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Loop-mediated isothermal amplification (LAMP) primer group for amplifying I-type herpes simplex virus and kit and application thereof

A primer set and kit technology, applied in the biological field, can solve the problems of LAMP primers being difficult to obtain, and achieve the effects of shortened detection time, strong specificity, and high sensitivity

Active Publication Date: 2014-06-18
HE EYE HOSPITAL SHENYANG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, since the sensitivity and specificity of LAMP are extremely dependent on the characteristics of the primers, good LAMP primers are not easy to obtain, so there is no method for direct detection of HSV-1 virus samples based on LAMP technology

Method used

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  • Loop-mediated isothermal amplification (LAMP) primer group for amplifying I-type herpes simplex virus and kit and application thereof
  • Loop-mediated isothermal amplification (LAMP) primer group for amplifying I-type herpes simplex virus and kit and application thereof
  • Loop-mediated isothermal amplification (LAMP) primer group for amplifying I-type herpes simplex virus and kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1: the accuracy of the LAMP primer set provided by the present invention

[0051] Utilize the LAMP primer set provided by the present invention to detect HSV-1 virus, experiment setting test sample combination negative control. The LAMP primer set includes primers with the sequences shown in SEQ ID NO: 1-6. The experimental groups are shown in Table 1:

[0052] Table 1 Experimental grouping and samples used

[0053] Sample to be tested 1

diseased corneal tissue

negative control 1

normal corneal tissue

Sample to be tested 2

Diseased vitreous cavity fluid

negative control 2

normal vitreous cavity fluid

Sample to be tested 3

HSV infected saliva

negative control 3

normal saliva

[0054] Each group of experiments set 10 repetitions.

[0055] The specific operation of LAMP experiment is as follows:

[0056] 1. Treatment of clinical samples: Take the corneal tissue of the diseased part and the nor...

Embodiment 2

[0072] Embodiment 2: the sensitivity of the LAMP primer set provided by the present invention

[0073] Take the concentration as 10 4 copies / μL of HSV-I virus DNA, 10-fold serial dilution of samples to make their titers 10 4 copies / μL, 10 3 copies / μL, 10 2 copies / μL, 10 1 copies / μL, 10 0 copies / μL, the sensitivity of the LAMP primer set provided by the present invention was tested using the above-mentioned diluted sample.

[0074] The experiment was carried out with the Fluorescent PCR HSV-1 Nucleic Acid Detection Kit purchased from Taipu Bioscience Co., Ltd. as a control. The detection of each concentration of virus was repeated three times.

[0075] For the experimental operation of LAMP, refer to Example 1 of the present invention.

[0076] The LAMP amplification system is:

[0077] Add the following components to a small PCR tube:

[0078]

[0079] Fluorescent PCR method was used to detect HSV-I according to the instructions provided in the kit. Ct value ≤ 36 a...

Embodiment 3

[0084] Embodiment 3: the specificity of the LAMP primer set provided by the present invention

[0085] Common clinical samples containing other viruses: adenovirus, herpes zoster virus, cytomegalovirus, HSV-2, Staphylococcus aureus, Fusarium in fungi and Acanthamoeba were used as negative controls, and normal saline was used as a negative control. As a blank control, HSV-1 was used as the experimental object, and the primers provided by the invention were specifically detected. For the experimental method of LAMP, refer to Example 1 of the present invention. Each group set 3 repetitions. The test results are shown in Table 3:

[0086] The specific detection result of table 3LAMP primer set

[0087]

[0088] The result shows that the primer set provided by the invention can only specifically amplify HSV-1, and does not produce amplification or color reaction to other pathogenic microorganisms. It can be seen that the LAMP primers provided by the present inventio...

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Abstract

The invention relates to the technical field of organisms, and particularly relates to a loop-mediated isothermal amplification (LAMP) primer group for amplifying an I-type herpes simplex virus and a kit and application thereof. The primer group comprises six primers of nucleotide sequences shown in SEQ ID NO:1-6. The invention also provides a kit for detecting a herpes virus I (HSV-I), and a method for detecting the HSV-I in a non-diagnostic target. An experiment proves that the primer provided by the invention is subjected to the HSV-I detection, and the accuracy can be up to 100%. No false positive phenomenon is generated in the process of utilizing a clinical common virus as a contrast to detect. Furthermore, the sensitivity of the primer provided by the invention in the HSV-I detection can be up to 101 copies / mu L. Thus, the LAMP primer group and the kit are applicable to quick diagnosis of diseases such as HSV-I viral keratitis, HSV-I viral conjunctivitis and HSV-I viral retinitis by clinics and primary physicians.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a LAMP primer set, kit and application for amplifying type I herpes simplex virus. Background technique [0002] Herpes simplex virus (HSV) belongs to the subfamily Alphavirinae of the family Herpesviridae, and the only host in nature is humans, and is divided into type I and type II. Among them, herpes simplex virus type I (HSV-I) often causes herpes simplex keratitis (HSK), HSV-I viral conjunctivitis, HSV-I viral retinitis and other diseases. Among them, HSK is the viral keratitis with the highest incidence rate, often causing corneal ulcers, corneal perforation, and blindness. The seropositive rate of HSV in normal adults is 90%, that is, most people have HSV latent in the trigeminal ganglion. When the body's immunity declines, the latent HSV will cause recurrent HSV keratitis. [0003] At present, commonly used detection methods of HSV-1 include: virus culture method, ELISA met...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6844C12Q1/70C12Q2531/119
Inventor 王卓实董昊徐玲
Owner HE EYE HOSPITAL SHENYANG
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