A broad spectrum PCR method for the detection of polyomaviruses and avoidance of contamination by cloning vectors.
Polyomaviruses induce tumours of different histological types when inoculated into experimental animals, but their aetiological role in the development of malignant tumours in humans remains questionable. We developed a degenerate PCR assay in an attempt to identify additional, presently unknown human polyomavirus types which may be involved in the malignant transformation of human tissues. Degenerate oligonucleotide primers were deduced from four different conserved amino acid motifs in the highly conserved viral capsid protein, VP1. Three different sets of primers were included for the each test. Bladder carcinomas, Hodgkin's lymphomas, meningiomas, Kaposi-tumours and -cell lines were analysed. No polyomavirus DNA sequences could be detected. A comparative analysis led to the recognition of the presence of SV40 DNA sequences in more than 200 vectors available in the EMBL and Genbank Databanks and commonly used in laboratories worldwide. The majority of primers used to detect polyomavirus sequences in human tumours are distributed throughout these regions also present in the vectors. Only a small stretch of 286bp in the overlapping region of the VP1, VP2 and VP3 genes is not present in the vector sequences. We propose to use this region for the design of additional non-contamination primers.
Deutsches Krebsforschungszentrum, Abteilung Tumorvirus-Charakterisierung, Heidelberg, Germany.
C
C
Völter
H
H
zur Hausen
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D
Alber
E M
EM
de Villiers
D003001:Cloning, Molecular; D004279:DNA, Viral / Q000737:chemistry; D005822:Genetic Vectors; D006801:Humans; D009369:Neoplasms / Q000209:etiology / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D011120:Polyomavirus / Q000302:isolation & purification*; D027601:Polyomavirus Infections / Q000150:complications; D014412:Tumor Virus Infections / Q000821:virology
D004279:DNA, Viral