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1. US20050142536 - Method and kit for the detection of a novel coronoavirus associated with the severe acute respiratory syndrome (SARS)

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Claims

1. A method for detecting Severe Acute Respiratory Syndrome-associated virus (SARS-associated virus), named HPAC (Human Pneumonia-Associated Coronavirus), by contacting a biological sample with a set of primers and a probe, incubating under conditions allowing amplification of nucleic acid using said primers, and determining binding of said probe to amplified nucleic acid, wherein detecting binding of said probe to amplified nucleic acid indicates the presence of SARS-associated virus, wherein the forward primer has a length of approximately 18 to 31 nucleotides and binds to a region defined by nucleotides 69 to 98 of the sequence shown as SEQ ID NO:1; and wherein the reverse primer has a length of approximately 18 to 31 nucleotides and binds to a region defined by nucleotides 123 to 168 of the sequence shown as SEQ ID NO:1; and wherein the probe has a length of approximately 18 to 35 nucleotides and binds to a region defined by nucleotides 89 to 132 of the sequence shown as SEQ ID NO:1; and wherein the probe is labeled with two dyes, one dye of which is a fluorescent reporter dye, and one dye of which is a quencher dye, and wherein at least one dye is a fluorescent dye.
2. The method of claim 1, wherein the amplification and detection are performed using real time RT-PCR.
3. Method according to claim 1, wherein the forward primer has the sequence shown as SEQ ID NO:2, the reverse primer has the sequence shown as SEQ ID NO:3, and wherein the probe has the sequence shown as SEQ ID NO:4.
4. Method according to claim 1, wherein the reporter dye is FAM, 6-FAM, 5-FAM and ALEXA-288.
5. Method according to claim 1, wherein the quencher dye is TAMRA, DABCYL or QSY.
6. Method according to claim 1, wherein detection is quantitative detection of the real time fluorescence signal intensity.
7. Method according to claim 1, wherein the biological sample is a body fluid.
8. Method according to claim 1, wherein the biological sample is sputum, feces or blood.
9. Kit for performing the method of claim 1.
10. Kit for detecting Severe Acute Respiratory Syndrome-associated virus (SARS-associated virus), named HPAC (Human Pneumonia-Associated Coronavirus), through real time RT-PCR, comprising a forward primer having a length of approximately 18 to 31 nucleotides, which forward primer binds to a region defined by nucleotides 69 to 98 of the sequence shown as SEQ ID NO:1; a reverse primer having a length of approximately 18 to 31 nucleotides, which reverse primer binds to a region defined by nucleotides 123 to 168 of the sequence shown as SEQ ID NO:1; and a probe having a length of approximately 18 to 35 nucleotides, which probe binds to a region defined by nucleotides 89 to 132 of the sequence shown as SEQ ID NO:1; and wherein the probe is labeled with two dyes, one dye of which is a fluorescent reporter dye, and one dye of which is a quencher dye, and wherein at least one dye is a fluorescent dye.
11. Kit according to claim 10, wherein the forward primer has the sequence shown as SEQ ID NO:2, the reverse primer has the sequence shown as SEQ ID NO:3, and wherein the probe has the sequence shown as SEQ ID NO:4.
12. Kit according to claim 10, wherein the reporter dye is FAM, 6-FAM, 5-FAM and ALEXA-288.
13. Kit according to claim 10, wherein the quencher dye is TAMRA, DABCYL or QSY.
14. Kit according to claim 10, further comprising enzymes and reagents required for performing a real time RT-PCR reaction.
15. Oligonucleotide having a length of approximately 18 to 31 nucleotides, which binds to a region defined by nucleotides 69 to 98 of the sequence shown as SEQ ID NO:1.
16. Oligonucleotide according to claim 14, having the sequence shown as SEQ ID NO:2.
17. Oligonucleotide having a length of approximately 18 to 31 nucleotides, which binds to a region defined by nucleotides 123 to 168 of the sequence shown as SEQ ID NO:1.
18. Oligonucleotide according to claim 16, having the sequence shown as SEQ ID NO:3.
19. A method for detecting Severe Acute Respiratory Syndrome-associated virus (SARS-associated virus), named HPAC (Human Pneumonia-Associated Coronavirus), by contacting a biological sample with a set of primers and a probe, incubating under conditions allowing amplification of nucleic acid using said primers, and determining binding of said probe to amplified nucleic acid, wherein detecting binding of said probe to amplified nucleic acid indicates the presence of SARS-associated virus, wherein the forward primer has a length of approximately 18 to 35 nucleotides and binds to a region defined by nucleotides about 1 to about 240 of the sequence shown as SEQ ID NO:1; and wherein the reverse primer has a length of approximately 18 to 35 nucleotides and binds to a region defined by nucleotides about 60 to about 300 of the sequence shown as SEQ ID NO:1; and wherein the probe has a length of approximately 12 to 40 nucleotides and binds to a region defined by nucleotides about 21 to about 279 of the sequence shown as SEQ ID NO:1; and wherein the probe is labeled with two dyes, one dye of which is a fluorescent reporter dye, and one dye of which is a quencher dye, and wherein at least one dye is a fluorescent dye.
20. A kit for performing the method of claim 18.