Multi-allelic molecular detection of SARS-associated coronavirus

a coronavirus and molecular detection technology, applied in the field of multiallelic molecular detection of sars-associated coronavirus, can solve the problems of failure to consider, failure to account for the possibility of continuous and/or multiple introduction of non-genetically identical sars-cov strains into the human population, and deletion of part of an important gene, so as to enhance the likelihood of fundamental genetic drift of the virus, minimize the likelihood, and enhance the intrinsic sensitivity of the assay

Inactive Publication Date: 2006-01-05
BIRCH BIOMEDICAL RES
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  • Summary
  • Abstract
  • Description
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AI Technical Summary

Benefits of technology

[0014] Multiple target sequences within the SARS-CoV S, E, M and N genes (Urbani and Tor2 strains) were identified. The S, M, and E genes encode structural proteins that are present on the outside of the virus, whereas the N gene encodes a structural protein that is required for viral RNA packaging inside the virion. The principle underlying the selection of four target sequences that uniquely identify SARS-CoV (rather than only one target sequence) is that the use of four different targets enhances the likelihood that the fundamental genetic drift of the virus will not l

Problems solved by technology

The main problems with current molecular diagnostic assays are: a) failure to consider the intrinsically polymorphic nature of coronaviruses, including the current SARS-CoV strains originated from the Tor2 and Urbani isolates—the ability of the virus to mutate and recombine during the period of time it is within the infected individual, and during horizontal transmission; and b) failure to account for the possibility of continuous and/or multiple introduction of non non-genetically identical SARS-CoV strains into the human population.
Coronaviruses, including SARS-CoV, are quite sloppy when it comes to replicating their g

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

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example 1

Design of SARS-CoV-Specific Molecular Beacons and Primers for Reverse Transcription and for PCR

[0051] Purpose: The overall rationale in the design of molecular beacons and oligonuclotides for our SARS assay is to construct mismatch-tolerant molecular beacons that are thermodynamically compatible to work in a five-amplicon multiplex assay.

[0052] Design: The molecular beacons were designed so that they are able to hybridize to their targets at the annealing temperature of the PCR, while unbound molecular beacons remain in the closed conformation. These basic aspects were achieved by using coronavirus gene-specific multiple alignments and thermodynamic considerations to select the target sequences, the identity and length of the PCR primers, the identity and length of the probe sequences (target recognition sequences), and the length of the arm sequences.

[0053] Materials: In order to theoretically calculate the melting temperatures of the PCR primers and the probe-target hybrids, wa...

example 2

Experimental Characterization of the Molecular Beacons and the Molecular Beacon-Target Complexes

[0057] Purpose: The overall rationale of these experiments is to evaluate the thermodynamic properties of the constructed molecular beacons prior to carrying out real-time PCR experiments.

[0058] Design: For each molecular beacon, we have determined two melting curves—one for beacon alone, and one for the beacon-target complex—by using the ABI Prism 7700 spectrofluorometric thermal cycler.

[0059] Materials: For each molecular beacon, melting curves were obtained by preparing two tubes containing 50 μL of 200 nM molecular beacon dissolved in 3.5 mM MgCl2 and 10 mM Tris-HCl, pH 8.0, and by adding a complementary oligonucleotide target to one of the tubes at a final concentration of 400 nM.

[0060] The fluorescence of each solution was determined as a function of temperature, using a thermal cycler with the capacity to monitor fluorescence. Temperature was decreased linearly with time from 8...

example 3

Uniplex SARS-CoV Viral and IPC PCR Amplifications Using SYBR Green to Detect the Amplicaons

[0064] Purpose: The overall rationale of these experiments is to evaluate the PCR primers and PCR conditions.

[0065] Design: For each SARS-CoV gene-specific and IPC amplification, a synthetic target DNA was used. PCR reactions were performed using a spectrofluorometric thermal cycler (Cepheid).

[0066] Materials: The PCR protocols are shown in the following exhibits:

[0067] for S Gene:

[0068] (A) SYBR Green-Based Detection of S Gene Amplicon (LK250) of SARS-Associated CoV

MixturePer reactiondH20 15 μl10X PCR Buffer (10X)2.5 μlMgCl2 (25 mM)4.0 μlPlat Taq DNA Pol (5 Uμl−1)0.3 μldNTP (25 mM)0.3 μlLK251 (10 pmole / μl)0.5 μlLK252 (10 pmole / μl)0.5 μlSybr Green DNA (25X)1.0 μlTarget DNA1.0 μlTOTAL25.0 μl 

[0069] Smart Cycler (Cepheid)

DNA Denaturation & Enzyme ActivationCycles: 1Target Temperature (° C.):95120 sec

[0070]

Primary AmplificationCycles: 35Denaturation:95° C.15 secAnnealing:53° C.15 secSpec...

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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

[0001] This Patent Application is a Non-Provisional Patent Application based on Provisional Patent Application Ser. No. 60 / 496,995 (Attorney Docket 42392-191812) filed Aug. 22, 2003, and Provisional Application Ser. No. 60 / 576,314 (Attorney Docket 42392-203685) filed on Jun. 3, 2004, the contents of which are incorporated herein by reference in their entiretyBACKGROUND OF THE INVENTION [0002] 1. Field of Invention [0003] This invention is directed to methods for the detection and / or quantitation of the SARS virus, reagents and test kits containing the same for use in the method. [0004] 2. Background of the Invention [0005] Severe acute respiratory syndrome (SARS) is one of the most recent emerging infectious diseases. The cause of SARS has been identified as a new coronavirus—a virus within the family Coronaviridae—designated as the “SARS coronavirus” (SARS-CoV) [1, 2] by the World Health Organization, following assessment of causation according to Koch's postulates, including monke...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68G01N33/53C12N7/00C12N7/01
CPCC12Q1/70G01N33/56983C12Q1/701
Inventor KOSTRIKIS, LEONDIOS
Owner BIRCH BIOMEDICAL RES
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