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Real-time fluorescent multiple PCR (Polymerase Chain Reaction) rapid detection kit for common pathogens leading to ocular infection

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

Active Publication Date: 2015-04-29
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 fluorescent multiple PCR (Polymerase Chain Reaction) rapid detection kit for common pathogens leading to ocular infection
  • Real-time fluorescent multiple PCR (Polymerase Chain Reaction) rapid detection kit for common pathogens leading to ocular infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] Example 1: Detection of HSV 1: A positive control of HSV 1 was used as a sample. For the acquisition of corresponding primer / probe combinations and positive controls, see Example 2. 2 μl of internal control (5 μM) was added to samples and negative control prior to extraction. Nucleic acids were extracted from samples using MagMAX Total Nucleic Acid Isolation Kit (Invitrogen). Each primer / probe combination (1.5 μl) and nucleic acid extracted from the sample or negative control (10 μl) were then added to TaqMan Fast Virus 1-Step Master Mix (Invitrogen, 13.5 μl). The final sample concentration was 5 μM and the primer / probe concentration was 0.4 μM. Subsequently, each reaction tube was subjected to a PCR reaction on an ABI 7500 (Applied Biosystems) real-time fluorescent PCR instrument according to the following procedures: 50°C for 15min (reverse transcription), 95°C for 10min (hot start); 95°C for 8s (denaturation), Maintain at 60°C for 34s (annealing / extension, collect...

Embodiment 2

[0013] Embodiment 2: A detection kit for detecting 5 kinds of 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 the same species to find conserved regions, and use Beacon Designer software to design multiplex PCR primers and Taqman probes for these conserved regions. The resulting primer / probe combinations were subjected to BLAST analysis on the NCBI website to ensure no cross-reaction with other microorganisms that may be present in the sample. Finally, its performance is verified by experiments. The same method was used for the design of the internal control.

[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: Contains adenovirus, Chlamydia trachomatis and murine cytomegalovirus (internal control)

[0019]

[0020] 2) Preparati...

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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 in particular relates to a real-time fluorescence multiple 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 affected sites include the eyelids, conjunctiva, cornea, and sclera. The most common inflammations 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 eyes with hands; direct or indirect infection by infected people; corneal foreign bodies caused by dust entering the eyes can also cause infection; mother-to-child transmission can also cause eye infections. local infection. [0004] The main infectious viruses are herpes simplex virus, varic...

Claims

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Application Information

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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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