Introduction of a fast and sensitive fluorescent in situ hybridization method for single-copy detection of human papillomavirus (HPV) genome.

Siadat-Pajouh, Majid; Ayscue, Andrea H.; Periasamy, Ammasi; Herman, Brian

doi:10.1177/42.11.7930533

Cited by 11 publications

(8 citation statements)

References 2 publications

(4 reference statements)

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“…Amplification of detection by use of an extra label of antisera after probe hybridization has not improved sensitivity [17] (J. Zehbe). The clinical use of fluorescence label [35] and the recently described biotinyl-tyramide deposition amplification method [20] must await further investigations.…”

Section: Resultsmentioning

confidence: 99%

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Detection of human papillomavirus in cervical intra-epithelial neoplasia, using in situ hybridization and various polymerase chain reaction techniques

Zehbe

Rylander

Edlund

et al. 1996

Vichows Archiv A Pathol Anat

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One hundred and forty-eight randomly chosen neutral-buffered formaldehyde-fixed cervical biopsies in which cervical intra-epithelial neoplasia (CIN) I-III had been diagnosed were tested for HPV (human papilloma virus) DNA by in situ hybridization (ISH) and polymerase chain reaction (PCR). For ISH, we utilized a biotinylated panprobe and type-specific, genomic probe sets. For PCR, we used the general primers GP5/GP6 and their recently described, elongated version GP5+/GP6+, and included the modification of hot-start PCR. Amplified DNA was detected by gel electrophoresis and slot blot hybridization. The positivity rate of ISH was 59% for all biopsies and 69%, 62% and 46% for CIN I, II and III, respectively. The sensitivity of GP5/GP6 was 74% with cold-start PCR and 78% with hot-start PCR. When GP5+/GP6+ was used, the sensitivity increased to 89% with cold-start PCR and to 95% with hot-start PCR. Based on the most sensitive PCR technique, HPV detection was 93%, 95% and 96% in CIN I, II and III, respectively. The number of HPV types decreased with the severity of the lesion, and HPV 16 was the predominant type. Multiple HPVs were rare and almost all HPV-positive cases could be typed. ISH and slot blot hybridization correlated well regarding HPV typing specificity. Our results confirm that distinct HPV types are present in a high proportion of cases of CIN. The sensitivity of ISH is lower than that of PCR. Furthermore, the modified general primers GP5+/GP6+ give a higher yield than GP5/GP6, while hot-start PCR increases sensitivity even further.

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Section: Resultsmentioning

confidence: 99%

“…At least 27 different HPV types have been associated with the genital tract mucosa. Of these types, HPV 16,18,31,33,35,39,45,51,52,56 and 58 have been found in cervical carcinomas [10,39,40], HPV 16 being the most prevalent type [50].…”

Section: Introductionmentioning

confidence: 99%

Detection of human papillomavirus in cervical intra-epithelial neoplasia, using in situ hybridization and various polymerase chain reaction techniques

Zehbe

Rylander

Edlund

et al. 1996

Vichows Archiv A Pathol Anat

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“…We continued our former study (25) to achieve the following: 1) collect more information on the physical state and the frequency of integration of HPV6, 11, 16, 18, 31, 33, 52b, and 58 DNA in benign and reactive cervical cells and SILs of 442 patients, 2) examine the integration profiles of different cancer‐associated HPV types in 125 of these patients, and 3) amplify E6/E7 mRNA transcripts by RT‐PCR analysis and use 2D gel electrophoresis to determine the physical state of viral DNA and the HPV16 DNA copy number in relation to the SiHa cell line. As described in several studies (30–35) , in this cervical carcinoma cell line, a single copy of HPV16 DNA is integrated in its genome and is therefore suitable for routine clinical determination of integration. We found that the viral loads of HPV16 E6/E7 are no prerequisite for the physical state of the integrated form of cervical lesion grades (Table 6).…”

Section: Discussionmentioning

confidence: 99%

Human papillomavirus DNA integration and messenger RNA transcription in cervical low- and high-risk squamous intraepithelial lesions in Austrian women

Manavi¹,

Hudelist

Fink-Retter³

et al. 2008

Int J Gynecol Cancer

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The human papillomavirus (HPV) plays an important role in the progression of cervical carcinoma. High-risk (HR) HPV types have been mainly identified in cytologic high-grade squamous intraepithelial lesions (HSILs) and histologic invasive carcinoma of the cervix. We examined cervical swabs of patients with abnormal Papanicolaou (Pap) smears, diagnosed as low-grade squamous intraepithelial lesions (LSILs) including atypical squamous cells of uncertain significance or HSILs. Low-risk (LR) HPV and HR-HPV types were identified by the Digene Hybrid Capture II test. Two-dimensional (2D) gel electrophoresis was used to specify the physical state of HPV DNA sequences. Expression of E6/E7 messenger RNA (mRNA) transcripts was analyzed by reverse transcriptase-polymerase chain reaction. Histopathologic results were correlated to the patients' physical status and HPV DNA mRNA transcripts. Pap smears with HPV infections of LR and HR types were correlated to the degree of squamous intraepithelial lesions (SILs). Comparing the physical states of HPV DNA sequences with the expression of HPV E6/E7 mRNA transcripts, all types were identified only as extrachromosomal in benign cervical smears, cervical intraepithelial neoplasia (CIN) I and II. HPV16 showed all physical states in CIN III/carcinoma in situ (CIS), whereas HPV18 only existed in mixed and integrated forms. HPV31/33/52b/58 appeared in all stages of lesions most commonly in extrachromosomal form; in integrated form, they were present only in CIN III/CIS. Although integration of some HR-HPV types is not always necessary for progression of SILs, the above-mentioned method is useful to analyze the physical state of HPV DNA sequences and predict the progression of SILs.

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“…Human cervical cancer cell lines were obtained from the American Type Cell Culture Collection (ATCC; Manassas, VA). Cell lines positive for HPV-16 or HPV-18 included HeLa, CaSki, and SiHa, which are well characterized with regard to genotype and number of integrated HPV genomes (Yee et al 1985;Baker et al 1987;Mincheva et al 1987;Siadat-Pajouh et al 1994;Plummer et al 1998;Meissner 1999). Controls included the HPV-39-positive ME180 and the HPV-negative C33A and HT3 cell lines.…”

Section: Cell Culturementioning

confidence: 99%

Single-copy Gene Detection Using Branched DNA (bDNA) In Situ Hybridization

Player

Shen

Kenny

et al. 2001

J Histochem Cytochem.

166

129

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We have developed a branched DNA in situ hybridization (bDNA ISH) method for detection of human papillomavirus (HPV) DNA in whole cells. Using human cervical cancer cell lines with known copies of HPV DNA, we show that the bDNA ISH method is highly sensitive, detecting as few as one or two copies of HPV DNA per cell. By modifying sample pretreatment, viral mRNA or DNA sequences can be detected using the same set of oligonucleotide probes. In experiments performed on mixed populations of cells, the bDNA ISH method is highly specific and can distinguish cells with HPV-16 from cells with HPV-18 DNA. Furthermore, we demonstrate that the bDNA ISH method provides precise localization, yielding positive signals retained within the subcellular compartments in which the target nucleic acid sequences are localized. As an effective and convenient means for nucleic acid detection, the bDNA ISH method is applicable to the detection of cancers and infectious agents. (J Histochem Cytochem 49:603-611, 2001)

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Introduction of a fast and sensitive fluorescent in situ hybridization method for single-copy detection of human papillomavirus (HPV) genome.

Cited by 11 publications

References 2 publications

Detection of human papillomavirus in cervical intra-epithelial neoplasia, using in situ hybridization and various polymerase chain reaction techniques

Detection of human papillomavirus in cervical intra-epithelial neoplasia, using in situ hybridization and various polymerase chain reaction techniques

Human papillomavirus DNA integration and messenger RNA transcription in cervical low- and high-risk squamous intraepithelial lesions in Austrian women

Single-copy Gene Detection Using Branched DNA (bDNA) In Situ Hybridization

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