ABSTRACT
Polymerase chain reaction (PCR)-based assays that use consensus primers to detect DNA of a broad spectrum of human papillomavirus (HPV) types in a single assay belong to the most frequently used methods to detect HPV in clinical specimens. Here, we describe in detail one of these assays, the so-called GP5+/6+ PCR method, which can be used to detect and type HPV DNA in crude extracts of cervical scrapes and biopsy specimens. Following PCR with GP5+ and GP6+ primers, the latter of which is biotinylated at its 5' end, the presence of DNA of any of the high-risk genotypes can easily be determined by an enzyme immunoassay (EIA). In this assay, PCR products are captured in streptavidin-coated wells of a microtiter plate, denatured by alkaline treatment, and hybridized to cocktails of digoxigenin-labeled oligonucleotides specific for high-risk or low-risk HPV types. The resulting hybrids can then be detected by alkaline phosphatase conjugated anti-digoxigenin polyclonal antibodies and substrate followed by optical density reading. Subsequently, EIA-positive PCR products can be typed by a reverse line blot genotyping procedure that, using a miniblotter device, enables typing of up to 39 samples with specific oligonucleotide probes for 37 different HPV (sub)types in a single assay.
Subject(s)
Cervix Uteri/virology , DNA, Viral/analysis , Papillomaviridae/classification , Papillomaviridae/isolation & purification , Base Sequence , DNA Primers , DNA, Viral/genetics , Female , Globins/genetics , Humans , Nucleic Acid Hybridization , Papillomaviridae/genetics , Polymerase Chain Reaction/methods , Sensitivity and SpecificityABSTRACT
Thallium acetate in concentrations of 500 to 1000 mg/l is tolerated in the culture by the most mollicutes of the orders Mycoplasmatales and Acholeplasmatales and by this reason it is added in the culture media as a selective element for the detection and propagation of mycoplasmas and acholeplasmas. Because of the high toxicity of thallium acetate and its accumulation in the environment, thallium acetate is not biodegradable, an alternative was searched. The results and analysis of tests with nine mollicute species are presented here. It is recommended to replace thallium acetate in the formulations where it is used and colistin sulfate is proposed as its substitute.
