Detection of human papillomavirus mRNA in cervical smears

• US 5,750,334 A
• Filed: 06/07/1995
• Issued: 05/12/1998
• Est. Priority Date: 11/11/1988
• Status: Expired due to Fees

• Current Assignee: DADE Behring Beteiligungs GmbH (Siemens AG)
• Original Assignee: Behring Diagnostics GmbH
• Inventors: Whitcomb, Jeannette, de Villiers, Ethel-Michelle, Cerutti, Peter, Zijlstra, Jacob
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US08/479,567
• Time in Patent Office: 1,070 Days
• Field of Search: 435/5, 435/6, 435/91.2, 935/77, 935/78, 536/24.33
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/708 for papilloma

Abstract

The invention relates to the direct detection of human papillomavirus MRNA and DNA. The recently developed polymerase chain reaction was modified in order to improve the sensitivity and specificity. By choosing appropriate oligonucleotide primers (amplimers) and reaction temperatures, individual HPV genes from among the total cell DNA can be identified and amplified to an extent such that non-isotopic detection is possible. In addition, use of reverse transcription permits selective amplification of spliced mRNA and thus permits indication of premalignant or malignant conditions and/or lesions.

Claims

1. A method for the direct detection of spliced human papillomavirus (HPV) mRNA in a cervical smear sample which comprises:

(a) combining the sample with reverse transcriptase in a reaction mixture and sonicating to release mRNA;

(b) synthesizing a cDNA by means of reverse transcription to establish the presence of E6 spliced MRNA;

(c) amplifying said cDNA in a polymerase chain reaction (PCR) by using;

i) deoxynucleotide triphosphate concentrations above 0.2 mM,

ii) amplimer concentrations above 0.5 μ

M, wherein said amplimers span the E6 splice sequence of the spliced MRNA to be detected, and

iii) cycle times of 1 to 2 minutes at two temperatures of 70°

C.-95°

C. and 50°

C.-70°

C., wherein the PCR is started above the melting temperature; and

(d) detecting said amplified cDNA, wherein said correspondent cDNA is distinguished from other amplified fragments by its size.

2. The method as claimed in claim 1, wherein said two temperatures are 89°

C. and 63°

C.

3. The method as claimed in claim 1, wherein the PCR is carried out with addition during step (b) of up to 20% v/v dimethyl sulfoxide.

4. The method as claimed in claim 2, wherein the PCR is carried out with addition during step (c) of up to 20% v/v dimethyl sulfoxide.