Method of early detection of HPV-associated carcinomas and extreme dysplasias caused by HPV

• US 6,027,891 A
• Filed: 01/28/1998
• Issued: 02/22/2000
• Est. Priority Date: 02/24/1995
• Status: Expired due to Fees

• Current Assignee: Deutsches Krebsforschungszentrum Stiftung des Offentlichen Rechts
• Original Assignee: Deutsches Krebsforschungszentrum Des Offentlichen Rechts
• Inventors: Emmerich, Florian, Von Knebel-Doberitz, Magnus, Worner, Stefan
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Taylor, Janell E.
• Application Number:
  US08/913,547
• Time in Patent Office: 755 Days
• Field of Search: 435/6, 435/5, 935/19
• US Class Current: 435/6.14
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/708 for papilloma

Abstract

This invention relates to a method of detection of an mRNA having HPV and cellular sequences in a body sample, comprising the following steps:

(a) obtaining a body sample;

(b) isolating mRNA from the body sample of (a);

(c) translating the mRNA of (b) into cDNA using a primer common for reverse transcription;

(d) amplifying the cDNA of (c) by a PCR reaction with a 5'"'"' HPV primer and a 3'"'"' primer having sequences of the primer of (c);

(e) cleaving the amplified cDNA of (d) with an endonuclease cleaving on the 5'"'"' side of the HPV polyadenylation sequence;

(f) amplifying the non-cleaved cDNA of (e) with the primers of (d) or with "nested" primers; and

(g) detecting the amplified cDNA of (f).

Furthermore, this invention concerns the use of such a method of early detection of HPV-associated carcinomas and extreme dysplasias caused by HPV, respectively.

Claims

1. A method of detecting an mRNA having HPV and cellular sequences in a body sample, comprising the following steps:

(a) obtaining a body sample,

(b) isolating mRNAs from the body sample of (a),

(c) reverese transcribing the mRNAs of (b) into cDNAs using a common primer,

(d) amplifying the cDNAs of (c) by a PCR reaction with a 5'"'"' HPV primer and a 3'"'"' primer having sequences of the primer of (c),

(e) cleaving the amplified cDNAs of (d) with an endonuclease, wherein said endonuclease cleaves at a restriction site on the 5'"'"' side of an HPV-polyadenylation sequence, such that cDNAs obtained from episomal HPVs are cleaved and cDNAs obtained from integrated HPVs are not cleaved,

(f) amplifying the non-cleaved cDNAs with the primers of (d) or with nested primers, and

(g) detecting the amplified cDNAs of (f).

2. The method according to claim 1 wherein the body sample is selected from the group consisting of a smear, a surface biopsy, an organ punctate and a biopsy, blood, sputum, urine, stool, liquor, bile, lymph and a gastrointestinal secretion.

3. The method according claim 1 wherein the 5'"'"' primer is a 5'"'"' primer for high-risk HPV types.

4. The method according to claim 1 wherein more than one 5'"'"' primers or more than one 3'"'"' primers are used.

5. The method according to claim 1 wherein in step (d) at least one of the following is used as a 5'"'"' primer:

space="preserve" listing-type="tabular">HPV 16;

5'"'"'- CGG ACA GAG CCC ATT ACA AT - 3'"'"' (SEQ. ID. NO;

     3) - HPV 18;

5'"'"'- TAG AAA GCT CAG CAG ACG ACC - 3'"'"' (SEQ. ID. NO;

     4) - HPV 31;

5'"'"'- TAT GAG CAA TTA TTA CCC GAC AGC - 3'"'"' (SEQ. ID. NO;

     5) - HPV 33;

5'"'"'- TTT ATA TCC TGA ACC AAC TGA CCT ATA CT - 3'"'"' (SEQ. ID. NO;

     6) - HPV 35;

5'"'"'- TAG ATT TGG AAC CCG AGG C - 3'"'"' (SEQ. ID. NO;

     7) - HPV 39;

5'"'"'- TCA CGA GCA ATT AGG AGA GTC A - 3'"'"' (SEQ. ID. NO;

8) - HPV 52;

5'"'"'- GCA ACC TGA AAC AAC TGA CCT AC - 3'"'"' (SEQ. ID. NO;

9) - as 3'"'"'primer;

- 5'"'"'-GAC TGC AGT CGA CAT CG-3'"'"' (SEQ. ID. NO;

     10).

6. The method according to claim 1 wherein in step (f) at least one of the following is used as a 5'"'"' primer:

space="preserve" listing-type="tabular">HPV 16;

5'"'"'-CCT TTT GTT GCA AGT GTG ACT CTA CG-3'"'"' (SEQ. ID. NO;

14) - HPV 18;

5'"'"'-GAG CAT TCC AGC AGC TGT TTC TGA A-3'"'"' (SEQ. ID. NO;

     15) - HPV 31;

5'"'"'-CAC GAG CAC ACA AGT AGA TAT TCG CAT ATT GCA-3'"'"' (SEQ. ID. NO;

     16) - HPV 33;

5'"'"'-TCA ACA GTA CAG CAA GTG ACC TAC GAA CCA-3'"'"' (SEQ. ID. NO;

     17) - HPV 35;

5'"'"'-AGA GGA GGA GGA AGA TAC TAT TGA CGG TCC A-3'"'"' (SEQ. ID. NO;

     18) - HPV 39;

5'"'"'-GTT AAT CAC CAA CAT CAA CTA CTA GCC AGA CGG-3'"'"' (SEQ. ID. NO;

     19) - HPV 52;

5'"'"'-CGG CCA GAT GGA CAA GCA GAA CA-3'"'"' (SEQ. ID. NO;

     20);

- and - 5'"'"'-GAC GTC GGA GTG CGA GCA TCG-3'"'"'(SEQ. ID. NO;

     21) - is used as a nested 3'"'"' primer.

7. A method for detecting HPV-associated carcinomas and extreme dysplasias caused by HPV comprising the steps of:

(a) obtaining a body sample,

(b) isolating mRNAs from the body sample of (a),

(c) reverese transcribing the mRNAs of (b) into cDNAs using a common primer,

(d) amplifying the cDNAs of (c) by a PCR reaction with a 5'"'"' HPV primer and a 3'"'"' primer having sequences of the primer of (c),

(e) cleaving the amplified cDNAs of (d) with an endonuclease, wherein said endonuclease cleaves at a restriction site on the 5'"'"' side of an HPV-polyadenylation sequence, such that cDNAs obtained from episomal HPVs are cleaved and cDNAs obtained from integrated HPVs are not cleaved,

(f) amplifying the non-cleaved cDNAs with the primers of (d) or with nested primers, and

(g) detecting the amplified cDNAs of (f).