Rhinovirus real-time fluorescent RT-PCR (reverse transcription-polymerase chain reaction) detection kit and application thereof

A RT-PCR, real-time fluorescence technology, applied in fluorescence/phosphorescence, microbial measurement/inspection, DNA/RNA fragments, etc., can solve the problems of cross-reactive antigens that are difficult to cover HRV serotypes, application restrictions, etc., to avoid the platform period effect, reduced reaction time, highly specific effect

Inactive Publication Date: 2013-05-22
湖北朗德医疗科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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

Method used

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  • Rhinovirus real-time fluorescent RT-PCR (reverse transcription-polymerase chain reaction) detection kit and application thereof
  • Rhinovirus real-time fluorescent RT-PCR (reverse transcription-polymerase chain reaction) detection kit and application thereof
  • Rhinovirus real-time fluorescent RT-PCR (reverse transcription-polymerase chain reaction) detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Development of rhinovirus one-step real-time fluorescence quantitative RT-PCR reagent

[0036] 1. Design of primers and probes: by using DNAman software for sequence comparison analysis of the existing rhinovirus nucleic acid sequences in the Genebank database, the conserved fragment of the rhinovirus genome ORF1-ORF2 junction was used as the amplification target site, according to The basic principle of primer probe design is to use software to manually design multiple pairs of primers and probes.

[0037] 2. Selection of samples: According to relevant domestic and foreign literature reports, you can choose nasal samples, oral samples, extracts containing nasal or oral samples or culture supernatants, such as sputum, nasopharyngeal swabs, etc. sample.

[0038] 3. Establishment and optimization of the reaction system

[0039] Sample preparation: 10 samples identified as positive for rhinovirus were used as HRV positive reference products, respectively HRV-1...

Embodiment 2

[0048] Example 2: Rhinovirus one-step fluorescent real-time quantitative RT-PCR detection kit and its use

[0049] 1. Prepare a kit including the following components: RNA extraction solution, RT-PCR amplification reaction solution, negative quality control substance, positive quality control substance, rhinovirus positive standard substance (quantitative reference substance), DEPC treated water.

[0050] 2. Collection, transportation and preservation of specimens

[0051] 2.1 Applicable specimen types: sputum, nasopharyngeal swab, etc.

[0052] 2.2 Specimen collection and pretreatment (pay attention to aseptic operation)

[0053] 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 coug...

Embodiment 3

[0072] Example 3: Clinical detection of rhinovirus one-step fluorescent real-time quantitative RT-PCR detection kit

[0073] 18 other sputum samples from patients with suspected rhinovirus infection were detected by the above method, among which 4 cases were positive for HRV detection, 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 HRV-positive samples. The specific results are shown in Table 1.

[0074] Table 14 Cases of HRV positive specimen virus concentration

[0075] Sample serial number

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Abstract

The invention belongs to the field of gene detection, and relates to a rhinovirus real-time fluorescent RT-PCR (reverse transcription-polymerase chain reaction) detection kit and an application thereof. The kit comprises a pair of oligonucleotide primers obtained by screening and an oligonucleotide probe, the minimum concentration of HRV (human rhinovirus) detected by a one-step method real-time fluorescent RT-PCR is 1.0*10<2>copies / mL, so that the sensitivity and specificity of the kit are very high. By the kit, the fast early detection and quantitative analysis of rhinovirus in samples such as sputum and nasopharyngeal swab are realized. The kit is short in detection period, high in efficiency, strong in specificity of detecting virus and high in accuracy, is capable of simultaneously realizing virus qualitative analysis and quantitative analysis, has higher sensitivity than ordinary PCR and immunological detection method, and is simple in operation, easy for popularization and good in experimental result repeatability.

Description

technical field [0001] The invention belongs to the field of gene detection, and relates to a rhinovirus real-time fluorescent RT-PCR detection kit and application thereof. The test kit of the present invention contains a pair of oligonucleotide primers and an oligonucleotide probe obtained by screening, and the minimum concentration of HRV detectable by one-step real-time fluorescent RT-PCR is 1.0×10 2 copies / mL, which shows that the sensitivity and specificity of this kit are very high. Background technique [0002] Rhinovirus (HRV) is the most common pathogen that causes viral respiratory tract infections in humans. Pelon first discovered the first strain of HRV in 1956. So far, more than 120 serotypes have been found, which is the virus with the most serotypes in humans. Research results show that about 50% of adult colds are caused by rhinovirus infection, while 10% to 25% of children's colds are caused by rhinovirus, and many of them develop into asthma. [0003] RhV...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11G01N21/64
Inventor 石康王琳琳朱世新
Owner 湖北朗德医疗科技有限公司
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