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Real-time fluorescence RT-PCR (reverse transcription-polymerase chain reaction) detection kit for human metapneumovirus and application thereof

A technology of RT-PCR and human metapneumovirus, which is applied in the field of real-time fluorescent RT-PCR detection kits for human metapneumovirus, can solve the problems of lack of cross-reactive antigens, application limitations, etc., to avoid false positives and environmental pollution, The effect of reducing the reaction time and simplifying the operation steps

Active Publication Date: 2013-12-11
湖北朗德医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] (2) Serological detection: including complement fixation test, hemagglutination inhibition test, indirect immunofluorescence and enzyme-linked immunoassay, etc., but due to the lack of suitable cross-reactive antigens to cover many hMPV serotypes, the application of these methods is greatly restricted limit

Method used

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  • Real-time fluorescence RT-PCR (reverse transcription-polymerase chain reaction) detection kit for human metapneumovirus and application thereof
  • Real-time fluorescence RT-PCR (reverse transcription-polymerase chain reaction) detection kit for human metapneumovirus and application thereof
  • Real-time fluorescence RT-PCR (reverse transcription-polymerase chain reaction) detection kit for human metapneumovirus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Development of one-step real-time fluorescent quantitative PCR reagent for human metapneumovirus

[0037] 1. The design of primers and probes: by using the DNAman software for sequence comparison analysis of the existing human metapneumovirus nucleic acid sequences in the Genebank database, the open reading frame M2-1 and M2-2 junction regions of the human metapneumovirus genome The conserved fragment of the DNA is the amplified target site. According to the basic principles of primer and probe design, multiple pairs of primers and probes are manually designed using software.

[0038] 2. Selection of samples: According to relevant domestic and foreign literature reports, samples such as sputum and nasopharyngeal swabs can be selected.

[0039] 3. Establishment and optimization of the reaction system

[0040] Sample preparation: 10 samples that were identified as positive for human metapneumovirus were used as hMPV positive reference substances, namely hMPV-1...

Embodiment 2

[0049] Example 2: Human metapneumovirus one-step fluorescent real-time quantitative RT-PCR detection kit and its use

[0050] 1. Prepare a kit including the following components: RNA extraction solution, RT-PCR amplification reaction solution, negative quality control substance, positive quality control substance, quantitative reference substance, and DEPC-treated water.

[0051] 2. Collection, transportation and storage of specimens

[0052] 2.1 Applicable specimen types: sputum, nasopharyngeal swab, etc.

[0053] 2.2 Specimen collection and pretreatment (pay attention to aseptic operation)

[0054] 2.2.1 Sputum sample collection: Morning sputum is better. Before collecting samples, you should rinse your mouth with clean water or cold boiled water or use a toothbrush (without toothpaste) to clean your mouth and teeth. If you have dentures, you should remove them (in order to reduce the normal oral flora Contaminated specimens), forcefully cough up the sputum in the deep par...

Embodiment 3

[0070] Example 3: Human metapneumovirus one-step fluorescence real-time quantitative RT-PCR detection kit clinical detection use

[0071] The above method was used to detect 18 sputum samples of other patients suspected of human metapneumovirus infection, among which hMPV detection results were positive in 4 cases, the virus fluorescence quantitative PCR amplification curve is shown in image 3 , according to the C of these 4 positive results t Values ​​combined with amplification curves were automatically analyzed by Roche LightCycler480 analysis software to obtain the virus concentrations of these 4 cases of hMPV positive samples. The specific results are shown in Table 1.

[0072] Table 1 Virus concentration of 4 cases of hMPV positive samples

[0073] Sample serial number

Ct value

hMPV virus concentration (copy number / μl)

sample 1

21.34

2.35×10 2

sample 2

20.98

2.92×10 2

sample 3

22.46

2.48×10 3

Sample ...

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Abstract

The invention relates to a real-time fluorescence RT-PCR (reverse transcription-polymerase chain reaction) detection kit for human metapneumovirus and application thereof, belonging to the field of gene detection. The kit provided by the invention has very high sensitivity and specificity. The kit provided by the invention implements quick early detection and quantitative analysis on human metapneumovirus in sputum, nasopharyngeal swab or any other sample. The invention has the advantages of short detection period, high efficiency, high specificity and accuracy for detecting virus, and favorable repetitiveness of experimental results, can simultaneously perform qualitative analysis and quantitative analysis on virus, and is simple to operate and easy to popularize; and the kit can detect the virus with the lowest concentration of 1.0*10<2> copies / mL, and thus, has higher sensitivity than a common PCR (polymerase chain reaction) and immunologic detection method.

Description

technical field [0001] The invention belongs to the field of gene detection, and relates to a real-time fluorescent RT-PCR detection kit for human metapneumovirus and its application. The kit of the present invention contains a pair of oligonucleotide primers and an oligonucleotide probe obtained by screening. The kit of the present invention has the characteristics of early, rapid, sensitive and specific, and can also be used for the detection of human metapneumovirus quantitative analysis. Background technique [0002] Human metapneumovirus (human metapneumovirus, hMPV) is a newly discovered respiratory pathogenic virus, which was first isolated in 2001 from the nasopharyngeal aspirate of an infant in the Netherlands. According to its morphology, biochemistry and genetic Because of its biological characteristics, hMPV was originally classified as avian metapneumovirus, which can cause upper respiratory tract infections in turkeys and other birds. Now it is generally belie...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11G01N21/64
Inventor 石康江城名朱世新
Owner 湖北朗德医疗科技有限公司
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