Development of reference materials to detect 15 different human papillomavirus genotypes

CCA-13417; No of Pages 6 Clinica Chimica Acta xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim

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Jee Eun Rhee a, Young Soon Kang c, Hyun Hee Seo c, Ju-yeon Choi a, Mee-Kyung Kee a, Tae-Jin Kim d, Sung Ran Hong b,⁎, Sung Soon Kim a,⁎⁎

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Article history: Received 28 November 2013 Received in revised form 29 January 2014 Accepted 17 February 2014 Available online xxxx

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Keywords: Human papillomavirus Reference material Genotype Proficiency

Division of AIDS, Center for Immunology and Pathology, Korea National Institute of Health, Chungcheong-Bukdo, Republic of Korea Department of Pathology, Cheil General Hospital & Women's Healthcare Center, College of Medicine, Kwandong University, Seoul, Republic of Korea Office of Genome Research and Development, Cheil General Hospital & Women's Healthcare Center, College of Medicine, Kwandong University, Seoul, Republic of Korea d Department of Obstetrics and Gynecology, Cheil General Hospital & Women's Healthcare Center, College of Medicine, Kwandong University, Seoul, Republic of Korea b

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Development of reference materials to detect 15 different human papillomavirus genotypes

Accurate human papillomavirus (HPV) typing is essential for evaluating and monitoring HPV vaccines in cervical cancer screening and in epidemiological surveys. In our country, different HPV DNA detection and genotyping methodologies have been established for diagnosing and monitoring HPV-related disease in clinical practice and for research. However, there is a lack of reference materials to standardize the methods for HPV detection and genotyping. In this study, we constructed candidate reference materials comprising 15 targets (13 types of high-risk HPV, two types of low-risk HPV). We evaluated whether the candidate reference materials could be used as the reference for HPV detection and genotyping using quantitative real-time polymerase chain reaction. Standard curves for the wide linear range (101–106 copies/μL) produced high correlation regression coefficient R2 of 0.99. The reaction efficiencies were 96.3% to 101.2% for the standard curves, indicating highly efficient reactions. Specific genotypes were detected in single or multiple mixed samples. Our results suggest that these reference materials may provide useful standards for standardizing quality assurance for different HPV-typing assays and for proficiency testing in diagnostic laboratories. © 2014 Published by Elsevier B.V.

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1. Introduction

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Cervical cancer is the second most common type of cancer among women aged 15–44 years worldwide, and human papillomavirus (HPV) infection is the cause of nearly 100% of cervical cancers [1–3]. The most important high-risk types are HPV-16 and -18, which account for about 70% of all invasive cervical cancers worldwide. HPV types 31, 33, 35, 45, 52, and 58 are associated with about 15% of cervical cancer cases [4]. The prophylactic vaccines against HPV-16 and -18 are Gardasil® (Merck and Co., NJ, USA; a quadrivalent vaccine for HPV-6, -11, -16, -18) and Cervarix® (GlaxoSmithKline Biologicals, Brentford, UK; a bivalent vaccine for HPV-16 and -18). These vaccines have been licensed by Korea Food and Drug Administration since 2007 and 2008, respectively. Both vaccines have been demonstrated to induce high titers of vaccine type-specific neutralizing antibodies and are effective in preventing

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⁎ Correspondence to: S.R. Hong, Department of Pathology, Cheil General Hospital & Women's Healthcare Center, College of Medicine, Kwandong University, Republic of Korea. Tel.: +82 2 2000 7661; fax: +82 2 2000 7779. ⁎⁎ Correspondence to: S.S. Kim, Division of AIDS, Center for Immunology and Pathology, Korea National Institute of Health, Republic of Korea. Tel.: +82 43 719 8410; fax: +82 43 719 8459. E-mail addresses: [email protected] (S.R. Hong), [email protected] (S.S. Kim).

persistent HPV infection and related cervical lesions [5–8]. These vaccines have increased the monitoring of HPV and have led to studies to document the effectiveness of the vaccines. Accurate and internationally comparable HPV DNA detection and genotyping assays play an essential role in epidemiological studies of HPV surveillance, vaccination impact monitoring, and prevention of the development of cervical cancer from infection. Diverse methods for genotyping HPV DNA are used as the primary tool to measure HPV disease burden and vaccine impact. The accurate detection and genotyping of HPV DNA in clinical samples are important in ensuring that all HPV laboratories obtain results that are consistent, meaningful, and comparable. In our country, different HPV DNA detection and genotyping methodologies have been established for diagnosing and monitoring HPVrelated disease in clinical practice and in research. However, we lack well-characterized reference materials to evaluate the performance of these laboratories and to determine the sensitivity and specificity of HPV DNA detection and quantification. In response to the need, we constructed recombinant HPV plasmid DNAs that encode the full-length L1 protein of 15 different HPV genotypes and evaluated whether these could be used as the reference materials for HPV genotyping. We assessed their suitability for use in assays to detect and genotype HPV DNA in HPV clinical laboratories and in the research field.

http://dx.doi.org/10.1016/j.cca.2014.02.013 0009-8981/© 2014 Published by Elsevier B.V.

Please cite this article as: Rhee JE, et al, Development of reference materials to detect 15 different human papillomavirus genotypes, Clin Chim Acta (2014), http://dx.doi.org/10.1016/j.cca.2014.02.013

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2.2. Construction of the specific genotype-targeted plasmid DNA

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2.2.1. Polymerase chain reaction (PCR) amplification The HPV L1 gene was amplified with specific genotype primers (Table 1). To amplify the 15 individual L1 genes, 1 U of Taq DNA polymerase, 250 μM dNTP, 1.5 mM MgCl2, 40 pmol of corresponding primers (Table 1), and 10 ng of cervical DNA were added to a 20 μL reaction volume. The optimized PCR program for the L1 gene was as follows: 94 °C for 5 min; then 30 cycles at 94 °C for 30 s for denaturation, 55 °C at 30 s for annealing; 72 °C for 1 min, and 30 s for extension; followed by 72 °C for 10 min. The amplified products were separated on a 1.5% agarose electrophoresis gel and purified using the QIAquickGel Extraction Kit (Qiagen, Hilden, Germany).

79 80 81 82 83 84 85 86 87

92 93 94 95 96 97 98 99 100 101 102 103

t1:1 t1:2

Table 1 Oligonucleotides used to amplify genotype-specific L1 genes.

For TaqMan real-time PCR, primers and probes corresponding to each specific genotype L1 were designed using Primer V3 software (Table 2) and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The probe contained a reporter dye 5′,6-carboxyfluorescein (for HPV-6, -16, -31, -45, -52, -58, and -66) or 5-hexachlorofluorescein (for HPV-11, -18, -33, -39, 51, -56, -59, and -68b) at the 5′ end and a quencher dye (NFQ-MGB) at the 3′ end. TaqMan real-time PCR was performed in a 20 μL final volume containing 1 μL of each serially diluted recombinant DNA (1 × 106, 1 × 105, 1 × 104, 1 × 103, 1 × 102, and 1 × 101 copies/μL), 10 μL of 2× LightCycler 480 Probe Master Mix (Roche), 0.5 μL of 10 pmol HPV genotype-specific forward primer, 0.5 μL of 10 pmol HPV genotype specific reverse primer, 0.1 μL of uracil DNA glucosidase (NEB Ltd., Hertfordshire, UK), 0.1 μL of HPV DNA type-specific TaqMan MGB probe, and 7.8 μL of H2O. The PCRs were all run on a LightCycler 480 II (Roche) using the following program: 95 °C for 10 s and 45 cycles at 95 °C for 10 s, 55 °C for 10 s, and 72 °C for 10 s. The fluorescence signals were measured once during each cycle at the extension step, and the data were then analyzed. Each reaction was repeated three times in triplicate.

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Extracted recombinant DNA was tested using the Linear Array HPV Genotyping Assay (Roche). The linear array genotyping system uses PCR amplification of DNA, followed by a reverse line blot hybridization assay to detect the amplified DNA products. The reaction volume

Size (bp)

GenBank accession no.

ATGTGGCGGCCTAGCGACAGCAC TTACCTTTTGGTTTTGGCGCGCTTAC ATGTGGCGGCCTAGCGACAGCAC TTACTTTTTAGTTTTGGTGCGCTTAC ATGCAGGTGACTTTTATTTACATCC TTACAGCTTACGTTTTTTGCGTTTAG ATGTGCCTGTATACACGGGTCCTG TTACTTCCTGGCACGTACACGCAC ATGTCTCTGTGGCGGCCTAGCGAGG TTACTTTTTAGTTTTTTACGTTTT ATGTCCGTGTGGCGGCCTAGTGAGG TTATTTTTTAACCTTTTTGCGTTTT ATGGCTCTGTGGCGGTCTAGTG TTATTTAGACACACGTTTACGTTTGTG ATGGCACACAATATTATTTATGGCC TTATTTCTTACTACGTATACGTACA ATGGCATTGTGGCGCACTAATGAC TTACTTTTTAACACGTTTACGTTTGGC ATGGTACAGATTTTATTTTACATCC TTACCTTTTAACCTTTTTCTTCTTT ATGATGTTACCCATGATGTATATATAC CTACCGCCTTTTACGTTTTGCTG ATGGTGCTGATTTTATGTTGCACC TTATTTTTTAACCTTTTTGCGTTTGGTGG ATGGCTCTATGGCGTTCTAGTGACA CTATTTTCTGGAAGACTTGCGACGC ATGGCGATGTGGCGGCCTAGTGACA CTATCGTTTTTTACGTTTAGCTGGT ATGGCATTGTGGCGCTCTAGCGAC TTACTTTGACACACGTTTACGTTTGTGC

1503

AF092932

1506

U55993

1596

K02718

1707

X05015

1515

JO4353

1500

M12732

1518

JN104070

1620

X74479

1515

M62877

1590

X74481

1605

X74483

1575

JX313772

1527

X77858

1512

U31794

1518

FR751039

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Oligonucleotide sequence (5′ → 3′)

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108 109 110 111 112

115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133

2.4. Linear array assay

HPV-6F HPV-6R HPV-11F HPV-11R HPV-16F HPV-16R HPV-18F HPV-18R HPV-31F HPV-31R HPV-33F HPV-33R HPV-39F HPV-39R HPV-45F HPV-45R HPV-51F HPV-51R HPV-52F HPV-52R HPV-56F HPV-56R HPV-58F HPV-58R HPV-59F HPV-59R HPV-66F HPV-66R HPV-68bF HPV-68bR

N

t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21 t1:22 t1:23 t1:24 t1:25 t1:26 t1:27 t1:28 t1:29 t1:30 t1:31 t1:32 t1:33

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Oligonucleotide

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Q3 t1:4

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105

2.2.2. Cloning and sequencing of the L1 gene into pGEM-T vector The full-length L1 gene of the 15 genotypes was purified from the agarose gels. Each purified DNA was ligated to pGEM-T Easy Vector (Promega, Madison, WI, USA) and transformed into Escherichia coli

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2.3. Real-time PCR

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To obtain the L1 gene of the 15 HPV genotypes (HPV-6, -11, -16, -18, -31, -33, -39, -45, -51, -52, -56, -58, -59, -66, and -68b), we used cervical DNA collected from the Korea HPV Cohort Study [9]. The Korea HPV Cohort Study is a multicenter, open, cross-sectional and prospective cohort study of participants in four metropolitan cities in Korea. The cohort recruits women who have been shown to be HPV positive with a cytological abnormality on a Pap smear; the HPV test results were obtained from the participating hospitals. Cervical DNA was extracted from exfoliated cervical cells after clinical examination. The target HPV-infected cervical DNAs of at least one of the 15 target genotypes collected in the cohort study were selected. We confirmed their genotypes using the Linear Array HPV genotyping test (Roche Diagnostics, Basel, Switzerland).

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2.1. HPV infectious samples

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JM109. The nucleotide sequences of the plasmid DNA fragment were determined by primer walking (Macrogen, Seoul, Korea). Comparisons of the nucleotide and deduced amino acid sequences were conducted using BLAST (Basic Local Alignment Search Tool, National Center for Biotechnology Information (NCBI)). The purified plasmids were then linearized with EcoRI for 3 h at 37 °C, and their expected size was confirmed.

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2. Materials and methods

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Please cite this article as: Rhee JE, et al, Development of reference materials to detect 15 different human papillomavirus genotypes, Clin Chim Acta (2014), http://dx.doi.org/10.1016/j.cca.2014.02.013

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t2:49 t2:50

HPV-39

HPV-45

HPV-51

HPV-52

HPV-56

HPV-58

HPV-59

HPV-66

HPV-68b

139

HEX

110

FAM

129

HEX

119

HEX

100

FAM

142

HEX

149

FAM

106

HEX

115

FAM

138

113

HEX

F

FAM

3.2. Validation of the candidate reference materials

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HPV-33

108

FAM

126

HEX

a Forward and reverse primers are shown as F and R, respectively. All probes are shown as P. b All probes were labeled with 5′,6-carboxyfluorescein (FAM) or 5-hexachlorofluorescein (HEX) as the reporter and 6-carboxytetramethylrhodamine as the quencher to maintain uniformity. The last column shows the labeled fluorescein used as the reporter for the probe of each genotype.

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3. Results

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3.1. Confirmation of constructed candidate as the reference material

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The ~1500-base pair (bp) full-length L1 gene was amplified through PCR (Fig. 1A), ligated into the pGEM-T easy vector, and transformed into E. coli JM109. The inserted DNA and the correct construction of the

140 141 142 143 144 145

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comprised 50 μL of Linear Array-HPV Master Mix and 50 μL of DNA. The PCR amplification reaction involved an initial activation step at 95 °C for 9 min, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 1 min, and extension at 72 °C for 1 min, with a final extension at 72 °C for 5 min [10]. The reaction uses PGMY 09/11 primers to detect HPV types. A total of 37 types of HPV are detectable (6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82, 83, 84, IS39, and CP6108) [11]. Type-specific oligonucleotide probes were used to differentiate hybridized HPV types. The HPV-52 probe hybridizes to HPV-33, -35, and -58.

138 139

153 154 155 156 157 158 159 160 161 162 163 164 165 166

3.2.1. Verification of the concentration-dependent identification of recombinant DNA from the 15 HPV genotypes To evaluate the sensitivity and specificity of the candidate reference materials, HPV DNA was detected using the TaqMan real-time PCR assay. The recombinant plasmids (ranging from 101 to 106 copies/μL) were prepared in 10-fold serial dilutions, and 1 μL of each serially diluted recombinant plasmid sample was used as the template. As shown in Table 3, the threshold cycle (Ct) for each genotype increased in inverse proportion to the concentration of the recombinant plasmid standard. The sample was judged as positive when the Ct value was 16 to 35 for the 15 different reference materials. Standard curves showed a good correlation regression coefficient R2 of 0.99 in three PCR runs. The reaction efficiencies were 96.3% to 101.2% for the standard curves, indicating highly efficient reactions. This means that the binding affinity and detection efficiency between the reference materials were high for the 15 genotypes and genotype-specific primers. To improve the confidence in the reference materials, we examined whether genotype-specific primers could target the genotypes specifically. Genotype-specific primers were added to a mixture of DNA of the 15 genotypes, and the HPV DNA was amplified in the real-time PCR assay. No nonspecific reactions or any internal cross-amplifications were observed, with the exception of HPV-6, -11, -45, and -51 (Table 4). The HPV-6 genotype was detected with HPV-11-specific primers, and HPV45 was detected with HPV-51-specific primers. This cross-detection might reflect some sharing of sequence similarity between HPV-6 and -11 and between HPV-45 and -51. To confirm this possibility, we have aligned (http://blast.ncbi.nlm.nih.gov) the nucleotide sequence between HPV-11 PCR product and HPV-6 L1 gene and between HPV-51 PCR product and HPV-45 L1 gene. Each sequence that aligned with 89% and 74% identity compared with other sequences is observed with no significance (data not shown). Additionally, we performed linear array assays to test whether this cross-detection can be avoided. No cross-hybridization was observed for the genotypes mentioned (Fig. 2). We assumed that the cross-reactivity observed was related to the interference of nonspecific PCR products generated during the process of TaqMan real-time PCR rather than the interference (cross-reaction) between HPV genotypes.

167 168

3.2.2. Single genotype identification of the recombinant DNA from 15 HPV genotypes To identify single HPV genotypes, the recombinant DNAs from the 15 HPV genotypes were diluted to obtain samples of high (1 × 105 copies/μL), medium (1 × 103 copies/μL), and low (1 × 101 copies/μL) concentrations and were subjected to TaqMan real-time PCR. In the identification of the 15 genotypes, the Ct value was 33.94 to 37.67 in samples at low concentration, 28.16 to 30.51 in samples at medium concentration, and 20.15 to 23.27 in samples at high concentration (Table 5). In all recombinant DNA samples of the 15 single HPV genotypes studied, specific HPV genotypes that matched the high, medium, and low concentrations were identified.

203

R O

HPV-31

HEX

P

HPV-18

108

D

HPV-16

FAM

E

HPV-11

84

T

F: GGGAACCTGTGCCTGATACA R: GCTCGGGGTGTTAACATATATACTACT P: AAGGGTAGTGGAAATCGCACGTCTG F: TGGAGGACTGGAACTTTGGT R: TTTCAGGTGTGGGTTTCTGA P: TCGCCTCCACCAAATGGTACACTG F: TAGGTCGTGGTCAGCCATTA R: TATCCACACCTGCATTTGCT P: AAGGATGGCCACTAATGCCCACA F: CCATATTGGTTACATAAGGCACA R: ATTGCCCAGGTACAGGAGAC P: TGCCAGCAAACACCATTGTTATGACC F: GGGTTCGTTTACCAGATCCA R: CCCGCGACCTACCTCTAA P: CCAACACAGGCCCAAACTAAGCG F: TTGGATGTAAGCCTCCAACA R: TGTCCACCATATCACCATCC P: TGCAGCACCTGCCAATGATTGT F: TGTCTGCAGATGTGTATGGG R: ATACAATTGGGCAGGAATGG P: CGTCACCCACCATACCACCACG F: AATAGGGCAGGTGTTATGGG R: GGGAATACACACAACTGCCA P: TTCACGCATATTAGCGCTAGTGCCTT F: GGCCGTGACCCTATAGAAAG R: GCTGATTGTTCCAGCAAATG P: TTATTGGCTCCACCGTGCGC F: TATGCAGGCAGTTCTCGATT R: TTATTAGGGTCCGGCAATTT P: CCCAAGGTGTCTGGCCTGCA F: AGTTGATGGCAAGCAAACAC R: CCCTGTGGTAACTTGTGTGG P: TCCAATGTTCACCCATAGCGGG F: GGGTAAAGGTGTTGCCTGTAA R: CATGCATCCAAACCCTGTAT P: TGCAGCTGCTACTGATTGTCCTCCA F: GACAAAGGGCACTGCTTGTA R: TCCTGCAACAATTTAAAGTCCA P: TGGAGGACAATCGCCCTGAACC F: GTGTTTAGGGTACGGTTGCC R: GGCCTACCTCCAAACCTACA P: CAGGCCCATACCAACCGTTCCT F: CAGTTTCCTTTAGGACGCAA R: CACACGTTTACGTTTGTGCTT P: CGGCGGACACCTGCCTGTAA

Labeled reporterb

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HPV-6

Size (bp)

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t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 t2:19 t2:20 t2:21 t2:22 t2:23 t2:24 t2:25 t2:26 t2:27 t2:28 t2:29 t2:30 t2:31 t2:32 t2:33 t2:34 t2:35 t2:36 t2:37 t2:38 t2:39 t2:40 t2:41 t2:42 t2:43 t2:44 t2:45 t2:46 t2:47 t2:48

Primer and probe sets in 5′ → 3′ orientationa

R

Q4 t2:4

Genotype

R

t2:3

~ 4500 bp recombinant product were confirmed by DNA sequencing and restriction enzyme digestion. The sequences of the constructs containing the L1 gene for 15 different genotypes were aligned by BLAST at the NCBI (data not shown). The double-stranded plasmid DNA template was subjected to restriction endonuclease digestion with EcoRI and was compared with the DNA ladder marker. Because the HPV L1 gene contains about 1.5 kb, the detected band in this region confirmed that the PCR reaction was performed correctly. The constructed plasmid DNA digested with EcoRI was resolved into two or three visible bands corresponding to the expected vector and DNA insert fragments of 3 kb and 1.5 kb for HPV-6, -11, -18, -31, -33, -45, -51, -56, -58, -59, -66, and -68b; and of 3 kb, 1.2 kb, and 0.3 kb for HPV-16, -39, and -52, respectively (Fig. 1B).

Table 2 Oligonucleotides and probes used in TaqMan real-time PCR analysis.

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t2:1 t2:2

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Please cite this article as: Rhee JE, et al, Development of reference materials to detect 15 different human papillomavirus genotypes, Clin Chim Acta (2014), http://dx.doi.org/10.1016/j.cca.2014.02.013

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3.0kb 1.5kb 1.0kb

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15

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0.5kb

P

Fig. 1. (A) For each genotype, the target L1 gene was amplified by PCR. The ~1500 bp L1 gene was amplified from samples from HPV-infected patients. M, DNA marker (Genedirex, 1 kb DNA Ladder RTU); lane 1, L1 gene for HPV-6; lane 2, L1 gene for HPV-11; lane 3, L1 gene for HPV-16; lane 4, L1 gene for HPV-18; lane 5, L1 gene for HPV-31; lane 6, L1 gene for HPV-33; lane 7, L1 gene for HPV-39; lane 8, L1 gene for HPV-45; lane 9, L1 gene for HPV-51; lane 10, L1 gene for HPV-52; lane 11, L1 gene for HPV-56; lane 12, L1 gene for HPV-58; lane 13, L1 gene for HPV-59; lane 14, L1 gene for HPV-66; and lane 15, L1 gene for HPV-68b. The molecular weights are shown on the left. (B) Digestion of genes cloned in pGEM-T vector by EcoR1. Restricted DNAs were loaded into 1% agarose gel. M, DNA marker (Roche, 100 bp ladder); lane 1, recombinant DNA for HPV-6; lane 2, recombinant DNA for HPV-11; lane 3, recombinant DNA for HPV-16; lane 4, recombinant DNA for HPV-18; lane 5, recombinant DNA for HPV-31; lane 6, recombinant DNA for HPV-33; lane 7, recombinant DNA for HPV-45; lane 8, recombinant DNA for HPV-51; lane 9, recombinant DNA for HPV-52; lane 10, recombinant DNA for HPV-56; lane 11, recombinant DNA for HPV-58; lane 12, recombinant DNA for HPV-59; lane 13, recombinant DNA for HPV-66; lane 14, recombinant DNA for HPV-68b; and lane 15, recombinant DNA for HPV-39. The molecular weights are shown on the left.

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2.6kb 1.5kb 1.0kb

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t3:1 t3:2

Table 3 TaqMan probe assay performance for 15 different HPV genotypes.

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one medium-concentration genotype and one medium-concentration genotype, and one low-concentration genotype and one highconcentration genotype. TaqMan real-time PCR sample sets and operation conditions were established as described in the Materials and methods section. The overall Ct values were 20.23 to 40 (Table 5). In samples of recombinant DNA of multiple HPV genotypes, when the specific genotype had high and medium concentrations (1 × 105 copies and 1 × 103 copies, respectively), the specific HPV genotype was identified appropriately regardless of the concentration of the complementary HPV genotype. However, in samples with low concentrations

t3:6 t3:7 t3:8 t3:9 t3:10 t3:11 t3:12 t3:13 t3:14 t3:15 t3:16 t3:17 t3:18 t3:19 t3:20

Ct value by copy numbera 1

t3:4

Q5 t3:5

10

HPV-6 HPV-11 HPV-16 HPV-18 HPV-31 HPV-33 HPV-39 HPV-45 HPV-51 HPV-52 HPV-56 HPV-58 HPV-59 HPV-66 HPV-68b a

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Genotype

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t3:3

C

O

222 223

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220 221

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218 219

C

224

3.2.3. Multiple genotype identification of the recombinant DNA from 15 HPV genotypes To identify the detected multiple HPV genotypes, the 15 HPV genotypes were divided into seven sets (6/56, 16/18, 31/33, 45/11, 52/51, 58/59, and 66/68b) by selecting those that showed no reciprocal nonspecific, false-positive, or false-negative responses. These were mixed to obtain high (1 × 10 5 copies), medium (1 × 103 copies), and low (1 × 101 copies) complementary concentrations, and subjected to TaqMan real-time PCR. In other words, we analyzed the sample sets of one high-concentration genotype and one low-concentration genotype,

216 217

35.95 34.76 35.87 35.96 35.61 36.02 35.89 34.87 34.75 33.49 36.61 34.75 37.12 35.93 35.28

2

3

4

5

Slope

R2

% efficiency

−3.36 −3.263 −3.36 −3.263 −3.326 −3.498 −3.436 −3.402 −3.375 −3.433 −3.401 −3.422 −3.512 −3.436 −3.374

0.999 0.999 0.999 0.999 0.999 0.999 0.997 0.998 0.999 0.999 0.999 0.997 0.995 0.997 0.999

99.2 101.25 99.2 101.25 99.95 96.55 97.7 98.4 98.9 97.8 98.4 98 96.3 97.7 98.95

6

10

10

10

10

10

32.58 31.96 31.84 33.02 31.77 32.49 33.43 31.87 31.7 30.34 33.6 32.45 32.2 33.48 32.3

28.94 28.77 28.32 29.52 28.39 29.03 30.12 28.91 28.41 27.07 30.06 28.8 28.56 30.07 29.09

25.61 25.39 24.89 25.96 24.98 25.56 26.75 25.54 25.03 23.68 26.76 25.52 25.5 26.6 25.65

22.21 21.97 21.49 22.49 21.7 21.91 23.12 22.21 21.66 20.07 23.37 22.06 21.38 23.14 22.23

18.92 19.1 18.15 19.63 18.4 18.56 19.74 18.73 18.28 16.84 19.94 18.71 18.27 19.76 18.99

Ct, cycle threshold values.

Please cite this article as: Rhee JE, et al, Development of reference materials to detect 15 different human papillomavirus genotypes, Clin Chim Acta (2014), http://dx.doi.org/10.1016/j.cca.2014.02.013

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Test primer

Q6 t4:4

t4:20

a

HPV-31

HPV-33

HPV-39

HPV-45

HPV-51

18.94 26.59

17.93

HPV-52

HPV-58

HPV-59

HPV-66

HPV-68b

17.5 18.72 18.46 17.4

22 22.94

21.98

(1 × 101 copies), the identification was hampered when the complementary HPV genotype had a high concentration (1 × 105 copies). These findings indicate that, in clinical samples, there is a high likelihood that a low-concentration genotype cannot be identified accurately because of the presence of a high-concentration genotype.

3

4

5

19.83 17.44

HPV infection is the most common sexually transmitted infection worldwide and is associated with both benign and severe clinical manifestations, including cancers of the cervix and the anogenital region. As a consequence, these infections represent a significant concern at both the individual and public health levels, and controlling HPV infection and related diseases through prevention strategies (both primary and secondary) remains a priority. Cytological screening for the early detection of cervical cancer precursors has been successful in many countries. However, the procedures contributing to the success of these screening programs are inefficient and unfeasible in parts of the world where the appropriate infrastructure is not available [12,13]. As an alternative to cytological analysis of cervical samples, nucleic acid-based assays are more sensitive in detecting HPV DNA, and, thus, the use of this methodology should be preferred as a primary screening test in newly implemented programs [14–16]. The detection of HPV DNA is also used in epidemiological studies of the geographical distribution of HPV genotypes and type-specific prevalence as well as for monitoring vaccine efficacy. However, there are diverse HPV DNA assays that show variable sensitivity and specificity in HPV DNA detection. The aim of this study was to evaluate candidate plasmid-based reference materials for 15 different HPV genotypes to determine their possible use in accurate HPV genotyping to improve the quality of HPV detection in HPV diagnostic laboratories. Our materials were not designed to assess the sample preparation steps, such as DNA extraction, purification, and centrifugation, and thus cannot be used for such studies. For proficiency testing using our developed reference material, it might provide the HPV infected cell lines such as SiHa, HeLa, and CasKi to assess the sample preparation steps in clinical laboratories. In a previous study, international standards for HPV genotyping were established by the WHO Expert Committee on Biological Standardization in 2008 for detecting and quantifying HPV-16 and -18 DNA with assigned potency in international units. These reference standards were evaluated through proficiency testing with commercial and in-house quantitative and qualitative assays from 19 laboratories in 13 countries [17]. The standard materials were confined to the HPV-16 and -18 genotypes. Other main high-risk genotypes such as HPV-52, -58, and -33 were not included. We have targeted genotypes to generate reference materials from the 10 most frequent HPV types among women with and without cervical lesions throughout the world, as identified in the 2010 WHO HPV and Related Cancers Summary Reports (WHO 2010) and the genotypes for WHO proficiency testing [18]. These include HPV-6, -11, -16, -18, -31, -33, -39, -45, -51, 52, -56, -58, -59, -66, and -68b. We evaluated

6

R

E

C

T

E

2

4. Discussion

20.84

R O

Ct, cycle threshold value.

1

HPV-56

17.71

R

238 239

17.58

HPV-18

N C O

236 237

18.32 24.32

HPV-16

U

235

HPV-11

F

HPV-6 HPV-11 HPV-16 HPV-18 HPV-31 HPV-33 HPV-39 HPV-45 HPV-51 HPV-52 HPV-56 HPV-58 HPV-59 HPV-66 HPV-68b

HPV-6

O

t4:5 t4:6 t4:7 t4:8 t4:9 t4:10 t4:11 t4:12 t4:13 t4:14 t4:15 t4:16 t4:17 t4:18 t4:19

Ct value for each genotypea

P

t4:3

Table 4 Specificity for HPV genotype in real-time PCR.

D

t4:1 t4:2

5

Fig. 2. Results for the linear array. The PCR linear array (PCR-LA) result for each sample was determined by comparing the band pattern with the PCR-LA reference guide. Lane 1, PCRLA HPV-negative control with no visible bands; lane 2, PCR-LA HPV-positive control with an HPV-16 band; lane 3, recombinant DNA positive for HPV-6; lane 4, recombinant DNA positive for HPV-45; lane 5, recombinant DNA positive for HPV-51; lane 6, recombinant DNA positive for HPV-11. Except for the positive control, all samples were negative for β-globin high- and β-globin low-molecular weight because of negative control and plasmid DNA.

Please cite this article as: Rhee JE, et al, Development of reference materials to detect 15 different human papillomavirus genotypes, Clin Chim Acta (2014), http://dx.doi.org/10.1016/j.cca.2014.02.013

240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 Q10 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284

6

Copy number

Q7 t5:4

HPV-6

HPV-11

HPV-16

HPV-18

HPV-31

HPV-33

HPV-45

HPV-51

HPV-52

HPV-56

HPV-58

HPV-59

HPV-66

HPV-68b

10 103 101

22.35 29.01 35.54

22.15 28.85 35.59

21.28 28.16 34.9

22.3 29.45 34.86

21.67 28.6 35.39

22.05 29.32 37.12

22.21 29.06 35.5

21.77 28.63 35.63

20.15 27.68 33.94

23 30.02 36.84

22.08 29.47 36.59

21.69 28.89 35.81

23.27 30.51 36.76

22.23 29.22 37.67

Copy number

Ct value for multiple genotypesa HPV-6/HPV-56

t5:12 t5:13 t5:14

105/101 103/103 101/105

HPV-31/HPV-33

HPV-16

HPV-18

HPV-31

HPV-33

HPV-45

HPV-11

HPV-52

HPV-51

HPV-58

HPV-59

HPV-66

HPV-68b

36.91 29.78 22.98

21.71 27.75 –

40 29.04 22

21.71 27.75 –

40 29.04 22

22.2 28.46 –

36.7 28.7 22.12

20.23 27.27 40

– 27.41 21.7

22.09 29.03 –

40 28.58 21.61

23.19 30.11 36.52

40 28.87 22.26

313

5. Conclusion

314

318 319

We have developed reference materials comprising 15 genotypes of HPV and have demonstrated that these are suitable for use as standards for variable copy numbers and single or multiple genotype amplification and detection of these 15 HPV genotypes. Our constructs can be used as the reference material for quality control and proficiency material for HPV genotyping in clinical laboratories.

320

Conflict of interest

306 307 308 309 310

315 316 317

321 322

C

E

R

304 305

R

302 303

O

300 301

C

298 299

N

296 297

U

294 295

The authors declare that they have no conflict of interest. Acknowledgments

323 Q11 This study was supported by a grant from the health promotion 324 against HIV/AIDS and STD (4800-4842-302) of the National Institute 325

References

[1] Walboomers JM, Jacobs MV, Manos MM, et al. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 1999;189:12–9. [2] ZurHausen H. Papillomaviruses in the causation of human cancers—a brief historical account. Virology 2009;384:260–5. [3] WHO/ICO Information Centre on HPV and Cervical Cancer (HPV Information Centre). Human papillomavirus and related cancers in the world, summary report. Available at www.hpvcentre.net; 2010. [4] Clifford GM, Smith JS, Plummer M, Munoz N, Franceschi S. Human papillomavirus types in invasive cervical cancer worldwide: a meta-analysis. Br J Cancer 2003;88:63–73. [5] Harper DM, Franco EL, Wheeler C, et al. Efficacy of a bivalent L1 virus-like particle vaccine in prevention of infection with human papillomavirus types 16 and 18 in young women: a randomised controlled trial. Lancet 2004;364:1757–65. [6] Paavonen J, Jenkins D, Bosch FX, et al. Efficacy of a prophylactic adjuvanted bivalent L1 virus-like-particle vaccine against infection with human papillomavirus types 16 and 18 in young women: an interim analysis of a phase III double-blind, randomised controlled trial. Lancet 2007;369:2161–70. [7] Brown DR, Kjaer SK, Sigurdsson K, et al. The impact of quadrivalent human papillomavirus (HPV; types 6, 11, 16, and 18) L1 virus-like particle vaccine on infection and disease due to oncogenic nonvaccine HPV types in generally HPV-naive women aged 16–26 years. J Infect Dis 2009;199:926–35. [8] Wheeler CM, Kjaer SK, Sigurdsson K, et al. The impact of quadrivalent human papillomavirus (HPV; types 6, 11, 16, and 18) L1 virus-like particle vaccine on infection and disease due to oncogenic nonvaccine HPV types in sexually active women aged 16–26 years. J Infect Dis 2009;199:936–44. [9] Lee WC, Lee SY, Koo YJ, et al. Establishment of a Korea HPV cohort study. J Gynecol Oncol 2003;24:59–65. [10] Stevens MP, Tabrizi SN, Quinn MA, Garland SM. Human papillomavirus genotype prevalence in cervical biopsies from women diagnosed with cervical intraepithelial neoplasia or cervical cancer in Melbourne, Australia. Int J Gynecol Cancer 2006;16:1017–24. [11] Gravitt PE, Peyton CL, Alessi TQ, et al. Improved amplification of genital human papillomaviruses. J Clin Microbiol 2000;38:357–61. [12] Louie KS, de Sanjose S, Mayaud P. Epidemiology and prevention of human papillomavirus and cervical cancer in sub-Saharan Africa: a comprehensive review. Trop Med Int Health 2009;14:1287–302. [13] Cronjé HS. Cervical screening strategies in resourced and resource-constrained countries. Best Pract Res Clin Obstet Gynaecol 2011;25:575–84. [14] Cuzick J, Arbyn M, Sankaranarayanan R, et al. Overview of human papillomavirusbased and other novel options for cervical cancer screening in developed and developing countries. Vaccine 2008;26:K29–41. [15] Naucler P, Ryd W, Tornberg S, et al. Efficacy of HPV DNA testing with cytology triage and/or repeat HPV DNA testing in primary cervical cancer screening. J Natl Cancer Inst 2009;101:88–99. [16] Jin XW, Sikon A, Yen-Lieberman B. Cervical cancer screening: less testing, smarter testing. Cleve Clin J Med 2011;78:737–47. [17] Wilkinson DE, Baylis SA, Padley D, et al. Establishment of the 1st World Health Organization international standards for human papillomavirus type 16 DNA and type 18 DNA. Int J Cancer 2009;126:2969–83. [18] Quint WGV, Pagliusi SR, Lelie N, Villiers EM, Wheeler CM, the World Health Organization Human Papillomavirus DNA International Collaborative Study Group. Results of the first world health organization international collaborative study of detection of human papillomavirus DNA. J Clin Microbiol 2006;44:571–9. [19] Ronken S, Mol A. The effect of ascorbic acid and retinoic acid on elastin and collagen gene expression in monolayers. Internal report BMTE 07.19. University of Technology Eindhoven, Department of Biomedical Engineering; 2007. [20] Lievens A, Bellocchi G, De Bernardi D, et al. Use of pJANUS™-02-001 as a calibrator plasmid for roundup ready soybean event GTS-40-3-2 detection: an interlaboratory trial assessment. Anal Bioanal Chem 2010;396:2165–73. [21] Wang X, Teng D, Yang Y, Tian F, Guan Q, Wang J. Construction of a reference plasmid molecule containing eight targets for the detection of genetically modified crops. Appl Microbiol Biotechnol 2011;90:721–31.

T

311 312

the candidate reference materials for 15 different genotypes based on quantitative real-time PCR, as described in Materials and methods. We found that the amplification efficiency was 96.3% to 101.25%, the slope was −3.263 to −3.512, and the regression coefficients R2 for all reactions were ≥0.995. The ideal slope is −3.32, which correlates with an amplification of 100%, and slopes in the range of − 3.60 to − 3.10 are generally considered acceptable for real-time PCR. These slope values correspond to amplification efficiencies between 90% and 110% [19]. The standard curves dependent on concentration for specific HPV genotype in our developed real-time PCR showed slopes and regression correlation coefficients within the acceptable range. The efficiencies of the other quantification systems using the plasmid DNA are 90.0 to 112.0%, and their R2 coefficients are 0.96 to 0.999 [20,21]. The high PCR efficiencies and the linear relationship between copy number and Ct value proved that the real-time PCR assay for the candidate reference materials based on plasmid DNA is well suited for quantitative measurements. Cross-contamination resulting from mixed plasmid DNAs of 15 different genotypes can be avoided in the detection of different genotypes using a linear array. The Ct values for real-time PCR for single or multiple mixed genotypes increased in relation to copy numbers. The quantitative real-time PCR assay established in this study can also be applied for genotyping in clinical samples with high accuracy and precision. In addition, the plasmid-based DNA standards developed in our study can be defined chemically in terms of the DNA concentration and can be assigned a copy number based on the molecular weight of the plasmid construct. This may be useful for proficiency testing in relation to the DNA concentration and preparation of variable panels such as single genotypes or multiple mixed genotypes.

292 293

HPV-66/HPV-68b

HPV-56

285

290 291

HPV-58/HPV-59

22.33 29.03 40

–, Ct value could not be determined. a Ct, cycle threshold value.

288 289

HPV-52/HPV-51

HPV-6

t5:15 t5:16

286 287

HPV-45/HPV-11

F

t5:11

HPV-16/HPV-18

O

t5:10

R O

5

P

t5:5 t5:6 t5:7 t5:8 t5:9

Ct value for single genotypea

D

t5:3

Table 5 Detection of specific HPV genotypes in single or multiple genotype.

E

t5:1 t5:2

J.E. Rhee et al. / Clinica Chimica Acta xxx (2014) xxx–xxx

of Health, Ministry of Health and Welfare, Republic of Korea.

Please cite this article as: Rhee JE, et al, Development of reference materials to detect 15 different human papillomavirus genotypes, Clin Chim Acta (2014), http://dx.doi.org/10.1016/j.cca.2014.02.013

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