PCR diagnosis of human papilloma virus type 16

Details PCR diagnosis of human papilloma virus type 16 PCR diagnosis of human papilloma virus type 16

1993-03-12

1996-03-26

Jones, W. Gary

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving virus or bacteriophage

435 6, 435 912, 536 2433, C07H 2100, C12Q 168, C12Q 170, C12P 1934

Patent

active

055019471

DESCRIPTION:

BRIEF SUMMARY
CROSS-REFERENCE TO RELATED APPLICATIONS

This application originates from International Application No. PCT/GB91/01212, filed Jul. 19, 1991, based on United Kingdom Application No. 901584.2, filed Jul. 19, 1990.


FIELD OF THE INVENTION

This invention relates to an improved method of detection of human papillomavirus (HPV), in particular HPV using the polymerase chain reaction (PCR).


BACKGROUND OF THE INVENTION

Papillomaviruses are a group of closely related viruses that produce a spectrum of diseases in Man and other mammals. At present there are in excess of 57 different types of human papillomaviruses. The definition of a new type is that it possesses less than 50% cross hybridisation with the DNA of known species in the liquid phase according to a standard protocol. A number of human diseases have been attributed to specific papillomavirus types; for example, plantar warts--papillomavirus type 1 and common warts--papillomavirus type 7.
Human papilloma viruses have also been implicated in the development of cervical cancer (zur Eausen, 1977). This was based on observations that HPV infection was present in a high percentage of patients with cervical intraepithelial neoplasia (CIN) and invasive carcinoma of the cervix (Walker et al, 1983). The availability of cloned DNA for specific HPV types has allowed the prevalence of HPV infection in disease tissue to be assessed using a range of hybridization studies. These studies have shown that between 60 and 90% of significant CIN lesions and invasive cancers contained HPV DNA sequences whereas the prevalence of HPV DNA amongst normal controls was between 9 and 23%. Molecular analysis has shown that certain types of HPV, such as HPV 16, 31, 33 and 35 are more strongly associated with malignant changes of the cervix and are able to immortalise primary human keratinocytes and transform established cell lines. Other types of HPV such as HPV 6 and 11 are associated with benign lesions and condyloma acuminata. These data support the hypothesis that HPV infection, in particular HPV 16, is indeed a causal or contributory agent to squamous cancer.
PCR (Saiki et al, 1988) is based on the enzymic amplification of a DNA target fragment by the use of two oligonucleotide primers that hybridise to opposite strands of the target sequence. The primers are oriented such that their 3' termini oppose each other.
Two recent studies (Young et al, 1989, Tidy et al, 1989) have used PCR to amplify specific HPV sequences from cervical tissue. Under the conditions used by these workers it has been shown that, in contrast to other hybridisation studies, that positive signals obtained for HPV types in the normal population is between 70-84%. Recently one study (Tidy et al, 1989) has been retracted in part (Tidy and Farrell, 1989) thus highlighting the problems associated with using PCR to identify the presence of HPV in the cervix.
Tidy et al amplified sequences within the upstream regulatory region (between nucleotides 7765 and 7775), whilst Young et al used oligonucleotide primers to amplify a region from nucleotides 421 to 540 within the E6 gene.
The prevalences of HPV 16 infection reported in these studies are surprisingly high when compared with data from Southern blot, filter in-situ hybridization, dot-blot studies and certain PCR studies (Van der Brule, 1989). The high prevalence identified in these PCR studies may be accounted for by the inability of the oligonucleotide primers to exhibit specificity for the target DNA sequence of interest and hence yielding an amplified DNA fragment for other papillomavirus types present in the sample.
A fundamental requirement for the PCR reaction is the availability of oligonucleotide primers that are specific for the DNA sequence to which they are complementary and which enable efficient amplification of target sequences. By "specific", it is meant that the sequences of the oligonucleotides are such that they do not hybridize to a significant degree to any DNA sequences present in the sample which is being analyzed by a PCR reaction other

REFERENCES:
Stratagene Catalog 1988 (p. 39).
European Patent 373352 (Behring Werke AG) published Jun. 20, 1990 Detection of human papilloma virus-by DNA amplification and analysis (English Abstract).
Seedorf et al. Human Papillomavirus Type 16 DNA Sequence, Virology (1985) 145: 181-185.
Gen Bank Loci PPH16 (citing Seedorf et al.), PPH33CG (Feb. 16, 1987), and PPH31A (Dec. 15, 1989) Fast DB Search and Gen Align Alignment.
Maitland, et al., Detection of human papillomavirus genes in human oral tissue biopsies and cultures by a polymerase chain reaction, (1989) Chem. Abs. 11:192.
Wagatsuma, et al., Analysis of integrated human papillomavirus type 16 DNA in cervical cancers; amplification of viral sequences together with cellular flanking sequences, (1990) Chem. Abs. 112:195.

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