1,721,051 research outputs found
A Multiplex Real-Time PCR-Platform Integrated into Automated Extraction Method for the Rapid Detection and Measurement of Oncogenic HPV Type-Specific Viral DNA Load from Cervical Samples
No full textThe persistent infection with most frequent high-risk (HR)-HPV types (HPV-16, -18, -31, -33, -45, -52, and -58) is considered to be the true precursor of neoplastic progression. HR-HPV detection and genotyping is the most effective and accurate approach in screening of the early cervical lesions and cervical cancer, although also the HR-HPV DNA load is considered an ancillary marker for persistent HPV infection. Here, it is described an in-house multiplex quantitative real-time PCR (qPCR)-based typing system for the rapid detection and quantitation of the most common HR-HPV genotypes from cervical cytology screening tests. First, a separate qPCR assay to quantify a single-copy gene is recommended prior to screening (prescreening assay) to verify the adequate cellularity of the sample and the quality of DNA extracted and to normalize the HPV copy number per genomic DNA equivalent in the sample. Subsequently, to minimize the number of reactions, two multiplex qPCR assays (first line screening) are performed to detect and quantify HPV-16, -18, -31, -33, -45, -52, and -58 (HPV-18 and -45 are measured together by single-fluorophore). In addition, a multiplex qPCR assay specific for HPV-18 and HPV-45 is also available to type precisely the samples found to be positive for one of the two strains. Finally, two nucleic acid extraction methods are proposed by using a 96-well plate format: one manual method (supported by centrifuge or by vacuum) and one automated method integrated into a robotic liquid handler workstation to minimize material and hands-on time. In conclusion, this system provides a reliable high-throughput method for the rapid detection and quantitation of HR-HPV DNA load in cervical samples
Applications of Artificial Intelligence in Microbiome Analysis and Probiotic Interventions—An Overview and Perspective Based on the Current State of the Art
No full textIdentification and quantification of oncogenic HPV nucleic acids by means of real-time PCR assays
No full textMethod for the identification and quantification of oncogenic HPV nucleic acids comprising:
a) first line screening by means of 5 independent SYBR Green I Real-time PCR assays to determine the total viral load and to identify the presence of one or more of 13 high risk HPV genotypes in the sample;
b) second line assays to be applied to samples positives for:
- 5 independent TaqMan Real-time PCR assays to determine the presence and the viral load of the most common oncogenic HPV types: HPV types: 16, 18, 31, 45, 33 group (including 33, 52, 58, 67 genotypes).
- 6 independent SYBR Green I RT Real-time PCR assays to determine the presence in the sample of the oncogenic transcripts E6/E7 of HPV types 16, 18, 31, 33, 45, 5
Is it true that pre-conization high-risk HPV DNA load is a significant factor of persistence of HPV infection after conization?
No full textAutomated extraction and quantitation of oncogenic HPV genotypes from cervical samples by a real-time PCR-based system
No full textAccurate laboratory assays for the diagnosis of persistent oncogenic HPV infection are being recognized increasingly as essential for clinical management of women with cervical precancerous lesions. HPV viral load has been suggested to be a surrogate marker of persistent infection. Four independent real-time quantitative TaqMan PCR assays were developed for: HPV-16, -31, -18 and/or -45 and -33 and/or -52, -58, -67. The assays had a wide dynamic range of detection and a high degree of accuracy, repeatability and reproducibility. In order to minimize material and hands-on time, automated nucleic acid extraction was performed using a 96-well plate format integrated into a robotic liquid handler workstation. The performance of the TaqMan assays for HPV identification was assessed by comparing results with those obtained by means of PCR using consensus primers (GP5+/GP6+) and sequencing (296 samples) and INNO-LiPA analysis (31 samples). Good agreement was found generally between results obtained by real-time PCR assays and GP(+)-PCR system (kappa statistic=0.91). In conclusion, this study describes four newly developed real-time PCR assays that provide a reliable and high-throughput method for detection of not only HPV DNA but also HPV activity of the most common oncogenic HPV types in cervical specimen
HHV-6A and HHV-6B in Autoimmune Disease
No full textA role for HHV-6 has been proposed in several autoimmune disorders (ADs), including multiple sclerosis (MS), autoimmune connective tissue diseases, and Hashimoto's thyroiditis. There are several potential mechanisms by which HHV-6 might induce autoimmune responses. HHV-6 could trigger autoimmunity through lysis of infected cells by exposing high amounts of normally sequestered cell antigens. Another potential trigger is represented by molecular mimicry, with the synthesis of viral proteins that resemble cellular molecules as a mechanism of immune escape. The virus could also induce aberrant expression of histocompatibility molecules, thereby promoting the presentation of autoantigens. CD46-HHV-6 interaction is a new attractive mechanism that has been proposed: HHV-6 could participate in neuroinflammation in the context of MS by promoting inflammatory processes through CD46 binding. Although HHV-6 has the ability to trigger all of the above-mentioned mechanisms, more studies are required to fully elucidate the possible role of HHV-6 as a trigger of AD. © 2014 Elsevier B.V. All rights reserved
Pityriasis rosea: An update with a critical appraisal of its possible herpesviral etiology
No full textPityriasis rosea is an acute, self-healing exanthem characterized by oval erythematous-squamous lesions of the trunk and limbs, that usually spares face, scalp, palms, and soles. Constitutional symptoms, which have the character of true prodromes; clinical features, which resemble those of the known exanthems; and many epidemiologic data all suggest an infectious origin. A host of infectious agents have been incriminated, but, recently, human herpesvirus 6 and 7 have been extensively studied. The goal of this review is to outline the epidemiologic, clinical, histologic, and ultrastructural features of pityriasis rosea, but mainly to stress its possible human herpesvirus nature. In addition, clues have been added to help the reader to go through the complex subtleties of the virologic investigation. © 2008 American Academy of Dermatology, Inc
Possible role of human herpesvirus 6 as a trigger of autoimmune disease
No full textHuman herpesvirus 6 (HHV-6) infection is common and has a worldwide distribution. Recently, HHV-6A and HHV-6B have been reclassified into two distinct species based on different biological features (genetic, antigenic, and cell tropism) and disease associations. A role for HHV-6A/B has been proposed in several autoimmune disorders (AD), including multiple sclerosis (MS), autoimmune connective tissue diseases, and Hashimoto's thyroiditis. The focus of this review is to discuss the above-mentioned AD associated with HHV-6 and the mechanisms proposed for HHV-6A/B-induced autoimmunity. HHV-6A/B could trigger autoimmunity by exposing high amounts of normally sequestered cell antigens, through lysis of infected cells. Another potential trigger is represented by molecular mimicry, with the synthesis of viral proteins that resemble cellular molecules, as a mechanism of immune escape. The virus could also induce aberrant expression of histocompatibility molecules thereby promoting the presentation of autoantigens. CD46-HHV-6A/B interaction is a new attractive mechanism proposed: HHV-6A/B (especially HHV-6A) could participate in neuroinflammation in the context of MS by promoting inflammatory processes through CD46 binding. Although HHV-6A/B has the ability to trigger all the above-mentioned mechanisms, more studies are required to fully elucidate the possible role of HHV-6A/B as a trigger of AD. © 2013 Francesco Broccolo et al
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