Oligonucleotide Microarray with RD-PCR Labeling Techniquefor Detection and Typing of Human Papillomavirus

Wei Min,1 Ma Wen-li,1 Zhang Bao,1 Li Ling,1 Sun Zhao-hui,1 Zheng Wen-ling2

1Institute of Molecular Biology, Southern Medical University, Guangzhou 510515, P.R. China2Southern China Genomics Research Center, Guangzhou 510800, P.R. China

Received: 18 July 2005 / Accepted: 5 September 2005

Abstract. Currently, screening for high-risk human papillomavirus (HPV) infection remains animportant health concern throughout the world, because of the close association between certain types ofHPV and cervical cancer. In this study, we explore the possibility of using �70mer oligonucleotidemicroarray for detection and genotyping of HPV. The �70mer type-specific oligonucleotide probes offour different types HPV were designed by using biological software Arraydesigner 2.0, which analyzedthe whole genome sequences of HPV and selected optimal probes. These probes were synthesized andprinted onto the surface of glass slides in order to prepare a low-density microarray. HPV samples werelabeled with fluorescence dyes Cy3 using a method of restriction display polymerase chain reaction(RD-PCR). HPV plasmid DNA was restricted with Sau3A I to produce multiple fragments that wereligated to adaptors subsequently and used as PCR template. PCR labeling was performed with thefluorescently labeled universal primer (Cy3-UP) whose sequence is designed according to the adaptor ofthe RD-PCR approaches. The labeled samples were hybridized with the oligonucleotide microarray. Thescanning results showed that HPV DNA hybridized specifically with multiple spots correspondingly toshow positive signals, whereas no signals were detected of all the negative and blank controls. Theseresults demonstrated that �70mer oligonucleotide microarray can be applied to HPV detection andgenotyping. The application of RD-PCR in the sample labeling can increase significantly the sensitivityof the assay and will be especially useful for the discriminate diagnosis of multiple pathogens.

Cervical cancer is one of the most common malignan-cies and the second leading cause of cancer mortality inwomen worldwide. Early identification and interventionwill have significant implications for the effectivetreatment and prevention of cervical cancer.

The link between genital human papillomavirus(HPV) infection with cervical cancer was first demon-strated in the early 1980s by Harold zur Hausen [17]. Upto the present, more than 120 types of HPV have beenrecognized according their genomic differences [11].Based on their association with cervical cancer andprecursor lesions, these HPVs can be grouped into high-risk and low-risk HPV types. Among the type of the

high-risk HPVs, HPV types 16 and 18 are the mostcommon within the malignant cells of cervical cancers[15], and HPV DNA can be found in more than 99% ofcervical squamous cell cancer cases. Therefore, detec-tion and genotyping of HPV infection, especially thehigh-risk HPV types, is very important to earlier pre-vention, diagnosis, and an efficient treatment of cervicalcancer. The rapid development of molecular biologytechnology during the last few decades has made it apowerful tool to detect HPV DNA sequences in clinicalspecimens. However, all of the existing techniques stillhave some limitations in sensitivity, ability to discrim-inate between different HPV types, and ability to rec-ognize multiple infections.

Here, we report a HPV oligonucleotide microarraydetection system that can detect multiple HPV typessimultaneously. The �70mer type-specific oligonu-

Correspondence to: Ma Wen-li; email: wenli@fimmu.com orwenling@fimmu.com

CURRENT MICROBIOLOGY Vol. 52 (2006), pp. 204–209DOI: 10.1007/s00284-005-0212-x Current

MicrobiologyAn International Journal

ª Springer Science+Business Media, Inc. 2006

cleotide probes of each HPV type (6, 11, 16, 18) weredesigned by the software Arraydesigner2.0 according tothe whole genome sequence of HPV and immobilizedonto the surfaces of glass slides. The HPV DNArecombinant plasmids were labeled with restrictiondisplay polymerase chain reaction (RD-PCR) employingfluorescent Cy3-labeled universal primers. The PCRproducts are then hybridized onto the gene chip, whichis then scanned by laser scanner.

Materials and Methods

HPV plasmids. Four types of HPV (6, 11, 16, 18) plasmids includingthe whole HPV genome were kindly provided by Dr. de Villier fromDeutsches Krebsforschnngszentrum (Heidelberg, Germany).

Chemicals and reagents. pMD-18 T vector, restriction enzymeSau3A I, Taq DNA polymerase and T4 DNA Ligase were purchasedfrom Takara Co. (Otsu, Japan). The universal primer (5¢-GTTTGGCTGGTCTCCATC-3¢), adaptor (SIP: 5¢-pGATCmCACACCAGCCAAACCCA-3¢; SIR: 5¢-GGTTTGGCTGGTGTG-3¢) were synthesizedby BIOASIA Co. (Shanghai, China). Cy3-labeled universal primer(5¢-Cy3-UGTTTGGCTGGTCTCCATC-3¢) was synthesized by GibcoCo. (USA). The �70mer oligo probes were synthesized by BIOASIACo. (Shanghai, China).

Design of type-specific HPV probes. The whole genome sequencesof officially recognized HPV types were obtained from the GenBank(with the GenBank accession number in parentheses): HPV6(NC_000904), HPV11 (NC_001525), HPV16 (NC_001526), HPV18(NC_001357). Here software Arraydesigner 2.0 and BLAST programwere applied to design the type-specific oligonucleotide probes on thebasis of following criteria: 1) Each 60�70mer oligonucleotidesequence should be checked for specificity by BLAST analysisensuring less than 20 consecutive homologous bases to any otherorganism. 2) Each oligo should be less than 70% identity to all othersequences in BLAST compare to avoid nonspecific hybridization. 3)The Tm value of all the probes should be consistent and within theaverage Tm value € 5�C of the whole genome. 4) The composition ofGC is within 40%–60%. 5) The contiguous single nucleotide cannot belonger than 6 bases. 6) The hairpin structure length cannot be longerthan 6. 7) These oligonucleotides cannot contain the recognizedsequence GATC of Sau3A I. According to these principles, 26oligonucleotide probes of four types of HPV have been selected (Thecomposition and characteristics of the oligo probes are shown inTable 1).

Design and fabrication of the microarray. A 16 · 12-spotmicroarray was designed including 26 type-specific HPV probes and6 control probes. There were two probes in each row and each probewas printed for six replicates repetitively to test the stability of thesystem. These oligo probes were spotted onto the CMT-GAPSTM

aminosilane-coated glass slides (Corning, New York, USA) at aconcentration of 1 lg/lL in spotting buffer (50% DMSO solution) byusing a Cartesian PixSys 5,500 robot (California, USA). The DNAprobes were then immobilized by a Bio-Rad UV Cross-linker(California, USA) with 65 mJ of energy. The arrays were stored in adesiccator at room temperature until later use in the hybridization.

Fluorescent labeling. The labeling of HPV DNA sample was carriedout by RD-PCR. Before PCR amplification, the two primersSIP (500 lg/lL) and SIR (500 lg/lL) were mixed in 10 mM Tris-

HCl (pH 7.6), 5 mM MgCl2 at 90�C for 5 min. Then the solution wascooled down to room temperature gradually in 30 min for annealing sothat the double-stranded universal adaptors were formed. Each HPVplasmids DNA was restricted with Sau3A I for 4 h to produce multiplefragments with a cohesive end of GATC on both sides. The digestedfragments were subsequently ligated to the adaptors and used as thePCR template. Cy3-labeled universal primer was designed accordingto the sequences of the adaptor and restriction site of Sau3A I.

Each 100-lL reaction contained 10 lL of 10 · PCR buffer(500 mM KCl, pH 8.3/100 mM Tris-HCl, 15 mM MgCl2 ), 10 ll ofdNTPs (2 mM each), 1 lL of template (200 lM), 1 lL of Taq DNApolymerase (5U/lL, TaKaRa), and 2 lL of 100 lM Cy3-labeled uni-versal primer. Amplification was performed in an ABI PCR System9700 with an initial denaturing temperature of 95�C for 5 min, fol-lowed by 30 cycles of 95�C for 30 s, 60�C for 30 s, 72�C for 1 min, anda final extension of 72�C for 5 min. PCR products were purified withQIAquick columns (Qiagen, Germany) according to the manufacturer�sprotocols. Then the labeled samples were dried in a Speed-Vac andresuspended in 50 lL ddH2O.

Prehybridization. The slide was incubated in prehybridization buffercontaining 25% formamide (Amresco), 5 · SSC, 0.1% sodium dodecylsulfate (SDS) for 45 min at 42�C. It was then washed in the MilliQwater and isopropanol, and dried in the air.

Hybridization and washing. Five microliters Cy3-labeled samplewas mixed with an equal volume of 2 · hybridization buffer (50%formamide, 10 · SSC, 0.2% SDS). The mixture was heated at 95�C for3 min, spun for 2 min to cool down. Three microliters of the mixturewas pipetted onto the slide, with a pretreated coverslip on the top of thearray. Then the slide was placed in a sealed hybridization chamber(Corning), and 20 lL of water was added to the chamber to maintainhumidity. The hybridization chamber was submerged in a 42�C waterbath for 12 h.

After hybridization, the slides were washed for 5 min at 42�C inlow-stringency wash buffer (2 · SSC/0.1% SDS), then for 10 min atroom temperature in high-stringency wash buffer (0.1 · SSC/0.1%SDS), finally for 1 min in 0.1 · SSC (repeated four times). The slideswere dried at room temperature.

Scanning and analysis. The hybridized microarrays were scannedusing ScanArray Lite laser scanner (GSI Lumoncis, Massachusetts,USA). The acquired image was analyzed to calculate the fluorescentintensity of each spot using Array-Pro Analyzer software. The averageintensity value of all negative control spots was applied as a criterion tojudge the positive signal. We identified a spot on the array as a positiveone when its fluorescent intensity was more than double of thecriterion, otherwise as a negative one.

Results

Design of the type-specific oligonucleotide probes. Usingan algorithm, the software Arraydesigner 2.0 usesBLAST search to design highly specific oligos forgenes. The oligos were BLAST realigned against publicdatabases available at NCBI to verify the specificity ofthe design. Eventually we obtained 26 type-specificoligo probes to prepare the microarray.

Fluorescent labeling of HPV DNA for hybrid-ization. HPV plasmid DNA was digested into manyfragments of different lengths by Sau3A I (Fig. 1a). After

W. Min et al.: HPV Detection with Oligonucleotide Microarray 205

Tab

le1.

Ser

ialnu

mbe

r,se

quen

ce,an

dch

arac

teristicsof

the

type

-spe

cific

olig

onuc

leot

ides

and

theirpo

sition

inth

em

icro

arra

y

No.

Typ

eSite

Seq

uenc

eTm

GC%

Hairp

ina

Dim

erb

1HPV-6

B1�

B6

CCCAACCACCCGTGGAGGCTAATGGACATATATTAATTTCTGCACCCACTATAACGTCACACCCT

89.5

47.7

35

2HPV-6

C1�

C6

CAGACCCTGCATTTCTTTCCACTCCTCAACGCTTAATTACATATGATAACCCTGTATATGAAGG

86.5.

40.6

45

3HPV-6

D1�

D6

TCGGTTGCCCTTGGCATACACTTTCCACCAATTTGTTACAACGTGTTGCCTGTTAATCCT

87.8

454

44

HPV-6

E1�

E6

GGCACTGGGCCTCCTCAAAGGCACCACATAAACATGCCATTGTAACTGTAACATATCATAGTGAG

88.9

46.2

44

5HPV-6

F1�

F6

TTGTTACAAAGTGGATATAGGGGACGGTCCTCTATTCGTACCGGTGTTAAGCGCCCTGCT

89.8

505

46

HPV-6

G1�

G6

GGGAACCTGTGCCTGATACTCTTATAATTAAGGGTAGTGGAAATCGAACGTCTGTAGGGA

87.8

454

57

HPV-6

H1�

H6

CATGTCAATTTAATGATGGAGATACCTGGCTGGGTTTGTGGTTGTTATGTGCCTTTATTG

85.7

403

48

HPV-6

I1�I6

GGGGTGTTGGGGTTATTATTAATGCACTATAGAGCTGTACAAGGCGATAAACACACCAAA

86.4

41.7

35

9HPV-1

1K1�

K6

ATGCCTATAAGAACCTAAAGGTTGTGTGGCGAGACAACTTTCCCTTTGCAGCGTGTGCCTGTTGCT

90.0

48.5

53

10HPV-1

1L1�

L6

GCACAGACGGAGACATCAGACAACTACAAGACCTTTTGCTGGGCACACTAAATATTGTGT

87.8

454

411

HPV-1

1M

1�M

6AACTAAATGTGCTAAGTGTAAATCAAACCGCAATACTACTGTGGATTATGTGTATATGTCAC

8333

.93

312

HPV-1

1N1�

N6

AACAAGTTTGCATTACCTGATTCATCCCTGTTTGACCCCACTACACAGCGTTTAGTATGG

87.2

43.3

44

13HPV-1

1O1�

O6

TGCTGAACCATTTGACCCTATCCCTGACCCTGTCCAACATTCTGTTACACAGTCTTATCTTACCTCCACA

87.8

45.7

44

14HPV-1

6B7�

B12

GCAATGTTTCAGGACCCACAGGAGCGACCCAGAAAGTTACCACAGTTATGCACAGAGCTG

90.5

51.7

34

15HPV-1

6C7�

C12

TGCAAGTGTGACTCTACGCTTCGGTTGTGCGTACAAAGCACACACGTAGACATTCGTACTTTGGA

89.5

47.7

45

16HPV-1

6D7�

D12

TGCGTACAAAGCACACACGTAGACATTCGTACTTTGGAAGACCTGTTAATGGGCACACTAGGAATTGTG

88.8

44.9

35

17HPV-1

6E7�

E12

ACATTAGGAAAACGAAAAGCTACACCCACCACCTCATCTACCTCTACAACTGCTAAACGCA

87.6

44.3

34

18HPV-1

6F7�

F12

GCGCCATGAGACTGAAACACCATGTAGTCAGTATAGTGGTGGAAGTGGGGGTGGTTGCAG

91.2

53.3

54

19HPV-1

6G7�

G12

GGACGCACTGGGTATATTCCATTGGGAACAAGGCCTCCCACAGCTACAGATACACTTGCTCCTGT

91.4

52.3

55

20HPV-1

6H7�

H12

TGCCACACCACTAAGTTGTTGCACAGAGACTCAGTGGACAGTGCTCCAATCCTCACTGCA

90.5

51.7

54

21HPV-1

6I7�12

CCTGCCACACCACTAAGTTGTTGCACAGAGACTCAGTGGACAGTGCTCCAATCCTCACTGCATT

9151

.65

522

HPV-1

8K7�

K12

ATGGAGTTAATCATCAACATTTACCAGCCCGACGAGCCGAACCACAACGTCACACAATGT

88.5

46.7

45

23HPV-1

8L7�

L12

AAACGACGATTTCACAACATAGCTGGGCACTATAGAGGCCAGTGCCATTCGTGCTGCAACCG

90.8

51.6

53

24HPV-1

8M

7�M

12ACACGGGTCCTGATATTACATTACCATCTACTACCTCTGTATGGCCCATTGTATCACCCACGGCCC

87.8

504

425

HPV-1

8N7�

N12

AGTGGCTAACCCTGAGTTTCTTACACGTCCATCCTCTTTAATTACATATGACAACCCGGC

87.8

453

426

HPV-1

8O7�

O12

GCCTACCAACAAGTGTCAGTGGCTAACCCTGAGTTTCTTACACGTCCATCCTCTTTAATTACA

87.8

44.4

44

aNum

berof

hairpi

nstru

ctur

ein

olig

opr

obe.

bNum

berof

dim

erstru

ctur

ein

olig

opr

obe.

206 CURRENT MICROBIOLOGY Vol. 52 (2006)

ligated with adaptors, the fragments were amplified withCy3-labeled universal primers (Fig. 1b). From the 1.5%agarose gel electrophoresis results, a dispersed bindingpattern containing multiple fragments from 250 bp to2000 bp was observed (Fig. 1c).

Detection and typing of microarray hybridizationresults. To explore the possibility of HPV detection andtyping using microarray, hybridization was performedusing Cy3-labeling HPV6, 11, 16, and 18 plasmidsamples separately. From the scanned images (Fig. 2a–d), we can see that each HPV type showed a specifichybridization pattern. The fluorescence-labeling HPVDNA hybridized with some corresponding probes on theslide and showed strong signals. The probes of negativecontrol (gene fragments of eukaryotic cells such as K562cells and plants genes such as rice) and blank control(50% DMSO) showed no hybridization signal. When the

array was hybridized with human genomic DNA sample,only the positive control probes showed fluorescencesignal (Fig. 2e). These results proved that these oligoprobes had a high specificity and a low homogeneity tohuman genome sequence. The hybridization signals ofsix spots of a same probe were consistent, and the resultsof three repetitive experiments had an enhancedconsistency. It signified that the �70mer oligomicroarray system had a satisfactory reproducibility.

Discussion

Plentiful studies have confirmed that cervical infectionby high-risk HPV types is a precursor event to cervicalcancer. It is agreed that early detection and subsequentlyearly treatment of HPV in precancerous lesions canprevent cancer progression [13]. However, HPV is verydifficult to culture in the laboratory from clinical spec-

Fig. 1. (a) Agarose gel electrophoresis ofrestricted fragments of HPV16, HPV18plasmids DNA digested by Sau3A I. (b, c)Agarose gel electrophoresis of amplifiedproducts of HPV16, HPV18 plasmids DNAwith Cy3-labeled universal primers. M:Marker DL 2000; 1: HPV 16 plasmid DNA;2: HPV 18 plasmid DNA.

Fig. 2. Scanning results of microarray hybridization. Scheme of the microarray: positive controls; negative controls; empty controls;HPV 6 probes; HPV16 probes; HPV11 probes; HPV18 probes. (a) Sample of HPV6; (b) sample of HPV11; (c) sample of HPV16; (d) sampleof HPV18; (e) sample of human DNA.

W. Min et al.: HPV Detection with Oligonucleotide Microarray 207

imens, and immunologic assays are not sufficient forearly detection of HPV infections. Traditionally, theprimary diagnostic tools have been cytology and his-tology. Among the conventional cytology methods, themethod for detection of high-risk HPV is still thePapanicolaou stained (Pap) smear. The Pap smearexamines the changes in cells of the transformed zone ofthe cervix. Such typical changes are often caused byHPV infection. However, the Pap smear has some lim-itations; for example, the false-negative rate is as high as20 to 30%, owing to inadequate sampling, contamina-tion samples, fixation methods, and laboratory error [1].

Recently, growing molecular methods have beenapplied to detect HPV DNA sequences in clinicalspecimens, including PCR-based methods and nucleicacid hybridization-based methods. Several studies usedPCR to date have used consensus primers (within HPVL1 ORF) to amplify a broad spectrum of HPV types in asingle PCR amplification [2, 4, 7, 10]. Then HPVgenotyping can be achieved when PCR products areanalyzed subsequently by sequence analysis, restrictionfragment length polymorphism, and hybridization withtype-specific probes using dot blot or microtiter plateformats [12, 14, 16]. Although these methods are sen-sitive enough to identify specific HPV types, there arestill some problems in clinical application. If integrationof HPV DNA into the genome of cervical cell disrupts ordeletes PCR primer target regions such as L1 ORF, thePCR amplification may get a false-negative result.Moreover, cross-contamination inevitably occurs in theprocedure of PCR amplification, which will produce afalse-positive result. The important factor is that untilnow, known assays still fail in order to identify all ofpossible HPV types simultaneously.

Ideally, a test for HPV should allow detection ofmultiple HPV types, identify individual types, and pro-vide quantitative information about the viral load ofeach individual type found. Moreover, it should be easyto perform, be highly reproducible, with a high speci-ficity and sensitivity, and amenable to high-throughputanalysis and automation. The emerging DNA micro-array technology is the most desirable method to meetall of these requests to date. This technique makes itpossible to examine the expression of thousands ofgenes simultaneously. The microarray system can rec-ognize single and multiple HPV infections, and identi-fies the corresponding HPV types easily, with manytype-specific oligonucleotides probes of HPV immobi-lized on the slide. This technology promises improve-ments in HPV diagnosis and eventual use for clinicalapplication.

We designed 26 type-specific 60–70mer oligonu-cleotides to represent the whole genome of HPV in this

research, and printed them onto the silanized slides,which were applied to hybridizing with the HPV frag-ments derived from the HPV plasmids DNA labeled byRD-PCR. The results showed that four types of HPVsamples can hybridize with part of the correspondingprobes and have obvious fluorescence signals. Therewas no signal on the negative and blank probe sites,showing a high specificity of the microarray. Thehybridizing results between low-risk HPV and high-riskHPV types are quite distinct from each other. Because ofthe higher sequence homology between HPV6 andHPV11, HPV16 and HPV18, there were some commonhybridization signals. From the results of analysissoftware (data not shown), we can screen out 10 type-specific oligo probes, including B1–6, E1–6, K1–6, N1–6, O1–6, B7–12, E7–12, I7–12, N7–12, O7–12.

At present, the detection and subtyping microarrayof HPV are employing the short oligo (20–25mer)microarrays [3, 5, 6]. The selection of a conservedregion of HPV and design of PCR primers are thecommitted steps, and the PCR products were hybridizedwith the short oligo microarray to detect and identify theHPV subtypes. It is very difficult to find a target regionthat is conserved in all of the possible HPV typesinvolved, so these studies have to design several sets ofprimers and perform multiple amplification, which is avery complicated procedure and increases the cross-contamination probability of samples and reagents.Although a short oligo probe has better specificity, itrepresents only a conserved region of HPV genome andwould get a false-negative result when there was deg-radation or absence in the region during the extraction,purification, and preservation procedure of viral nucleicacid. The probes designed in our experiments containedthe whole genome sequence of HPV, combining withthe RD-PCR labeling technique invented by our labo-ratory [9], which can enhance the hybridization sensi-tivity at the same time as ensuring a high specificity.

RD-PCR technique was initially a method for geneseparating and differential display. It can also be appliedto sample labeling and hybridization of gene chips [8].In this study, the HPV DNA samples were labeled byRD-PCR with fluorescently labeled universal primersand hybridized onto the HPV microarray. First, the newprotocol increases greatly the incorporation rate of flu-orescently labeled nucleotides and thus evidentlyenhances the hybridization signals. Second, by theRD-PCR labeling technique, the hybridization conditionwould be easier to control and the signal-to-noise ratio islower because fragments prepared by these methodswere more uniform in length. Furthermore, amplifica-tion and labeling of different gene targets in the samesample can be performed simultaneously with only one

208 CURRENT MICROBIOLOGY Vol. 52 (2006)

primer, which is especially useful for the detection of themiscellaneous infections of different pathogens.

ACKNOWLEDGMENTS

This work is supported by the National Natural Science Foundation(36990032) and the regional Key Project of the Guangzhou City. TheHPV plasmids used in this study were kindly provided by Dr. de Villierfrom Deutsches Krebsforschnngszentrum (Heidelberg, Germany).

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W. Min et al.: HPV Detection with Oligonucleotide Microarray 209