Detection and identification of human papillomavirus using a PCR-restriction fragment mass polymorphism assay

Lee, Jin Kyung; Hong, Young Jun; Um, Tae Hyun; Lee, Eun Hee; Chi, Hyun‐Sook; Koh, Jae Soo; Yim, Hyeon Woo; Joο, Young Eun

doi:10.3892/mmr.2011.475

Cited by 3 publications

(2 citation statements)

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“…Furthermore, previous studies have shown that the HC II HPV test cross-reacts with at least 15 HPV genotypes not included in its current high-risk probe cocktail set (13, 14). In this study, HPV genotypes were identified by using RFMP assay which is comparable to HC II with regard to detection of high-risk HPV infection, but is also able to identify 38 HPV genotypes in a single assay, which offers high analytical and clinical sensitivity and the advantage of reliable detection of multiple HPV infections, and it showed a good correlation with direct sequencing data (7, 9). Several studies on the prevalence of overall HPV infection in Korean women have reported rates ranging from 7.2% to 44.8%, and the prevalence of HR-HPV infection ranging from 7.2% to 34.3% (15-20).…”

Section: Discussionmentioning

confidence: 92%

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Prevalence and Distribution of Human Papillomavirus Infection in Korean Women as Determined by Restriction Fragment Mass Polymorphism Assay

Lee

Sook

et al. 2012

J Korean Med Sci

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The development of a prophylactic vaccine that targets human papillomaviruses (HPV) 6, 11, 16, and 18 to prevent cervical cancer has increased interest in the ethnic and geographical distributions of HPV genotypes. We investigated HPV prevalence and type distribution by restriction fragment mass polymorphism (RFMP) testing a total of 60,775 specimens (aged 18-79 yr, median 44) taken from liquid-based cytology. Overall HPV positive rate of total patients was 34.2%. Among the positive patients, 87.7% was single type infections, and 12.3% was multiple HPV types. HPV-16 was the most prevalent genotype observed in 2,307 (26.0%), followed by type 52 in 2,269 (25.5%), type 58 in 1,090 (12.3%), type 18 in 633 (7.1%), type 56 in 436 (4.9%). The pattern of high risk-HPV positive rate according to age showed U-shape with a peak in HPV prevalence among women less than 30 yr of age, and a second peak among the older females aged 70 to 79 yr. The leading four high-risk HPV genotypes were HPV-16, HPV-52, HPV-58, and HPV-18 in descending order. In conclusion, this study provides the most representative prevalence and type-specific distribution of HPV among Korean women, and demonstrates that the epidemiology of HPV infection is different from that of other regions of the world.

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Section: Discussionmentioning

confidence: 92%

“…The restriction fragmentation mass polymorphism (RFMP) assay (GeneMetrix Co., Yongin, Korea), which utilizes PCR to amplify the HPVL1 gene followed by enzyme restriction and mass measurement using MALDI-TOF, offers a method to detect 38 types of HPV. This new method has manifested sensitivity and reliability comparable to HC II (7-9). …”

Section: Introductionmentioning

confidence: 92%

Prevalence and Distribution of Human Papillomavirus Infection in Korean Women as Determined by Restriction Fragment Mass Polymorphism Assay

Lee

Sook

et al. 2012

J Korean Med Sci

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A Novel Method for Multiplex Genotyping in a Single Reactor Using GTPlex-PyroSeq

Oh¹,

Hoehn²,

Moon³

et al. 2012

The Journal of Molecular Diagnostics

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Herein, we describe a novel multiplex genotyping method, GTPlex-PyroSeq. This method consists of two phases: multiplex PCR followed by a single reaction of pyrosequencing. This study demonstrates how GTPlex-PyroSeq can be adapted for the determination of multiple human papillomavirus (HPV) genotypes. A biotinylated consensus primer, GP6+, and 15 high-risk HPV type-specific primers are used for multiplex PCR. Each type-specific primer has a 5'-tag unique ID sequence connected to a pyrosequencing primer binding region. The unique ID sequence is composed of three parts: i) a single nucleotide ID representing a specific genotype; ii) a sign post; and iii) an end mark. This design allows multiple genotype determination under an ID sequence-dependent nucleotide dispensation order during pyrosequencing. Following initial studies using HPV plasmids and cell lines, we evaluated the clinical utility and effectiveness by comparing our assay with direct sequencing and HPV DNA chip analysis of 80 samples from high-risk, HPV-positive patients. We found in single-type infections, 100% concordance with direct sequencing (70 of 80 perfect matches) and 97.5% concordance with HPV DNA chip data (50 of 80 perfect matches). Additionally, our system was superior to direct sequencing in detection of multiple infections (12 of 80), with a limit of detection of 100 copies. The scalability of this multiplex system, with its open-platform design and ability to use various sample types, makes the GTPlex applicable for use in multiple settings.

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Molecular Diagnosis of Human Papillomavirus

Dutra¹,

Foroni²,

Couto³

et al. 2012

Human Papillomavirus and Related Diseases - From Bench to Bedside - Research Aspects

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Please use Adobe Acrobat Reader to read this book chapter for free. Just open this same document with Adobe Reader. If you do not have it, you can download it here. You can freely access the chapter at the Web Viewer here. Human Papillomavirus and Related Diseases-From Bench to Bedside-Research Aspects 206 reactions: a primary reaction that occurs on the targeted DNA sequence and a secondary reaction that produces a fluorescent signal. In the primary reaction, two types of sequence specific oligonucleotides-a probe oligonucleotide and an Invader oligonucleotide-bind to the DNA target sequence. When these oligonucleotides overlap by at least one base pair on the target sequence, an invasive structure forms that acts as a substrate for the Cleavase® enzyme. The enzyme cleaves the 5' portion (flap) of the probe at the position of the overlap. The probes are present in large molar excess and cycle rapidly on and off the target sequence so that many cleaved 5' flaps are generated per target sequence. The cleaved flaps then bind to a universal hairpin fluorescence resonance energy transfer (FRET) oligonucleotide creating another invasive structure that the Cleavase enzyme recognizes as a substrate. The enzyme cleaves the FRET oligonucleotides between the fluorophore and quencher and produces a fluorescence signal as the cleaved flaps cycle on and off. For each copy of target, the combined primary and secondary reactions result in 10 6-10 7 fold signal amplification per hour. The flap sequences and FRET oligonucleotides are universal since they are not complementary to the targeted sequence (Technologies, 2009). Oligonucleotides that bind to the human histone 2 gene (HIST2H2BE) are also present in all three oligonucleotides mixtures. This serves as an internal control, producing a semi-quantitative signal from cellular DNA present in the sample (Day et al., 2009). The Cervista™ HPV 16/18 uses de same chemistry for the identification of the two most highly oncogenic and persistent HPV types. It does not allow the exact determination of HPV type(s) present in the clinical specimen, but rather express the result as negative or positive (Day et al., 2009, Poljak and Kocjan, 2010). Sensitivity of the Cervista HPV HR test for detection of ≥CIN2 among women with ASCUS cytology was 92.8% (84.1-96.9) and the negative predictive value (NPV) was 99.1% (98.1-99.6). Sensitivity for detection of ≥CIN3 among women with ASCUS cytology was 100% (85.1-100) and the NPV was 100% (99.4-100). HPV 16/18 genotyping test sensitivity of ≥CIN2 was 68.8 (56.6-78.8) and NPV was 96.0% (93.9-97.4). HPV 16/18 genotyping test sensitivity of ≥CIN3 was 77.3 (56.6-89.9) and NPV was 99.0% (97.7-99.6)(Einsteina et al., 2010). The reproducibility, tested at three different testing centers, resulted in an overall inter-run reproducibility agreement of 98.8% [1-sided 95% Confidence Lower Limit = 96.9%] and an overall inter-site reproducibility agreement of 98.7% [1-sided 95% Confidence Lower Limit = 97.9%] (Day et al., 2009). The Cervista HPV HR test showed no...

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Detection and identification of human papillomavirus using a PCR-restriction fragment mass polymorphism assay

Cited by 3 publications

References 14 publications

Prevalence and Distribution of Human Papillomavirus Infection in Korean Women as Determined by Restriction Fragment Mass Polymorphism Assay

Prevalence and Distribution of Human Papillomavirus Infection in Korean Women as Determined by Restriction Fragment Mass Polymorphism Assay

A Novel Method for Multiplex Genotyping in a Single Reactor Using GTPlex-PyroSeq

Molecular Diagnosis of Human Papillomavirus

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