Virus detection method, primers therefor and screening kit

• US 20060057561A1
• Filed: 06/13/2002
• Published: 03/16/2006
• Est. Priority Date: 06/14/2001
• Status: Abandoned Application

• Current Assignee: Cardiff University
• Original Assignee: Cardiff University
• Inventors: Hart, Keith W
• Application Number:
  US10/480,852
• Publication Number:
  US 20060057561A1
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/70 involving virus or bacteriophage immunoassay for viruses G01N33/56983

Abstract

Discloses a novel detection and typing method for viruses, such as human papillomaviruses, based on real-time PCR using self-probing amplicon fluorescent primers. The method comprises: (IA) contacting the sample with a self-probing amplicon (‘virus self-probing amplicon’) comprising (i) a virus primer capable of hybridising to at least one target viral nucleic acid sequence and undergoing amplification thereof under primer amplification conditions to form a virus primer extension product; (ii) a virus probe comprising a nucleic acid sequence complementary to a target sequence of the virus primer extension product and capable of hybridisation thereto, provided that the self-probing amplicon is adapted to ensure that the virus probe is unresponsive to amplification under the primer amplification conditions; and (iii) a member of a virus signalling system, which system is capable of causing a detectable signal to be effected on hybridisation of the virus probe sequence to the virus primer extension product, whereby presence or absence of the target viral nucleic acid sequence in the sample is indicated by the detectable signal; (IB) amplifying the product of step (IA) under the primer amplification conditions to an extent enabling the detectable signal to be effected after step(II); and (II) separating the virus primer extension product from the target viral nucleic acid sequence; allowing the virus probe to hybridise to the target sequence of the virus primer extension product; and monitoring the signalling system. This method is quick, simple, specific, sensitive, and capable of estimating viral load per cell. The results of over 100 HPV typing reactions performed on cell lines, biopsies and cervical cytobrush samples are given which, when compared to the current reference HPV detection and typing technique, present a kappa value of 0.89. The method is also applicable to other viruses, such as SV40.

Claims

1-55. -55. (canceled)

56. A self-probing amplicon comprising a nucleic acid sequence selected from the group consisting of:

(i) SEQ IDs NOS;

1 to 9;

(ii) SEQ ID NO;

11;

(iii) SEQ ID NOS;

12 to 17;

(iv) SEQ ID NOS;

18 to 20;

or

(v) the group of self-probing amplicons for use in SV40, JC or BK determination consisting of ScSV40 primer, ScJC primer, ScBK primer, Sc SV40 probe, Sc JC probe, ScBK probe, SV40 P1, SV40 P2, SV40 P3, SV40 P4, JC P1, JC P2, JC P3, JC P4, BK P1, BK P2, BK P3, or BK P4, and any mixture thereof.

59. A self-probing amplicon comprising a nucleic acid sequence comprising:

(a) a primer component; and

(b) a probe component;

wherein the primer component comprises a sequence selected from the group consisting of SEQ ID NOS;

32 to 40, SEQ ID NOS;

47 to 52, or SEQ ID NO;

61; and

wherein SEQ ID NO;

61 is a primer component of SEQ ID NOS;

19-20.

60. A self-probing amplicon comprising a nucleic acid sequence comprising:

(a) a primer component; and

(b) a probe component;

wherein the probe component comprises a sequence selected from the group consisting of SEQ ID NOS;

21 to 29, SEQ ID NO;

31, SEQ ID NOS;

41 to 46, or SEQ IDS NOS;

59-60; and

wherein SEQ ID NOS;

59-60 are the probe component of SEQ ID NOS;

19-20.

61. A diagnostic kit for the detection, typing, or determination of viral cell load or viral integration state comprising:

(a) at least one virus self-probing amplicon, housekeeping self-probing amplicon, or tailed primer; and

(b) instructions for use of the kit.