Development of a novel multiplex type-specific quantitative real-time PCR for detection and differentiation of infections with human papillomavirus types HPV2, HPV27, and HPV57

Abstract

Introduction:The present study describes the development and evaluation of the first multiplex type-specific quantitative realtime PCR (RT-PCR), enabling simple, rapid, sensitive, and specific concurrent detection and differentiation of human papillomavirus (HPV) types HPV2, 27, and 57 in a single PCR reaction. Results: The HPV2/27/57 multiplex RT-PCR with a dynamic range of seven orders of magnitude (discriminating 10 to 10 8 viral genome equivalents/reaction) has an analytical sensitivity of at least 10 viral copies of each targeted HPV type/reaction, and no cross-reactivities were observed among the included targets. All three primer/probe combinations were efficient in amplifying 500 copies of targeted DNA in a background of 10 8 , 10 7 , 500, 100, and 10 copies of non-targeted viral DNA/reaction, and the performance of the HPV2/27/57 multiplex RT-PCR was additionally not affected by the presence of background human genomic DNA. When testing DNA isolates obtained from fresh-frozen tissue specimens of various children's warts, the results of the HPV2/27/57 multiplex RT-PCR were completely in line with the results of the conventional Low-risk Alpha-PV PCR. Conclusion: The newly developed HPV2/27/57 multiplex RT-PCR is an appropriate test for use in routine clinical laboratory settings and for studies focusing on the molecular epidemiology, pathogenesis, and natural history of HPV2/27/57-related lesions.

Determination of Causative Human Papillomavirus Type in Tissue Specimens of Common Warts Based on Estimated Viral Loads

“…In this study, performed on the largest sample collection of CWs unambiguously diagnosed by histopathological assessment, HPV DNA detection rate was 176/185 (95.1%), which is in line with similar studies, in which HPV positivity ranged from 64.3 to 100% (Gross et al, 1982;Chen et al, 1993;Egawa, 1994;Lei et al, 2009;Sun et al, 2010;de Koning et al, 2011;Šterbenc et al, 2017). High HPV DNA prevalence in this study was most probably due to the use of optimal quality samples (fresh-frozen tissue), highly sensitive HPV detection and genotyping methods (Hošnjak et al, 2016;Šterbenc et al, 2017), and a relatively wide spectrum of HPV types tested (n = 26). Multiple HPVs were detected in 71/185 (38.4%) CWs, which is substantially higher in comparison to the majority of similar studies (Gross et al, 1982;Chen et al, 1993;Egawa, 1994;Lei et al, 2009;Sun et al, 2010;de Koning et al, 2011).…”

“…The integrity of extracted DNA was verified by real-time polymerase chain reaction (RT-PCR) amplification of a 268-bp fragment of human beta-globin gene, as described previously (Jancar et al, 2009). In order to determine the presence of HPV, 100 ng of each DNA sample was first tested for the presence of HPV2, 27, and 57, using type-specific quantitative multiplex RT-PCR, allowing sensitive detection and differentiation of HPV2, 27, and 57 in a single PCR reaction (Hošnjak et al, 2016). HPV2/27/57 RT-PCR-negative samples were additionally tested using a consensus primer set (Odar et al, 2014) in combination with Sanger sequencing, preferentially allowing detection of 23 low-risk Alpha-PVs (LR-HPVs): HPV2,3,6,7,10,11,13,27,28,29,32,40,42,43,44,55,57,74,77,91,94,117, and 125 etiologically associated with various mucosal and cutaneous warts.…”

Risk factors for the development of high‐grade dysplasia and carcinoma in patients with laryngeal squamous cell papillomas: Large retrospective cohort study

“…In order to explore the role of TP53 mutagenesis in the malignant transformation of LSP, a total of 22 samples, including matched samples (before and after malignant transformation) of LSCC ex‐LSP patients (n = 7) and eight control samples of LSP with low‐grade dysplasia (LG‐D) cases, were tested. Briefly, total DNA was extracted from FFPE tissue sections (14 LSCC ex‐LSP samples and 5 control samples) and fresh‐frozen specimens (3 control samples), using the QIAamp DNA Mini Kit, as described previously, 28,29,35 which was followed by the quantification of the extracted DNA using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, Massachusetts) on the Qubit Fluorimeter (Thermo Fisher Scientific) according to the manufacturer's instructions. The quality of the genomic DNA for TP53 typing was subsequently assessed based on delta C t scores using RT‐PCR from the TruSeq FFPE DNA library Prep QC Kit (Illumina, San Diego, California) and was calculated as the difference between the C t values of samples and control DNA with numbers of 0 to 2 indicating DNA of good quality, 2 to 4 indicating DNA of medium quality, and 4 to 6 indicating DNA of poor quality.…”

Clinical, Histopathological, and Virological Evaluation of 203 Patients With a Clinical Diagnosis of Molluscum Contagiosum

“…Likewise, it is highly recommended to confirm a clinical diagnosis of MC-like lesions located in children’s anogenital area using more objective methods (preferably virological). Namely, MC-like lesions could represent anogenital warts etiologically linked with sexually transmitted HPV6/11, which in many countries requires investigation into suspected sexual abuse and consequent legal action (reviewed in 13 ). Virological testing can also reliably distinguish between anogenital and common warts in children’s anogenital area, indicating no further investigation or legal action.…”

“…Presence of MCV DNA was determined using MCV fluorescence resonance energy transfer (FRET)-based real-time polymerase chain reaction (MCV RT-PCR), allowing very sensitive (at least 3.3 viral copies/reaction) and specific detection of MCV, as well as a reliable differentiation of MCV1 and MCV2 in a single reaction [ 8 ]. All MCV DNA-negative isolates obtained from the torso and extremities were further tested for the presence of HPV2, HPV27, and HPV57, which are etiologically associated with more than 65% of common (skin) warts, using multiplex type-specific quantitative HPV2/27/57 RT-PCR, allowing sensitive and specific concurrent detection and differentiation of the 3 HPV types in a single PCR reaction [ 13 ]. All MCV-negative DNA isolates obtained from the anogenital region were further tested for the presence of HPV6 and HPV11, which are etiologically associated with more than 90% of anogenital warts, using a FRET-based RT-PCR (HPV6/11 RT-PCR) [ 14 ].…”