ABSTRACT
BACKGROUND: It has been shown that women positive for HPV 16 and HPV 18 have an increased risk of high-grade cervical intraepithelial neoplasia (CIN) compared with women positive for other high-risk (HR) HPV types. In addition, HPV 18 and HPV 45 have been closely linked to aggressive and difficult to detect adenocarcinomas. OBJECTIVES: To develop a test based on the Hybrid Capture technology capable of specifically detecting the most important carcinogenic HPV types; 16, 18, and 45. STUDY DESIGN: The assay is based on Hybrid Capture technology utilizing a mixture of short type-specific oligoribonucleotides to detect HPV types 16, 18, or 45. The assay utilizes no target amplification and shares workflow and critical reagents with the Digene HC2 HPV screening assay. Studies to evaluate specificity, performance of the test in comparison to HC2, and capability to detect a single genotype in the presence of multiple infections are described. Specificity was evaluated analytically using a panel of HR- and LR-HPV types to illustrate cross-reactivity. Performance in comparison to the HC2 test was evaluated by testing aliquots of the same prepared samples by the genotyping test and HC2. Ability to detect a single genotype during multiple infections was modeled by detecting HPV 16 plasmid in the presence of HPV 6 or HPV 31 at high copy numbers. RESULTS: The proposed genotyping assay specifically detects HPV 16, 18, and 45 with an analytical sensitivity of 5,000 copies per assay. The assay is highly specific and does not detect other tested high-risk or low-risk types at 10(8) copies per reaction. Utility of the genotyping test was demonstrated using clinical samples collected in Digene Specimen Transport Medium (STM) and results were confirmed by PCR. CONCLUSIONS: The target-amplification free assay provides a genotyping method for highly specific detection of HPV 16, 18, and 45 without the complexity of PCR technology.
Subject(s)
Cervix Uteri/virology , DNA, Viral/genetics , Molecular Diagnostic Techniques/methods , Nucleic Acid Hybridization/methods , Papillomaviridae/classification , Papillomaviridae/isolation & purification , Papillomavirus Infections/diagnosis , Virology/methods , Cross Reactions , Female , Genotype , Human papillomavirus 16/classification , Human papillomavirus 16/genetics , Human papillomavirus 16/isolation & purification , Human papillomavirus 18/classification , Human papillomavirus 18/genetics , Human papillomavirus 18/isolation & purification , Humans , Papillomaviridae/genetics , Papillomavirus Infections/virology , Sensitivity and SpecificityABSTRACT
Anaplasma ovis (Rickettsiales: Anaplasmataceae) is a tick-borne pathogen of sheep, goats and wild ruminants. The genetic diversity of A. ovis strains has not been well characterized due to the lack of sequence information. In this study, we evaluated bighorn sheep (Ovis canadensis) and mule deer (Odocoileus hemionus) from Montana for infection with A. ovis by serology and sequence analysis of the msp4 gene. Antibodies to Anaplasma spp. were detected in 37% and 39% of bighorn sheep and mule deer analyzed, respectively. Four new msp4 genotypes were identified. The A. ovismsp4 sequences identified herein were analyzed together with sequences reported previously for the characterization of the genetic diversity of A. ovis strains in comparison with other Anaplasma spp. The results of these studies demonstrated that although A. ovismsp4 genotypes may vary among geographic regions and between sheep and deer hosts, the variation observed was less than the variation observed between A. marginale and A. phagocytophilum strains. The results reported herein further confirm that A. ovis infection occurs in natural wild ruminant populations in Western United States and that bighorn sheep and mule deer may serve as wildlife reservoirs of A. ovis.
