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Real-time fluorescence multiplex PCR rapid detection kit for common pathogens of eye infection

An eye infection, real-time fluorescence technology, applied in the field of nucleic acid detection, can solve problems such as affecting disease diagnosis, and achieve the effect of short detection time and high accuracy

Active Publication Date: 2016-09-07
华美生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Each set of primers / probes cannot have cross-homology to other target and non-target nucleic acid sequences to prevent false positive results and affect the diagnosis of diseases

Method used

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  • Real-time fluorescence multiplex PCR rapid detection kit for common pathogens of eye infection
  • Real-time fluorescence multiplex PCR rapid detection kit for common pathogens of eye infection

Examples

Experimental program
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Effect test

Embodiment 1

[0012] Example 1: Detection of Herpes Simplex Virus 1: Herpes simplex virus 1 positive control was used as a sample. The acquisition of the corresponding primer / probe combination and positive control is shown in Example 2. 2 [mu]l of internal control (5 [mu]M) was added to samples and negative controls prior to extraction. Nucleic acids were extracted from the samples using the MagMAX Total Nucleic Acid Isolation Kit (Invitrogen). To TaqMan Fast Virus 1-Step Master Mix (Invitrogen, 13.5 μl) were then added each primer / probe combination (1.5 μl) and nucleic acid (10 μl) extracted from the samples or negative controls, respectively. The final sample concentration was 5 μM and the primer / probe concentration was 0.4 μM. Then, each reaction tube was subjected to PCR reaction on an ABI 7500 (Applied Biosystems) real-time PCR instrument according to the following procedures: 50°C for 15min (reverse transcription), 95°C for 10min (hot start); 95°C for 8s (denaturation), 60°C for 34...

Embodiment 2

[0013] Example 2: Detection kit for 5 pathogens of ophthalmic infection at one time

[0014] 1) Design primer / probe combination: According to the nucleic acid sequence of each pathogen, perform sequence alignment among species to find conserved regions, and use Beacon Designer software to design primers and Taqman probes for multiplex PCR for these conserved regions. Each primer / probe combination obtained was subjected to BLAST analysis on the NCBI website to ensure that it did not cross-react with other microorganisms that may be present in the sample. Finally, its performance is verified by experiments. The internal control was designed in the same way.

[0015] The designed primers / probes are as follows:

[0016] Reaction 1: Contains herpes simplex virus 1, 2 and herpes zoster virus

[0017]

[0018] Reaction 2: containing adenovirus, chlamydia trachomatis and murine cytomegalovirus (internal control)

[0019]

[0020] 2) Preparation of each positive control: firs...

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Abstract

The invention belongs to the technical field of nucleic acid detection, and particularly relates to a real-time fluorescent multiple PCR (Polymerase Chain Reaction) rapid detection kit for common pathogens leading to ocular infection. According to the rapid detection kit, the problem of cross linkage among different primers / probes is avoided, each set of primer / probe is prevented from generating cross homology on other target / non-target nucleic acid sequences, various common pathogens leading to ocular infection can be detected simultaneously in one reaction, the detection time required is short, the accuracy is high, and a powerful detection tool is provided for the clinic and detection laboratories.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid detection, and particularly relates to a real-time fluorescent multiplex PCR rapid detection kit for common pathogens of eye infection. Background technique [0002] Eye infection refers to the local tissue inflammation caused by bacteria, viruses, fungi, parasites and other pathogens invading the human eye. The most commonly infected sites include the eyelids, conjunctiva, cornea, and sclera. The most common inflammatory conditions are conjunctivitis and blepharitis. [0003] There are many reasons for infection, including bacterial or viral infection caused by not paying attention to eye hygiene and rubbing the eyes with hands; direct or indirect infection by an infected person; corneal foreign bodies caused by dust entering the eyes can also cause infection; mother-to-child transmission can also cause eye infections. Department of infection. [0004] The main infectious viruses are herp...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12Q1/04
CPCC12Q1/689C12Q1/701C12Q1/705C12Q2600/16
Inventor 贾昕张胜楠李明梁红李科
Owner 华美生物工程有限公司
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