Assay system and methods for detecting SARS-CV

• US 20050003340A1
• Filed: 07/01/2003
• Published: 01/06/2005
• Est. Priority Date: 07/01/2003
• Status: Active Grant

• Current Assignee: Asiagen Corporation
• Original Assignee: Asiagen Corporation
• Inventors: Chou, George Chin-Sheng
• Granted Patent: US 7,393,638 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/701 Specific hybridization probes

Abstract

The present invention relates to an assay system and methods for detecting SARS coronavirus (SARS-CV) from the samples (especially for urine) of suspected patient in the control of SARS to provide updated information of prognosis as well as the criteria for discharging a recovered patient from a hospital. The present invention also relates to an apparatus for performing the integration of thermal and magnetic control in the same apparatus to largely reduce the time of hybridization less than 20 minutes and the whole process of SARS-CV detection is less than 5 hours.

Claims

1. A method for detecting SARS coronavirus (SARS-CV) DNA from the samples of suspected patient comprising:

(a) hybridizing the SARS-CV cDNA with SARS-CV-specific probes in hybridization tube wherein the probe linked to magnetic bead;

(b) transferring hybridization tubes to magnetic wells for washing;

(c) adding blocking solution into the tubes;

(d) adding avidin enzyme complex or streptavidin enzyme complex into the tubes;

(e) performing washing reaction to remove interfering material by the aid of magnetic field;

(f) suspending magnetic beads; and

(g) detecting the luminescent or color change after adding substrate of enzyme.

2. The method of claim 1, wherein the sample is urine.

3. The method of claim 1, wherein the SARS-CV DNA are obtained from the PCR amplification.

4. The method of claim 3, wherein biotinylated primer pairs used in the PCR amplification are.

5. The method of claim 1, wherein the sequence of the probe in the step (a) is

6. The method of claim 1, wherein the streptavidin enzyme complex in the step (d) is streptavidin horseradish peroxidase (SA-HRP).

7. The method of claim 1, wherein the magnetic field in step (e) is magnetic rack.

8. The method of claim 1, wherein the suspension of magnetic beads of the step (f) is achieved by vortexing.

9. The method of claim 1, wherein the detection of the step (g) is performed by luminometer or spectrophotometer.

10. The method of claim 2, which further identify other assays for SARS-CV in a patient to provide updated information of prognosis as well as the criteria for discharging a recovered patient from a hospital.

11. An apparatus for performing the dissociation of nucleic acid double strands, hybridization, washing, the separation of magnetic beads and thermal control in the same apparatus, comprising:

(a) the means for fitting reaction containers;

(b) the means for controlling the temperature of the containers; and

(c) the means for controlling the magnetic force of the containers, wherein the means for controlling the temperature of the containers are connected to the means for fitting reaction containers, and the means for controlling the magnetic force of the containers are connected to the means for fitting reaction containers.

12. The apparatus of claim 11, wherein the means for controlling the temperature of the containers to heat the containers to perform the dissociation of nucleic acid double strands according to temperature change.

13. The apparatus of claim 11, wherein the means for controlling the magnetic force of the containers to perform the magnetic change of magnetic bead to facilitate hybridization, washing and the separation of magnetic beads in the containers.

14. An assay system for detecting SARS coronavirus (SARS-CV) DNA from the samples of suspected patient, the system comprising:

(i) a diagnostic kit for detecting amplified SARS coronavirus (SARS-CV) DNA in the presence of hybridization buffer comprising;

(a) a probe linked to magnetic bead;

(b) avidin enzyme complex or streptavidin enzyme complex; and

(c) enzyme substrate;

(ii) an apparatus for performing the dissociation of nucleic acid double strands, hybridization, washing, the separation of magnetic beads and thermal control in the same apparatus, comprising;

(a) the means for fitting reaction containers;

(b) the means for controlling the temperature of the containers; and

(c) the means for controlling the magnetic force of the containers,

wherein the means for controlling the temperature of the containers are connected to the means for fitting reaction containers, and the means for controlling the magnetic force of the containers are connected to the means for fitting reaction containers;

(iii) a magnetic rack to bind the magnetic bead on the wall of the containers; and

(iv) a detector.

15. The assay system of claim 14, wherein the sequence of probe in the kit is amine-TAGAGGGCTGTCATGCAACT (SEQ ID NO:

3).

16. The assay system of claim 14, wherein the streptavidin enzyme complex in the kit is streptavidin horseradish peroxidase (SA-HRP).

17. The assay system of claim 14, wherein the kit further comprises hybridization buffer, washing buffer and blocking buffer.

18. The assay system of claim 14, wherein the detector is luminometer or spectrophotometer.

19. The assay system of claim 14 which further identify other assays for SARS-CV in a patient to provide updated information of prognosis as well as the criteria for discharging a recovered patient from a hospital.

20. The assay system of claim 14 which reduces the time of hybridization less than 20 minutes and the whole process of SARS-CV detection less than 5 hours.