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

A technology of RT-PCR and detection kit, which is applied in the directions of fluorescence/phosphorescence, microbiological determination/inspection, DNA/RNA fragments, etc. It can solve the problems of inability to cover HAstV serotypes, lack of cross-reactive antigens, and low sensitivity. Achieve the effects of avoiding plateau effect, avoiding false positive and environmental pollution, and high sensitivity

Active Publication Date: 2013-12-11
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

[0005] (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, it cannot cover many HAstV serotypes, and the sensitivity is low, making these The application of the method is very limited

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Development of one-step real-time fluorescent quantitative PCR reagent for human astrovirus

[0035] 1. Design of primers and probes: by using DNAman software to perform sequence alignment analysis on the existing human astrovirus nucleic acid sequences in the Genebank database, the conserved fragment of the ORF1-ORF2 junction region of the human astrovirus genome was used as the amplification target According to the basic principles of primer-probe design, multiple pairs of primers and probes were manually designed using software.

[0036] 2. Selection of samples: According to relevant literature reports at home and abroad, samples such as stool and anal swab can be selected.

[0037] 3. Establishment and optimization of the reaction system

[0038] Sample preparation: 10 samples that were identified as positive for human astrovirus were used as HAstV positive reference products, namely HAstV-1, HAstV-2, HAstV-3, HAstV-4, HAstV-5, HAstV-6, HAstV- 7. HAstV-...

Embodiment 2

[0047] Example 2: Human astrovirus one-step fluorescent real-time quantitative RT-PCR detection kit and its use

[0048] 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.

[0049] 2. Collection, transportation and storage of specimens

[0050] 2.1 Applicable specimen types: stool, anal swab, etc.

[0051] 2.2 Specimen collection and pretreatment (pay attention to aseptic operation)

[0052] 2.2.1 Stool specimen collection: Stool collection should be carried out by specially trained personnel. The specimen collector collects 5g (5ml) of stool and puts it in a sterile stool sampling cup (without adding any reagents).

[0053] 2.2.2 Anal swab specimen collection: soak cotton swab in normal saline, insert it into the anus 2-3cm, wipe it from the folds around the anus, or gently rotate...

Embodiment 3

[0067] Example 3: Human astrovirus one-step fluorescence real-time quantitative RT-PCR detection kit clinical detection use

[0068] The above method was used to test 18 stool samples of other patients suspected of human astrovirus infection, among which 4 cases were positive for HAstV, and the virus fluorescence quantitative PCR amplification curve is shown in image 3 According to the Ct values ​​of these 4 cases of positive results combined with the amplification curve, the virus concentration of these 4 cases of HAstV positive samples was automatically analyzed by Roche LightCycler480 analysis software. The specific results are shown in Table 1.

[0069] Table 1 Virus concentration of 4 cases of HAstV positive samples

[0070] Sample serial number Ct value HAstv virus concentration (copy number / μl) sample 1 23.14 2.45X10 2 sample 2 21.89 2.2g×10 2 sample 3 21.46 2.58×10 3 Sample 4 21.86 2.48×10 2

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Abstract

The invention belongs to the field of gene detection, and relates to a real-time fluorescence RT-PCR (reverse transcription-polymerase chain reaction) detection kit for human astrovirus and application thereof. 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 astrovirus in excrement, anus 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 astrovirus 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 detection of human astroviruses. quantitative analysis. Background technique [0002] Astrovirus (Astrovirus) was discovered by Appleton and Higgins in 1975 from stool samples of children with diarrhea using electron microscopy, and it has been proved to be one of the important pathogens causing diarrhea in young infants, the elderly and immunocompromised persons. The only pathogen found to cause both sporadic and outbreaks of acute gastroenteritis. There are 5 to 6 star-shaped protrusions on the surface of the virus particles under the electron micro...

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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