult to adapt to clinical needs, low DNA yield, sample loss , to avoid non-specific amplification, high amplification efficiency, and good 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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  • Primer set for detecting I-type herpes simplex virus and kit
  • Primer set for detecting I-type herpes simplex virus and kit
  • Primer set for detecting I-type herpes simplex virus and kit

Examples

Experimental program
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Effect test

preparation example Construction

[0040] 2. Preparation of the kit

[0041] Preparation of direct PCR detection of herpes simplex virus type I kit (5 samples):

[0042] (1) DNA amplification reagent, 1 tube, containing 15 amplification reactions (1 positive reaction, 1 experimental sample reaction, and 1 negative reaction are required for each sample), required reagents, 10.5 μL / part, each Contains 10 μL 2×MightyAmp buffer, 0.5 μL MightyAmp DNA polymerase; (MightyAmp buffer and corresponding DNA polymerase is a PCR reaction system produced by TaKaRa Company, catalog number is DR071)

[0043] (2) Forward and reverse primer combination, 1 tube, containing 15 copies, 2 μL / part, each contains 5 μM of the concentration of the forward primer and the reverse primer of herpes simplex virus type I, and the specific sequence of the primer is shown in SEQ ID NO: 1~2.

[0044](3) Positive control nucleic acid, 1 tube, containing 5 μL of a plasmid containing the target DNA of herpes simplex virus type I, named pUC-HSV-1....

Embodiment 1

[0057] The sensitivity detection of embodiment 1 kit of the present invention

[0058] Gradiently dilute the known titer type 1 herpes simplex virus suspension to 10 4 ,10 3 ,10 2 ,10 1 ,10 0 PFU / μL, using the DNA amplification reagent and primer combination in the kit of the present invention to amplify, evaluate the sensitivity of direct PCR to the detection of type I herpes simplex virus, the specific method is as follows:

[0059] 1) 10.5 μL of DNA amplification reagent, 1 μL of herpes simplex virus type I suspension of each gradient, 2 μL of forward and reverse primer combinations, and sterilized distilled water to 20 μL;

[0060] 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.

[0061] 3) After the PCR reaction is completed, add loading buffer solution t...

Embodiment 2

[0063] Embodiment 2: the specific detection of kit of the present invention

[0064] Utilize other common viruses in ophthalmology such as adenovirus, herpes zoster virus, cytomegalovirus, another serotype of herpes simplex virus type II, other common pathogens in ophthalmology such as Staphylococcus epidermidis in bacteria, Fusarium solani in fungi The primers of herpes simplex virus type I were specifically detected by bacteria and Acanthamoeba and corneal epithelial cells often mixed in corneal scrapings.

[0065] The specific operation is the same as that in Example 1, and the results show that the primer combination of type I herpes simplex virus in the kit of the present invention only specifically amplifies type I herpes simplex virus, and does not produce amplification for other pathogenic microorganisms, indicating that the primers of the present invention There will be no false positives during the test.

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Abstract

The invention relates to a primer set for detecting I-type herpes simplex virus and a kit. Primer sequences of the primer set 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. According to the invention, viruses are detected by a method of designing and synthesizing forward and reverse primers capable of amplifying I-type herpes simplex virus specific DNA according to a conserved sequence of I-type herpes simplex virus genome DNA and utilizing a special PCR (Polymerase Chain Reaction) amplification reagent to direct perform PCR reaction without carrying out extraction on DNA of a sample. According to the invention, the step of extracting DNA of a template is omitted, one important link of losing the sample size is reduced and the highest utilization degree of limited clinical samples is ensured; and meanwhile, detection time is greatly shortened, a detection result can be obtained in 2 hours, and the primer set 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 set and a kit for detecting type I herpes simplex virus. Background technique [0002] Herpes simplex virus belongs to the representative herpes virus of the subfamily Alphaherpesviridae of the family Herpesviridae. It is divided into two serotypes, type I and type II, according to the difference in antigenicity. Currently, the envelope glycoprotein G is considered to be type I and type II. A glycoprotein with the greatest difference between the two can induce the body to produce type-specific antibody responses. Herpes simplex virus can cause a variety of human diseases, and common viral keratitis is mainly caused by the infection of the cornea by type I herpes simplex virus. Herpes simplex virus type I is transmitted from person to person through secretion of body fluids and intimate co...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 赵格孙士营袁青翟华蕾谢立信
Owner SHANDONG EYE INST
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