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Method of diagnosing SARS corona virus infection

A coronavirus and antibody technology, applied in the field of diagnosing SARS coronavirus infection, can solve problems such as time-consuming and difficult RNA extraction methods

Inactive Publication Date: 2006-07-19
AGENCY FOR SCI TECH & RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] However, existing protocols for PCR do not escape their limitations
Despite their high sensitivity, such assays have inherent problems: scientists and clinicians around the world are uncertain which samples from patients (respiratory samples, saliva, stool, blood or conjunctival fluid) provide the best source of reproducible RNA. Preparation; the RNA extraction method is not simple, and if it is not done well, it may prepare an RNA preparation that cannot be used in the reverse transcription step of converting viral RNA into DNA; if it is necessary to perform confirmatory experiments with several pairs of primers, extraction, reverse transcription and the whole process of PCR is time consuming
To date, there is still no standardized test for the diagnosis of SARS, whether such a test is antigen- or antibody-based

Method used

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  • Method of diagnosing SARS corona virus infection
  • Method of diagnosing SARS corona virus infection
  • Method of diagnosing SARS corona virus infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0125] Example 1: Use of SARS CoV protein as a diagnostic marker

[0126] Materials and methods

[0127] Materials: Unless otherwise specified, all reagents used in this study were purchased from Sigma (St. Louis, Mo.). All cell lines were purchased from the American Type Culture Collection (Manassas, Va.) and cultured at 37°C in 5% CO 2 , Dulbecco's modified Eagle medium, the medium contains 1g glucose / liter, 2mM L-glutamine, 1.5g sodium bicarbonate / liter, 0.1mM non-essential amino acids, 0.1mg streptavidin / ml , 100 U of penicillin and 5% fetal bovine serum (HyClone, South Logan, Utah).

[0128] Construction of plasmids: For transient expression in mammalian cells, the vector used was pXJ40HA for tagging the N-terminus of the protein with one hemagglutinin (HA) epitope (15); The vector used for tagging at the C-terminus of the protein is pXJ40-3-HA. For expression of glutathione S-transferase (GST) fusion proteins in bacteria, the gene was cloned in-frame with GST into pG...

Embodiment 2

[0151] Example 2: ELISA and immunochromatographic assays

[0152] Materials and methods

[0153] Recombinant proteins: Materials and methods used to obtain recombinant proteins are as described in Example 1 above. For a study, use Superdex TM Gst-U274 was further purified on an S-200 HR10 / 30 column on an AKTA Fast Protein Liquid Chromatography (FPLC) system (Amersham). The buffer used contained 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 6 M urea and 1 mM β-mercaptoethanol at a flow rate of 0.5 ml / min, and 1 ml of fragments were collected. Fragments 12 and 13 were joined together and dialyzed against phosphate buffered saline (PBS) overnight with at least 3 buffer changes.

[0154] Serum samples: 74 convalescent serum samples were collected from patients admitted to Tan Tock Seng Hospital or Singapore General Hospital. Ninety-one control sera were obtained from healthy local donors working at the Institute of Molecular and Cell Biology in Singapore. All samples were collected ...

Embodiment 3

[0166] Example 3: Evaluation of ELISA and Immunochromatographic Assays

[0167] determination

[0168] Materials and methods

[0169] Serum samples: Serum samples were collected from patients clinically suspected of SARS according to the WHO definition (18) and from patients admitted between March 18 and May 24, 2003, in three acute regional hospitals in Hong Kong. A total of 227 serum samples from these patients were assayed with the IF assay (9) and confirmed to have IF titers >1:10-1:2560 dilution. At the same time, 385 serum samples from 385 healthy donors collected in Hong Kong were used as controls. In addition, 1066 sera from healthy donors purchased from BioClinical Partner Inc. (Franklin, MA) were included in this study as additional healthy controls. Genelabs Diagnostics (GLD, Singapore) also used archived serum samples from various previous studies for disease control; this included 50 samples from patients with non-SARS-associated fever (identified as dengue fev...

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Abstract

The present invention relates to methods of detecting the presence or absence of antibody to SARS coronavirus in a sample, based on the discovery that the S, M, E, N and U274 proteins of SARS coronavirus are antigenic. Such methods may be used to diagnose whether a patient has been infected by or exposed to SARS coronavirus. Antibodies directed against S, M, E, N and U274 proteins of SARS are also provided.

Description

[0001] Cross-references to related literature [0002] This application claims priority to US Provisional Application No. 60 / 477,059, filed June 10, 2003, which is incorporated herein by reference. technical field [0003] The present invention mainly relates to the method for diagnosing virus infection, especially the method for diagnosing SARS coronavirus infection. Background technique [0004] The emerging severe acute respiratory syndrome (SARS) is a severe respiratory disease of global significance. Infected patients can show symptoms of atypical pneumonia, and early and precise distinction between SARS and atypical pneumonia is the key to the successful control of this highly contagious disease. [0005] The highly contagious nature of the SARS virus and its high mortality rate can be devastating and costly in an increasingly globalized world. When the SARS epidemic spread beyond its origin in China's Guangdong Province in 2003, the World Health Organization (WHO) w...

Claims

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Application Information

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IPC IPC(8): G01N33/68C07K16/10G01N33/569
CPCG01N2333/165C07K16/10G01N33/56983C07K16/1002
Inventor 陈燕如吴佩燕伯特拉姆·克林顿·菲尔丁申硕林成义洪万进阮一骏卫嘉玲
Owner AGENCY FOR SCI TECH & RES
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