Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Patent
1992-07-16
1994-11-15
Parr, Margaret
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
435 6, 435 912, 536 2432, 536 2433, 935 78, C12Q 170, C07H 2104, C12P 1934
Patent
active
053647580
DESCRIPTION:
BRIEF SUMMARY
The invention relates to primers and primer pairs for use in a polymerase chain reaction (PCR) for amplification of DNA of genital human papillomavirus (HPV) genotypes. The invention also relates no a method for amplification of genital EPV DNA and a method of analysing a sample, such as a cervical smear,. for the presence of genital HPV genotypes.
At present, testing for cervical carcinoma and pre-stages thereof is mostly done by the detection of morphologically abnormal cells in cell smears of the cervix. This involves staining of smears by means of the Papanicoloau technique. By means of a light microscope, the pathologist detects premalignant and malignant cells. Then morphologically abnormal cells are classified into PAP classes I-V according to the severity of the cell lesion. With PAP I, only normal cells are found, while with PAP V, actual cancer cells are observed. This cytomorphological method, however, has some inherent disadvantages. In addition to great inter-observer variations, abnormal cells may be missed by "sample error" or by the microscopist. Furthermore, the PAP III class poses many problems as regards the gynaecological policy no be followed. With PAP III, which is found quite frequently, cancer may develop in the course of time, but persistence or regression to the normal condition may also occur. For this reason, therefore, there is a great need for better prognostic markers for cervical carcinoma.
In recent years, a great deal of research has been done into the causes of cervical carcinoma. This has revealed in particular the implication of the sexually transmittable human papiilomavirus (HPV). To date, 24 types of this virus have been associated with lesions of the cervical mucosa. The distinction between these HPV types is based on a difference in the base-sequence of the HPV DNA. Broadly, a 50% difference in base-sequence has been found to exist between the various HPV types. Studies have shown that the HPV types 6 and 11 are found in particular in benign genital lesions such as genital verrucae and in normal to mild dysplastic smears. These are called "low-risk" HPV types. HPV types 16 and 18 in particular and to a lesser extent 31, 33 and 35 occur in severely dyspiastic and "malignant" cervical smears (PAP IIIa higher) and are accordingly called "high-risk" HPV types. As yet, fairly little is known about the other 18 genital HPV types. It looks like the early detection of the various HPVs in cervical smears may in the future be an important prognostic factor in predicting the biological behaviour of cervical cells.
At present, there are various methods to determine the various human papillomavirus genotypes. The most sensitive method is the polymerase chain reaction (PCR), sometimes referred to as DNA amplification method, which in principle is capable of detecting one HPV DNA molecule in a smear. In this method, to a clinical sample (in this case a cervical smear), after an isolation of the DNA present therein (or in the case of the present invention, optionally after a short pretreatment) under specific conditions, 2 small synthetic pieces of single-stranded DNA, whose base-sequence is complementary to a part of one of the two strands of a given HPV genotype, are added. These DNA pieces, called primers, in virtue of their base-sequence, flank one HPV type-specific DNA fragment (the target DNA) of some 100 to 500 bases, in such a way that the enzyme Taq DNA polymerase present in the reaction mixture starts on the two primers and synthesizes the two strands in overlapping direction. The product obtained is called "amplimer". The procedure in which the target is synthesized once is called a cycle. Such a HPV-specific cycle comprises three steps, each taking place at a different temperature: the HPV target DNA is denatured at 94.degree. C. (1 min), followed by a primer annealing step at 58.degree. C. (2 min) and polymerization at 72.degree. C. (1.5 min). By repeating such a cycle 30-40 times, the number of amplimers increases by a factor 2.sup.30-40, so than an amount of HPV typ
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Meijer Christophorus J.
Snijders Petrus J.
van den Brule Adrianus J.
Walboomers Jan M.
Myers Carla
Parr Margaret
Stichting Researchfonds Pathologie
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