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Real-time fluorescence detection method for detecting various pathogens and application

A fluorescence and fluorophore technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of increased detection difficulty, detection errors, time-consuming and labor-intensive, etc.

Inactive Publication Date: 2017-10-17
INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because there are many types of microorganisms such as viruses and bacteria, and they mutate quickly, detection errors (such as false positives and false negatives) are prone to occur.
In order to detect multiple types of viruses or different virus subtypes or strains of the same type of virus, separate tests are often required, which increases the difficulty of detection and is time-consuming and laborious

Method used

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  • Real-time fluorescence detection method for detecting various pathogens and application
  • Real-time fluorescence detection method for detecting various pathogens and application
  • Real-time fluorescence detection method for detecting various pathogens and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0118] Example 1. Construction of real-time fluorescent RT-PCR detection of H7N9 influenza virus based on fluorescent primers and high-fidelity DNA polymerase system

[0119] The HA fragment of influenza A virus H7N9 was selected as the RNA template, which were wild-type N9-WT and mutant N9-Mu1 sequences, respectively. There is only one single nucleotide mutation site in the two sequences, and the mutant type has only one A→G point mutation compared with the wild type. Design forward fluorescent primers and reverse ordinary primers, wherein the reverse ordinary primers are located downstream of the mutation site and are completely complementary to the wild-type and mutant templates at the corresponding positions; the forward fluorescent primers are completely complementary to the wild-type templates, and its 3' The end is located at the single nucleotide mutation site, that is, it contains a mismatch with the mutant type (as shown in Table 1). The 5' end of the forward fluore...

Embodiment 2

[0132] Embodiment 2, the impact of the amount of non-high-fidelity DNA polymerase on the system

[0133]Select the plasmid DNA (wild type) cloned from the HA sequence of Influenza A, and transcribe it into RNA in vitro, as a real-time fluorescent quantitative RT-PCR template, using a forward fluorescent primer exactly the same as in Example 1 And reverse general primer, add a large amount of high-fidelity enzyme to the system, add different amounts of non-high-fidelity DNA polymerase, such as 0, 0.2, 0.5, 1, 1.2, 1.5U / 25μl system, and add nuclease-free Water is the most negative control (NTC).

[0134] The reaction system is as follows

[0135]

[0136] The reaction conditions are as in Example 1.

[0137] According to the above reaction system and reaction conditions in ROCHE The reaction was carried out on a 96 real-time fluorescent quantitative PCR instrument, and the buffer solution in the reaction system and the Q5 high-fidelity enzyme were all products of NEB Comp...

Embodiment 3

[0139] Example 3, the tolerance of the detection system based on fluorescent primers and high-fidelity DNA polymerase to different mutation sites

[0140] The present invention selects human gene β-Actin (ACTB) for PCR amplification and in vitro transcription to obtain RNA standard (wild-type ACTB-WT), and designs a mutant template with only a single nucleotide mutation (A→G) sequence (ACTB-Mu1), mutant template sequence (ACTB-Mu2) with only a single nucleotide mutation (A→U), mutant template sequence with a single nucleotide mutation (A→C) (ACTB- Mu3). Forward fluorescent primers and reverse ordinary primers were designed respectively, wherein the reverse ordinary primers were located downstream of the mutation site and completely matched with the wild-type and mutant templates at the corresponding positions; the 3' ends of the four forward fluorescent primers were located at the point mutations ( As shown in table 2). The 5' end of the forward fluorescent primer was modifi...

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Abstract

The invention provides a real-time fluorescence detection method for detecting various pathogens and application. Specifically, the invention provides a novel simple real-time fluorescence detection (RT-) Polymerase Chain Reaction (PCR) method, a reaction system and application of the reaction system in single and multiple detections of the pathogens (in particular viruses) and quantitative detection of gene expression. The tail end 3' is provided with a fluorescence or quenching group-modified fluorescence primer, so that the end 3' is not required to be mispaired with a template; under the action of a 3'-5' excision enzyme correction function of high-fidelity DNA polymerase and any trace non-high-fidelity DNA polymerase, the fluorescence or quenching group at the end 3 is cut off, so that real-time fluorescence detection of pathogen nucleic acids and DNA and mRNA of human genes is realized. The real-time fluorescence detection method is simple in design, quick in operation, high in sensitivity and high in accuracy, and can be widely applied to relevant fields of quick and multiple detection of infectious pathogens, the quantitative detection of the gene expression and the like.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid detection, and more specifically relates to a fluorescence real-time detection method and application for detecting various pathogens. The present invention also relates to quantitative and / or qualitative detection kits using the method. Background technique [0002] Pathogen detection is to detect pathogens in the early stage of disease infection, provide timely treatment, and help prevent diseases. Pathogen diagnosis is generally divided into bacterial, viral and fungal diagnosis. [0003] There are multiple methods for virus detection, including virus culture, electron microscope observation, and virus antigen and antibody detection. Although viral culture is the gold standard for detection, the process is complex and takes several days. Electron microscopy can determine the family and genus by observing the shape of the virus, but it requires high technical personnel and is expensive. ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/6851C12Q1/6858C12Q2531/113C12Q2561/113C12Q2563/107C12Q2537/143
Inventor 张驰宇张梦玲
Owner INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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