Touchdown General Primer (GP5+/GP6+) PCR and optimized sample DNA concentration support the sensitive detection of human papillomavirus

Evans, Mark; Adamson, Christine S.-C.; Simmons-Arnold, Linda; Cooper, Kumarasen

doi:10.1186/1472-6890-5-10

Cited by 64 publications

(57 citation statements)

References 29 publications

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“…Therefore, the possibility exists that HPV detected by PCR in some TSCC could be background rather than driver HPV. Since the sensitivity of the PCR technique supports the detection of either driver or passenger HPV [22,29], the clinical significance of 'PCR-only HPV positive' TSCC demands further study.…”

Section: Discussionmentioning

confidence: 99%

“…Detection of this fragment was taken as evidence of HPV positivity. HPV genotyping of the *140 bp fragments was performed by dot blot hybridization [22].…”

Section: Hpv Genotypingmentioning

confidence: 99%

“…DNA samples (*100 ng) were assayed for HPV by touchdown GP5?/6? PCR, as previously described [22]. Following PCR, an aliquot of the product was screened for the presence of a *140 bp amplicon by agarose gel electrophoresis.…”

Section: Hpv Genotypingmentioning

confidence: 99%

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Discrimination of ‘Driver’ and ‘Passenger’ HPV in Tonsillar Carcinomas by the Polymerase Chain Reaction, Chromogenic In Situ Hybridization, and p16INK4a Immunohistochemistry

Evans

Matthews

Kandil

et al. 2011

Head and Neck Pathol

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Human papillomavirus (HPV) positive tonsillar squamous cell carcinoma (TSCC) is associated with a favorable clinical outcome. However, the HPV detected in a given tumor may be causal (driver HPV) or an incidental bystander (passenger HPV). There is a need to discriminate these forms of HPV in TSCCs to understand their impact on HPV as a biomarker for use in TSCC patient management. This study has compared the polymerase chain reaction (PCR), chromogenic in situ hybridization (CISH), and p16INK4a immunohistochemistry in the assessment of HPV status in TSCC. Archival specimens of TSCC from thirty patients were investigated. HPV was detected by PCR in 25/30 (83.3%) tumors; HPV16 (70.0%) and HPV52 (6.7%) were the most common types. HPV was corroborated by CISH in 22/25 (88.0%) specimens; integrated HPV was implicated by the presence of punctate signals in each of these cases. p16INK4a staining was found in 20/22 (90.9%) HPV PCR positive samples; two PCR/CISH HPV positive cases were p16INK4a negative and two HPV negative samples were p16INK4a positive. These data suggest that a minority of HPV positive TSCCs are positive for passenger HPV and that two or more assays may be required for diagnosing driver HPV status. INK4a and CISH negative, also requires further investigation.

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Section: Discussionmentioning

confidence: 99%

“…Detection of this fragment was taken as evidence of HPV positivity. HPV genotyping of the *140 bp fragments was performed by dot blot hybridization [22].…”

Section: Hpv Genotypingmentioning

confidence: 99%

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Discrimination of ‘Driver’ and ‘Passenger’ HPV in Tonsillar Carcinomas by the Polymerase Chain Reaction, Chromogenic In Situ Hybridization, and p16INK4a Immunohistochemistry

Evans

Matthews

Kandil

et al. 2011

Head and Neck Pathol

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“…assays were performed on each sample: a 'touchdown' (TD) protocol [13], and a Slow Ramping (SR) method [14]. HPV type was determined by dot blot hybridization with oligonucleotide probes [12,13] In Situ Hybridization (ISH) ISH was carried out on 34/35 FFPE sections (papilloma lesion from one specimen was exhausted during sectioning for DNA extraction). A tyramide-based ISH assay (GenPoint TM , Dako North America, Carpinteria, CA) was performed using a Dako biotinylated wide spectrum HPV probe and 3-amino-9-ethylcarbazole (AEC) as the substrate [15].…”

Section: Dna Extraction and Polymerase Chain Reaction (Pcr)mentioning

confidence: 99%

In Situ Hybridization Signal Patterns in Recurrent Laryngeal Squamous Papillomas Indicate that HPV Integration Occurs at an Early Stage

Brooks

Evans

Adamson

et al. 2011

Head and Neck Pathol

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Laryngeal papillomas are benign tumors that frequently recur and can compromise airways. We investigated HPV genotype, physical status, and protein expression in juveniles versus adults. Thirty-five laryngeal papilloma specimens were obtained from ten juveniles (1-16 years) and eleven adults (24-67 years). In cases of recurrent papillomatosis (7 juveniles, 7 adults), the first and last papillomas were assayed. HPV type was determined by GP5?/6? PCR and dot blot hybridization. In situ hybridization (ISH) was performed on 34 specimens; the data were recorded in terms of diffuse (episomal HPV) and punctate (integrated HPV) signal patterns. Immunohistochemistry for the HPV L1 capsid protein, a marker of HPV productive status, was performed on 32 samples. All samples tested HPV positive: HPV 11 in 2/10 (20.0%) juveniles and 5/11 (45.5%) adults; HPV 6 in 7/10 (70%) juveniles and 5/11 (45.5%) adults; and HPV 6/11 double infection was noted in one juvenile and one adult. ISH signals (punctate ± diffuse) were detected among 7/10 (70.0%) juveniles and 7/11 (63.6%) adults. L1 staining was detected in 1/9 (11.1%) juveniles and 6/10 (60.0%) adults (P = 0.06). These data support the idea that integration of low-risk HPV types into the cell genome is an early and common event in the etiology of juvenile and adult recurrent laryngeal papillomas. Productive HPV infections may be more common in adults; accordingly, constant laryngeal re-infection by HPV shed from a productive lesion may contribute to adult recurrent lesions, whereas the mechanism of papilloma recurrence in juveniles may be more attributable to HPV integration.

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“…First round of PCR was carried out using primers MY09/11 (Baay et al1996) [13] and second round PCR was performed using GP5+/6+ (Evans et al 2005) [14] ..First round PCR was performed at a 20 µl reaction volume using 2x PCR master mix containingTaq DNA Polymerase (Promega, USA), forward and reverse primers (MY09/MY11) at 0.5 µM final concentration, 2 µl sample and nuclease free water was added to make a final reaction volume of 20 µl. Amplification of DNA by conventional PCR was performed in a thermal cycler (Veriti 9902, Applied Biosystem, Singapore) PCR.…”

Section: ) Pcr Amplification and Amplicon Detectionmentioning

confidence: 99%

Detection of Human Papilloma Virus Dna From Dry Paper Cervical Smear- A Hospital Based Study

Śarmā¹,

Mahanta²,

Borkakoty³

et al. 2013

LSMR

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Touchdown General Primer (GP5+/GP6+) PCR and optimized sample DNA concentration support the sensitive detection of human papillomavirus

Cited by 64 publications

References 29 publications

Discrimination of ‘Driver’ and ‘Passenger’ HPV in Tonsillar Carcinomas by the Polymerase Chain Reaction, Chromogenic In Situ Hybridization, and p16INK4a Immunohistochemistry

Discrimination of ‘Driver’ and ‘Passenger’ HPV in Tonsillar Carcinomas by the Polymerase Chain Reaction, Chromogenic In Situ Hybridization, and p16INK4a Immunohistochemistry

In Situ Hybridization Signal Patterns in Recurrent Laryngeal Squamous Papillomas Indicate that HPV Integration Occurs at an Early Stage

Detection of Human Papilloma Virus Dna From Dry Paper Cervical Smear- A Hospital Based Study

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