Methods and compositions for detecting sars virus

• US 20070037140A1
• Filed: 05/09/2003
• Published: 02/15/2007
• Est. Priority Date: 05/09/2003
• Status: Abandoned Application

• Current Assignee: CapitalBio Corporation, Tsinghua University
• Original Assignee: Capital Biochip Company, Ltd., Tsinghua University
• Inventors: Cheng, Jing, Wang, Jianwei, Hong, Tao, Zhou, Yiming, Cheng, Yun, Li, Ze, Tao, Shengce, Shan, Quanzhong
• Application Number:
  US10/556,182
• Publication Number:
  US 20070037140A1
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/701 Specific hybridization probes

Abstract

This invention relates generally to the field of virus detection. In particular, the invention provides chips, probes, primers, kits and methods for amplifying and detecting SARS-CoV nucleotide sequence. The clinical and other uses of the present chips, probes, primers, kits and methods are also contemplated.

Claims

1. A chip for assaying for a coronaviruse causing the severe acute respiratory syndrome (SARS-CoV), which chip comprises a support suitable for use in nucleic acid hybridization having immobilized thereon at least two oligonucleotide probes complementary to at least two different nucleotide sequences of SARS-CoV genome, each of said two different nucleotide sequences comprising at least 10 nucleotides.

2. The chip of claim 1, wherein the at least two different nucleotide sequences of SARS-CoV genome comprises:

a) a nucleotide sequence of at least 10 nucleotides located within a conserved region of SARS-CoV genome and a nucleotide sequence of at least 10 nucleotides located within a variable region of SARS-COV genome;

or

b) a nucleotide sequence of at least 10 nucleotides located within a structural protein coding gene of SARS-CoV genome and a nucleotide sequence of at least 10 nucleotides located within a non-structural protein coding gene of SARS-CoV genome.

3. The chip of claim 1, which further comprises:

a) at least one of the following three oligonucleotide probes;

an immobilization control probe that is labeled and does not participate in any hybridization reaction when a sample containing or suspected of containing of a SARS-CoV is contacted with the chip, a positive control probe that is not complementary to any SARS-CoV sequence but is complementary to a non-SARS-CoV-sequence contained in the sample and a negative control probe that is not complementary to any nucleotide sequence contained in the sample; and

b) a blank spot.

4. The chip of claim 1, which comprises at least two oligonucleotide probes complementary to two different nucleotide sequences of at least 10 nucleotides, respectively, located within a conserved region of SARS-CoV genome, located within a structural protein coding gene of SARS-CoV genome or located within a non-structural protein coding gene of SARS-CoV genome.

5. The chip of claim 1, wherein:

a) the conserved region of SARS-CoV genome is a region located within the Replicase 1A or 1B gene or the Nucleocapsid N) gene of SARS-CoV;

b) the structural protein coding gene of SARS-CoV genome is a gene encoding the Spike glycoprotein (S), the small envelope protein (E) or the Nucleocapsid protein (N);

or

c) the non-structural protein coding gene of SARS-CoV genome is a gene encoding the Replicase 1A or 1B.

6. The chip of claim 1, wherein the variable region of SARS-CoV genome is a region located within the Spike glycoprotein (S) gene of SARS-CoV.

7. The chip of claim 1, which comprises at least two of the following four oligonucleotide probes:

two oligonucleotide probes complementary to two different nucleotide sequences of at least 10 nucleotides located within the Replicase 1A or 1B gene of SARS-CoV, an oligonucleotide probe complementary to a nucleotide sequence of at least 10 nucleotides located within the N gene of SARS-CoV and an oligonucleotide probe complementary to a nucleotide sequence of at least 10 nucleotides located within the S gene of SARS-CoV.

8. The chip of claim 7, wherein one of the two different nucleotide sequences located within the Replicase 1A or 1B gene of SARS-CoV comprises a nucleotide sequence that:

a) hybridizes, under high stringency, with a Replicase 1A or 1B nucleotide sequence, or a complementary strand thereof, that is set forth in Table 3;

or

b) has at least 90% identity to a Replicase 1A or 1B nucleotide sequence comprising a nucleotide sequence, or a complementary strand thereof, that is set forth in Table 3.

9. The chip of claim 8, wherein one of the two different nucleotide sequences located within the Replicase 1A or 1B gene of SARS-CoV comprises a nucleotide sequence that is set forth in Table 3.

10. The chip of claim 7, wherein the nucleotide sequence located within the N gene of SARS-CoV comprises a nucleotide sequence that:

a) hybridizes, under high stringency, with a N nucleotide sequence, or a complementary strand thereof, that is set forth in Table 3;

or

b) has at least 90% identity to a N nucleotide sequence comprising a nucleotide sequence, or a complementary strand thereof, that is set forth in Table 3.

11. The chip of claim 1O, wherein the nucleotide sequence located within the N gene of SARS-CoV comprises a nucleotide sequence that is set forth in Table 3.

12. The chip of claim 7, wherein the nucleotide sequence located within the S gene of SARS-CoV comprises a nucleotide sequence that:

a) hybridizes, under high stringency, with a S nucleotide sequence, or a complementary strand thereof, that is set forth in Table 3;

or

b) has at least 90% identity to a S nucleotide sequence comprising a nucleotide sequence, or a complementary strand thereof, that is set forth in Table 3.

13. The chip of claim 12, wherein the nucleotide sequence located within the S gene of SARS-CoV comprises a nucleotide sequence that is set forth in Table 3.

14. The chip of claim 3, wherein the label of the immobilization control probe is selected from the group consisting of a chemical, an enzymatic, an immunogenic, a radioactive, a fluorescent, a luminescent and a FRET label.

15. The chip of claim 3, wherein the non-SARS-CoV-sequence is spiked in the sample to be assayed.

16. The chip of claim 15, wherein the spiked non-SARS-CoV-sequence is a sequence of Arabidopsis origin.

17. The chip of claim 7, which comprises two oligonucleotide probes complementary to two different nucleotide sequences located within the Replicase 1A or 1B gene of SARS-CoV, an oligonucleotide probe complementary to a nucleotide sequence located within the N gene of SARS-CoV, an oligonucleotide probe complementary to a nucleotide sequence located within the S gene of SARS-CoV, an immobilization control probe that is labeled and does not participate in any hybridization reaction when a sample containing or suspected of containing of a SARS-CoV is contacted with the chip, a positive control probe that is not complementary to any SARS-CoV sequence but is complementary to a non-SARS-CoV-sequence contained in the sample and a negative control probe that is not complementary to any nucleotide sequence contained in the sample.

18. The chip of claim 17, which comprises multiple spots of the two oligonucleotide probes complementary to two different nucleotide sequences located within the Replicase 1B gene of SARS-CoV, the oligonucleotide probe complementary to a nucleotide sequence located within the N gene of SARS-CoV, the oligonucleotide probe complementary to a nucleotide sequence located within the S gene of SARS-CoV, the immobilization control probe, the positive control probe and the negative control probe.

19. The chip of claim 1, further comprising an oligonucleotide probe complementary to a nucleotide sequence of a coronaviruse not related to SARS-CoV.

20. The chip of claim 19, wherein the coronaviruse not related to the SARS is the Group I, II or III coronaviruse or is a coronaviruse that infects an avian species, an equine species, a canine species, a feline species, a porcine species, a calf species, a bovine species, a murine species, a puffinosis species, a rat species, a turkey species or a human species.

21. The chip of claim 3, wherein at least one of the oligonucleotide probe comprises, at its ′

5 end, a poly dT region to enhance its immobilization on the support.

22. The chip of claim 1, wherein at least one of the oligonucleotide probes is complementary to a highly expressed nucleotide sequence of SARS-CoV genome.

23. The chip of claim 1, wherein the support comprises a surface that is selected from the group consisting of a silicon, a plastic, a glass, a ceramic, a rubber, and a polymer surface.

24. A method for assaying for a SARS-CoV in a sample, which methods comprises:

a) providing a chip of claim 1;

b) contacting said chip with a sample containing or suspected of containing a SARS-CoV nucleotide sequence under conditions suitable for nucleic acid hybridization; and

c) assessing hybrids formed between said SARS-CoV nucleotide sequence, if present in said sample, and said at least two oligonucleotide probes complementary to two different nucleotide sequences of SARS-CoV genome, respectively, to determine the presence, absence or amount of said SARS-CoV in said sample,

whereby detection of one or both said hybrids indicates the presence of said SARS-CoV in said sample.

25. A method for assaying for a SARS-CoV in a sample, which methods comprises:

a) providing a chip of claim 2;

b) contacting said chip with a sample containing or suspected of containing a SARS-CoV nucleotide sequence under conditions suitable for nucleic acid hybridization; and

c) assessing hybrids formed between said SARS-CoV nucleotide sequence, if present in said sample, and

i) said oligonucleotide probe complementary to a nucleotide sequence located within a conserved region of SARS-CoV genome and an oligonucleotide probe complementary to a nucleotide sequence located within a variable region of SARS-CoV genome, respectively;

or

ii) said oligonucleotide probe complementary to a nucleotide sequence located within a structural protein coding gene of SARS-CoV genome and an oligonucleotide probe complementary to a nucleotide sequence located within a non-structural protein coding gene of SARS-CoV genome,

to determine the presence, absence or amount of said SARS-CoV in said sample,

whereby detection of one or both said hybrids indicates the presence of said SARS-CoV in said sample.

26. A method for assaying for a SARS-CoV in a sample, which methods comprises:

a) providing a chip of claim 3;

b) contacting said chip with a sample containing or suspected of containing a SARS-CoV nucleotide sequence under conditions suitable for nucleic acid hybridization; and

c) assessing;

(i) hybrids formed between said SARS-CoV nucleotide sequence, if present in the sample, and the oligonucleotide probe complementary to a nucleotide sequence within a conserved region of SARS-CoV genome and an oligonucleotide probe complementary to a nucleotide sequence located within a variable region of SARS-CoV genome, respectively;

(ii) a label comprised in the immobilization control probe, or a hybrid(s) involving the positive control probe and/or the negative control probe; and

(iii) a signal at said blank spot

to determine the presence, absence or amount of said SARS-CoV in a sample.

27. The method of claim 26, wherein the chip comprises two oligonucleotide probes complementary to two different nucleotide sequences located within the Replicase 1A or 1B gene of SARS-CoV, an oligonucleotide probe complementary to a nucleotide sequence located within the N gene of SARS-CoV, an oligonucleotide probe complementary to a nucleotide sequence located within the S gene of SARS-CoV, an immobilization control probe, a positive control probe and a negative control probe and the presence of the SARS-CoV is determined when:

a) a positive hybridization signal is detected using at least one of the two oligonucleotide probes complementary to two different nucleotide sequences located within the Replicase 1A or 1B gene of SARS-CoV, the oligonucleotide probe complementary to a nucleotide sequence located within the N gene of SARS-CoV and/or the oligonucleotide probe complementary to a nucleotide sequence located within the S gene of SARS-CoV;

b) a positive signal is detected from the immobilization control probe;

c) a positive hybridization signal is detected using the positive control probe;

d) a positive hybridization signal is not detected using the negative control probe; and

e) a positive hybridization signal is not detected at the blank spot.

28. The method of claim 27, wherein detecting a positive hybridization signal using at least one of the two oligonucleotide probes complementary to two different nucleotide sequences located within the Replicase 1A or 1B gene of SARS-CoV, or the oligonucleotide probe complementary to a nucleotide sequence located within the N gene of SARS-CoV, while not detecting a positive hybridization signal using the oligonucleotide probe complementary to a nucleotide sequence located within the S gene of SARS-CoV indicates mutation of the SARS-CoV.

29. The method of claim 24, which is used to positively identify SARS-CoV infected patients from a population of patients who have SARS-like symptoms.

30. The method of claim 24, wherein the chip of claim 19 is used and the method is used to positively identify SARS-CoV infected patients from patients who have been infected with a coronaviruse not related to the SARS.

31. The method of claim 24, wherein the chip of claim 22 is used and the method is used to diagnose early-stage SARS patients.

32. The method of claim 31, wherein the early-stage SARS patients have been infected with SARS-CoV from about less than one day to about three days.

33. The method of claim 24, wherein the SARS-CoV nucleotide sequence is a SARS-CoV RNA genomic sequence or a DNA sequence amplified from an extracted SARS-CoV RNA genomic sequence.

34. The method of claim 33, wherein the SARS-CoV RNA genomic sequence is extracted from a SARS-CoV infected cell using the QIAamp Viral RNA kit, the Chomczynsiki-Sacchi technique or TRIzol.

35. The method of claim 34, wherein the SARS-CoV RNA genomic sequence is extracted from a SARS-CoV infected cell using the QIAamp Viral RNA kit.

36. The method of claim 33, wherein the SARS-CoV RNA genomic sequence is extracted from a sputum or saliva sample, a lymphocyte of a blood sample.

37. The method of claim 33, wherein the SARS-CoV RNA genomic sequence is extracted from nasopharyngeal, oropharyngeal, tracheal, bronchaleolar lavage, pleural fluid, urine, stool, conjunctiva, tissue from human, mouse, dog, rat, cat, horse, avian, earth, water, air.

38. The method of claim 33, wherein the SARS-CoV RNA genomic sequence is amplified by PCR.

39. The method of claim 38, wherein a label is incorporated into the amplified DNA sequence during the PCR.

40. The method of claim 38, wherein the PCR comprises conventional, multiplex, nested PCR or RT-PCR.

41. The method of claim 38, wherein the PCR comprises a two-step nested PCR, the first step being a RT-PCR and the second step being a conventional PCR.

42. The method of claim 38, wherein the PCR comprises a one-step, multiplex RT-PCR using a plurality of 5′

and 3′

specific primers, each of the specific primers comprising a specific sequence complementary to its target sequence to be amplified and a common sequence, and a 5′ and

a 3′

universal primer, the 5′

universal primer being complementary to the common sequence of the 5′

specific primers and the 3′

universal primer being complementary to the common sequence of the 3′

specific primers, and wherein in the PCR, the concentration of the 5′ and

3′

universal primers equals to or is higher than the concentration of the 5′ and

3′

specific primers, respectively.

43. The method of claim 42, wherein the 3′

universal primer and/or the 5′

universal primer is labeled.

44. The method of claim 43, wherein the label is a fluorescent label.

45. The method of claim 38, wherein the PCR comprises a multiplex nested PCR.

46. The method of claim 38, wherein the PCR is conducted using at least one of the following pairs of primers set forth in Table 4.

50. A kit for amplifying a SARS-CoV nucleotide sequence, which kit comprises:

a) a primer of claim 42;

and

b) a nucleic acid polymerase that can amplify a SARS-CoV nucleotide sequence using said primer of claim 42.

51. The kit of claim 50, wherein the nucleic acid polymerase is a reverse transcriptase.

47. An oligonucleotide primer for amplifying a SARS-CoV nucleotide sequence, which oligonucleotide primer comprises a nucleotide sequence that:

a) hybridizes, under high stringency, with a target SARS-CoV nucleotide sequence, or a complementary strand thereof, that is set forth in Table 1;

or

b) has at least 90% identity to a target SARS-CoV nucleotide sequence comprising a nucleotide sequence, or a complementary strand thereof, that is set forth in Table 1.

48. The primer of claim 47, which comprises DNA, RNA, PNA or a derivative thereof.

49. The primer of claim 47, which comprises a nucleotide sequence, or a complementary strand thereof, that is set forth in Table 1.

52. An oligonucleotide probe for hybridizing to a SARS-CoV nucleotide sequence, which oligonucleotide probe comprises a nucleotide sequence that:

a) hybridizes, under high stringency, with a target SARS-CoV nucleotide sequence, or a complementary strand thereof, that is set forth in Table 2;

or

b) has at least 90% identity to a target SARS-CoV nucleotide sequence comprising a nucleotide sequence, or a complementary strand thereof, that is set forth in Table 2.

53. The probe of claim 52, which comprises DNA, RNA, PNA or a derivative thereof.

54. The probe of claim 52, which comprises a nucleotide sequence, or a complementary strand thereof, that is set forth in Table 2.

55. The probe of claim 52, which is labeled.

56. The probe of claim 55, wherein the label is selected from the group consisting of a chemical, an enzymatic, an immunogenic, a radioactive, a fluorescent, a luminescent and a FRET label.

57. A kit for hybridization analysis of a SARS-CoV nucleotide sequence, which kit comprises:

a) a probe of claim 52;

and

b) a means for assessing a hybrid formed between a SARS-CoV nucleotide sequence and said probe.