Multiplex PCR (Polymerase Chain Reaction) assay method for simultaneously assaying multiple respiratory viruses and probe set and kit thereof

A detection method and respiratory technology, applied in the directions of microorganism-based methods, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems that products cannot be widely used and high prices, and achieve low cost, short time consumption, and low interference. Effect

Active Publication Date: 2017-12-15
CHILDRENS HOSPITAL OF FUDAN UNIV +1
View PDF2 Cites 11 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although these high-throughput detection solutions reduce the detection cost compared with the single-pathogen detection system, making it possible for clinical application, the prices of interna

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Multiplex PCR (Polymerase Chain Reaction) assay method for simultaneously assaying multiple respiratory viruses and probe set and kit thereof
  • Multiplex PCR (Polymerase Chain Reaction) assay method for simultaneously assaying multiple respiratory viruses and probe set and kit thereof
  • Multiplex PCR (Polymerase Chain Reaction) assay method for simultaneously assaying multiple respiratory viruses and probe set and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Embodiment 1: detection pre-processing

[0091] 1) Specimen pretreatment

[0092] Add an equal volume of normal saline to the nasopharyngeal secretions and shake fully for 1 minute. Pipette 1ml of the supernatant into a 1.5ml EP tube, centrifuge at 13000rpm for 10min, discard the supernatant and keep the precipitate. Add 200 μL of normal saline to the precipitate and mix well by pipetting for later use.

[0093] 2) Viral DNA / RNA extraction

[0094] Use the magnetic bead method virus DNA / RNA nucleic acid extraction kit together with the nucleic acid extraction instrument to quickly extract the DNA / RNA of viruses in clinical respiratory specimens. The specific steps are carried out according to the instructions.

[0095] 3) Reverse transcription PCR

[0096] Using TAKARA's PrimeScript TM The reverse transcription kit performs reverse transcription PCR on the extracted viral nucleic acid, and the specific steps are carried out according to the instructions.

Embodiment 2

[0097] Embodiment 2: Multiplex PCR detection and result interpretation

[0098]A Roche LC480II fluorescent quantitative PCR instrument was used, and the reverse transcription PCR obtained in Example 1) was used as a template to carry out multiple regeneration PCR reactions. The amplification program was set at 95°C for 5min; 95°C for 30s, 55°C for 30s, and 72°C for 30s for 50 cycles, and the fluorescence signals of FAM, HEX, ROX, and CY5 channels were collected simultaneously during the 55°C annealing stage. The melting curve analysis program was set as: 35°C for 5 minutes; the temperature was gradually raised to 85°C, and the fluorescence signal was collected 10 times for every 1°C increase during the process;

[0099] In the multiplex PCR reaction step, the multiplex PCR reaction system is a 25 μL reaction system, including:

[0100] 2.5 μL mTaq PCR Buffer 10×, 2.0 μL dNTP with a concentration of 2.5 mM, 0.5 μL mTaq DNA polymerase with a concentration of 5 U / μL, 2.0 μL temp...

Embodiment 3

[0109] Example 3: Studying the Effect of 5' Exonuclease Activity on the Melting Curve of the Probe

[0110] Taking the detection of adenovirus as an example, we compared the effects of DNA polymerases with or without 5' exonuclease activity on the amplification curve and probe melting curve analysis. The Taq DNA polymerase PCR reaction with 5' exonuclease activity adopts the SuperReal PreMix (Probe) kit produced by Beijing Tiangen Biochemical Technology Co., Ltd., and the reaction system is: 25 μL of the reaction system contains 1 × SuperRealPreMix, 0.1 mM upstream Primer, 1mM downstream primer, 0.2mM probe, 2μL template, make up to 25μL with sterile water. The DNA polymerase lacking 5' exonuclease activity uses mTaq DNA polymerase (Beijing Kangwei Century Co., Ltd.), and the reaction system is: 25μL reaction system contains 1×mTaq PCR Buffer, 0.2mM dNTP, 2.5U mTaqDNA Polymerase , 0.1 mM upstream primer, 1 mM downstream primer, 0.2 mM probe, 2 μL template, filled to 25 μL w...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a multiplex PCR (Polymerase Chain Reaction) assay method for simultaneously assaying multiple respiratory viruses, which includes the following steps: design of probes, design of upstream and downstream primer sequences, multiplex PCR reaction and dissociation curve analysis. By designing Taqman probes for the conserved group sequences of the respiratory viruses and adopting different fluorophores when Tm phase difference is less than or equal to 6 DEG C, the method can assay a variety of respiratory viruses with high throughput and high accuracy. The invention further provides a multiplex PCR assay probe set and a corresponding kit for simultaneously assaying multiple respiratory viruses, which are convenient to apply and have a great market and application prospect.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, and in particular relates to a method for simultaneously detecting multiple respiratory viruses, a probe group and a kit thereof. Background technique [0002] Diseases caused by respiratory infections have caused a huge disease burden to the global health system and are one of the leading causes of death of children under 5 years old in developing countries including China. In 2013, 6.3 million children under the age of 5 died globally, of which 935,000 (14.8%) died of pneumonia. In addition to increasing mortality, respiratory infections are a major cause of increased morbidity among children under 5 years of age. In economically developed countries, 3% to 18% of hospitalized children are caused by lower respiratory tract infection. At the same time, children's respiratory tract infection also brings a huge economic burden. A multi-center study in Germany shows that the average annu...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2537/143C12Q2561/101
Inventor 徐锦柳鹏程钟华清徐梦华卢丽娟贾然李杨霞王伟伟
Owner CHILDRENS HOSPITAL OF FUDAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap