Multi-allelic molecular detection of SARS-associated coronavirus

A SARS virus and coronavirus technology, applied in the field of multi-allelic molecular detection of SARS-related coronaviruses, can solve problems such as inability to explain multiple infected people, SARS virus infection, and failure to take into account the polymorphism of coronaviruses

Inactive Publication Date: 2007-01-31
BIRCH BIOMEDICAL RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main problems with existing molecular diagnostic assays are: a) failure to take into account the inherently polymorphic nature of coronaviruses, including the current SARS-CoV isolates originating from the Tor2 and Urbani isolates - i.e. the period during which the virus survives in an infected individual and mutation and recombination capabilities during horizontal transmission; b) does not account for the possibility of non-genetically identical SARS-CoV isolates serially and / or repeatedly infecting populations
Genetic deletion likely helped SARS virus jump from its animal host to humans

Method used

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  • Multi-allelic molecular detection of SARS-associated coronavirus
  • Multi-allelic molecular detection of SARS-associated coronavirus
  • Multi-allelic molecular detection of SARS-associated coronavirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 - Molecular Beacons Specific to SARS-CoV,

[0052] And the design of primers required for reverse transcription and PCR

[0053] Purpose: The overall rationale for designing the molecular beacons and oligonucleotides required for this SARS assay was to construct thermodynamically compatible mismatch-tolerant molecules for use in a 5-amplicon multiplex.

[0054] Design: The Molecular Beacon is designed such that it hybridizes to its target at the PCR annealing temperature, while the unbound Molecular Beacon remains in a closed conformation. These basic aspects are achieved by using coronavirus gene-specific multiple comparisons and thermodynamic considerations to select target sequences, identity and length of PCR primers, identity and length of probe sequences (target recognition sequences), and The length of the arm sequence.

[0055] Materials: In order to theoretically calculate the melting temperature of PCR primers and pr...

Embodiment 2

[0059] Example 2 - Experimental Properties of Molecular Beacons and Molecular Beacon-Target Complexes

[0060] Purpose: The general principle of this set of experiments is to evaluate the thermodynamic properties of the constructed molecular beacons before performing real-time PCR experiments.

[0061] Design: For each molecular beacon, we have measured two melting curves using an ABI Prism 7700 Fluorescence Spectrophotometer Thermal Cycler - one for the molecular beacon alone and one for the beacon-target complex curve.

[0062] Materials: For each molecular beacon, its melting curve can be obtained by the following steps: Prepare two tubes of 50 μL dissolved in 3.5 mM MgCl 2 and 200 nM Molecular Beacons in 10 mM Tris-HCl, pH 8.0, to one of the tubes was added a complementary oligonucleotide target to a final concentration of 400 nM.

[0063] The fluorescence of each solution was determined as a function of temperature using a thermal cycler that can monitor fluorescence. ...

Embodiment 3

[0067] Embodiment 3-Using SYBR Green to detect amplicon

[0068] Uniplex SARS-CoV virus and IPC PCR amplification

[0069] Purpose: The general principle of this set of experiments is to evaluate PCR primers and PCR conditions.

[0070] Design: Synthetic target DNA was used for each SARS-CoV gene-specific amplification and IPC amplification. PCR reactions were performed using a spectrofluorometer thermal cycler (Cepheid).

[0071] Materials: The PCR protocol is as follows:

[0072] For S gene:

[0073] (A) Detection of the S gene amplicon (LK250) of SARS-associated CoV based on SYBR Green

[0074] mixture per reaction system

[0075] dH 2 O 15 μL

[0076] 10X PCR Buffer (10X) 2.5μL

[0077] MgCl 2 (25mM) 4.0μL

[0078] Plat Taq DNA Pol (5 0.3μL

[0079] UμL -1 )

[0080] dNTP (25mM) 0.3μL

[0081] LK251 (10pmole / μL) 0.5μL

[0082] LK252 (10pmole / μL) 0.5μL

[0083] Sybr Green DNA (25X) 1.0μL

[0084] Target DNA 1.0μL

[0085] 25.0 μL total ...

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Abstract

The subject invention relates to a multiple-allelic RT-real-time polymerase chain reaction (PCR) assay for coronaviruses including the SARS virus. Multiple target sequences within the SARS-CoV, S, E, M and N genes are identified. The use of the four different targets enhances the likelihood that the fundamental genetic drift of the virus will not lead to a false negative result. Multiplex assays format for the assay are envisioned. Thus, the present invention allows for early diagnosis of a SARS infection. The assay would be useful in the context of monitoring treatment regimens, screening potential anti SARS agents, and similar applications requiring qualitative and quantitative determinations.

Description

field of invention [0001] The present invention mainly relates to a detection and / or quantification method of SARS virus, as well as a reagent used in the method and a detection kit comprising the reagent. Background of the invention [0002] Severe acute respiratory syndrome (SARS) is a newly emerged acute infectious disease. The cause of SARS has been identified as a new coronavirus of the Coronaviridae family. The World Health Organization (WHO) named the virus as "SARS coronavirus" (SARS-CoV) [1, 2]. Coronavirus is an enveloped positive-sense single-stranded RNA virus with a genome length of about 30kb, which is the largest among all RNA virus genomes and can replicate in the cytoplasm of host cells without going through a DNA intermediate. Coronaviruses have been reported to cause the common cold in humans, and respiratory, digestive, neurological diseases and hepatitis in animals. Human coronaviruses are usually difficult to culture in vitro, while most animal coron...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCY02A50/30
Inventor L·G·科斯特日克斯
Owner BIRCH BIOMEDICAL RES
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