Comparison of a one step and a two step polymerase chain reaction with degenerate general primers in a population based study of human papillomavirus infection in young Swedish women

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0095-1137/92/040987-06$02.00/0

Comparison of

a

One-Step and

a

Two-Step

Polymerase Chain

Reaction with Degenerate General Primers in

a

Population-Based

Study of Human Papillomavirus Infection in

Young

Swedish

Women

MAGNUS EVANDER,1* KARIN EDLUND,' ELISABETH BODEN,2AKE

GUSTAFSSON,l

MONICAJONSSON,3 ROGER KARLSSON,3 EVA RYLANDER,2ANDGORANWADELL'

Departmentsof Virology,1Obstetrics and Gynecology,2andFamily Medicine,3 University of Umeac, S-901 85 Umea, Sweden

Received 15 July 1991/Accepted 26 December 1991

The prevalence ofhuman papillomavirus (HPV) infection in cervical cell scrapesfrom young women was

determined by polymerase chain reaction (PCR) by using generalprimer pairs localized within the Li region. With aone-stepgeneral PCR, 5.9%Yo (35 of 590) ofyoung women in apopulation-based studywerefoundto contain HPV DNA. Theproportion of HPV-positivewomen increased withage, from 1.4% (1 of 69) among

womenaged19yearsto9.2% (13 of 142)among womenaged25years.Among the cervicalscrapesfromwomen

with normal cytology, 5.6% (30 of 539) harbored HPVDNA.A total of 5 of 19 (26.3%) ofthewomen with pathological signswerepositive forHPVDNA.Byatwo-stepPCR, using nested general primers, 20.3% (118 of581)of allwomenwereshowntocontainHPVDNA. The proportion of HPV-positivewomenalso increased

withage,from 17.4% (12 of 69)among womenaged 19yearsto31.9%o(43 of 135)among womenaged25years, when thetwo-step PCRwas used. Some 19.2% (102 of 530) of cervical scrapes from women with normal cytologycontained HPVDNA. Among thewomenwith pathologicalsigns, 16 of19(84.2%)werepositive for HPV DNA. The HPV DNA-positive specimensweredemonstratedtocontain HPVtype6, 11, 16, 18, 31, 33,

35, 39, 40, 45, 55,or56.ThemostprevalentHPVtypeswere6(2.0%o)and 16(2.7%).Morethanonetypewas

found in16 specimens. SixtyHPV-positive samplescould notbe typed. Human papillomaviruses (HPVs) are double-stranded

DNAviruses thatcausevariousproliferative diseases in the infected epithelium (18). Over 60differenttypes have been isolated from human tissue. Different types of HPV are

associated with specific lesions. Several types, HPVtypes 6, 11, 16, 18, 31, 33 to 35, 39, 40, 42 to 45, and51 to 59, infect thegenitaltract (4). HPVtypes16, 18, 31,and 33 are mainlyassociated withmalignantlesions of the cervix(2, 6, 12, 14), while HPVtypes 6 and 11 are generally limited to low-grade lesions which rarely progress to malignancy (3, 14).

In large cancer-screening programs, about 2 to 3% of Papanicolaou (Pap) smears from essentially asymptomatic

womenshow abnormalcytologies (16). Nearlyallsquamous

cell abnormalities in thePap smears (koilocytosis, cervical intraepithelial neoplasiatypeI, higher-grade lesions)appear

to be associated with HPV infections (24). Koutsky et al. (11) have estimated that genital tract HPV infections are prevalent in approximately 10% of the men and women in the 15-to 49-yearage groupin theUnited States and thata majority of these infections are subclinical. They further suggestthat thetrue prevalencemaybesignificantly higher

because of the lowersensitivityofthecommonlyused HPV detection methods(dot blot,Southernblot, and filter in situ hybridization).

During the last few years, studies based on polymerase

chain reaction (PCR) technology have been performed in which type-specific as well as general HPV primers have been used. HPV DNA was demonstrated in 5 to 49% of

cytologically normal women (1, 17, 25-28). HPV DNA

sequences were present in 80 to 100% of patients with

* Correspondingauthor.

cervicalcancersand in 60to90% ofpatientswithhigh-grade cervicalintraepithelial neoplasia.HPVtype16wasthemost

frequently present type, but unknown HPVs have been detectedbyPCR inaportion (maximumof 10to15%)of the

cases(24).

We determined the prevalence of HPV infection in a population-based studyofyoung Swedishwomenbyuseof

ageneral primer-based PCR. We also compared the sensi-tivityofaone-stepgeneral primer-based PCR with that ofa

two-stepgeneral primer-basedPCR.

MATERLALS AND METHODS

Populationandspecimencollection. Allwomenaged 19, 21,

23, and 25 yearswhowere inhabitants ofa primary health

care areainUmea, Sweden, accordingtothepublic record,

were asked to participate in the study during the period September 1989 to September 1990. Among the women

asked to participate, 70 could not be reached. Cervical

scrapesfrom 602womenweretakenbythesameregistered

midwife; 590 of the scrapes were analyzed for presence of HPVDNA. At thesametime,asecond cellsmear wastaken from558 of thewomen forcytologicalevaluation.

Cytology. A total of 558 Pap smears were taken for cytological evaluation. Theywere evaluated at the

Depart-mentofCytology, University HospitalofUmea,and abnor-mal results were categorized according to the Bethesda system.

DNApreparation. Cervical cellswerecollectedby scrap-ingacotton-tippedswaboverthe entire surface of theportio vaginalis. Subsequently,the swabwassuspendedinaplastic

tubewith 1.5 ml of STE(0.1MNaCl, 10mMTris-HCl [pH 8.0], 1 mM EDTA) and centrifuged to pelletthe cells. The

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DNAwas

prepared

asdescribed

previously (8).

For

ampli-fication,

20

RI

of thesolutionwasuseddirectly.

PCR.

Amplification

of HPV DNAwascarriedoutin three

ways. First, a consensus primer pair, MY11-MY09, which spans nucleotides 6722 to 7170 in HPV type 6 and the

corresponding regions

of the other

genital

HPVs (15), was used inaone-step

amplification

of the DNAprepared from the cervical scrapes

(40 cycles) (Fig.

1 and 2A). Second,

another

general primer pair,

GP5-GP6, which spans nucleo-tides 6764 to 6902 in HPV type 6 and the

corresponding

regions

of other

genital

HPVs

(23),

was used in combination with theMY11-MY09

primer

pairinanestedgeneral primer

two-step

amplification

of the HPV DNA

(20 cycles

for

MY11-MY09,

30

cycles

for

GP5-GP6) (Fig.

1and

2B). Third,

the GP5-GP6

primer pair

was used ina one-step

amplifica-tion of the HPV DNA

(40 cycles).

All

primers

were

synthe-sizedon aBeckman DNA SM automated DNA

synthesizer.

For a

typical

one-step

amplification

reaction, 20 ,ul of DNA prepared from cervical cells was mixed with PCR buffer

[16.6

mM

(NH4)2SO4,

67 mM Tris-HCl (pH 8.8) at

25°C,

6.7 mM MgCl2, 10 mM

1-mercaptoethanol];

the four

deoxynucleotides

dATP, dlTP, dCTP, and dGTP at final

concentrations of 50

puM

each; 12pmolof either the

MY11-MY09 or the GP5-GP6

primer pair;

and 2 U of Taq DNA

polymerase (Amplitaq;

Cetus, Berkeley, Calif.) to a final volume of 50

plI.

This mixturewasoverlaid with 2to3 drops

of mineral oil. Thermal

cycling

of the

amplification

mixture

(denaturation, annealing,

and

extension)

was

performed

in a

programmable

heatblock

(Techne

PHC-2;TecheLtd.,

Cam-bridge,

United

Kingdom)

for a total of 40

cycles.

Denatur-ation was

performed

at

94°C

for 30 s,

annealing

was per-formedat

45°C

for 30 s, and extensionwasperformedat72°C

for 30sfor all

amplifications.

For a

typical

two-step amplification, 0.2 pmol of the

MY11-MY09

primer pair

wasmixed with thePCR

buffer,

the four

deoxynucleotides

at a final concentration of 100

puM

each,

2U of

Taq

DNA

polymerase,

and 20

pul

ofthe DNA

prepared

from the cervical scrapes. The mixture was over-laid with mineral

oil,

andatotalof 20

cycles

was

performed

as described above. A total of 20

pmol

of the GP5-GP6

primer pair

wasthen

added, together

with 2 U ofTaqDNA

polymerase,

tothe reaction

mixture,

and 30 additional

cycles

were

performed.

All clinical

specimens

were also

amplified

with the

P-globin

primers

PCO3 and PCO4

(19)

to exclude

false-negative

results.

Samples

that were

negative

for

3-globin

amplification

were extracted with

phenol

and

precipitated

with

ethanol,

andasecond

13-globin

PCRwasperformed.All 3-globin-positive samples (590 of 602 samples) were

ampli-fied with the HPV

general primers.

All PCRswere

performed

sothat every third

sample

was

a

negative

control. All such controls were

negative.

To

minimizethe riskofcontamination,strict precautionswere taken

during sample collection, preparation

of

DNA,

and PCR.

Detection and hybridization. For

detection,

40% of the

amplified

DNA

(20

p.l)

was

separated

on a 2.0% NuSieve

GTG

plus

1.0% SeaKemMEagarose

gel

(FMC Bioproducts,

Rockland, Maine)

by electrophoresisandstained with ethid-ium bromide. Two

32P-end-labeled

internaloligonucleotide

probes,

MY1019andMY18(15),wereused forhybridization

of theMY11-MY09one-step

amplification

productsfrom223 ofthe

samples.

For HPV typing, 10% of the amplified DNA (5

p.l)

was boundtoa

nylon

filterbyslotblotting by usingaMinifold II

Slot-Blotter

(Schleicher

& Schuell

GmbH, Dassel,

Germa-ny). The entire genomes ofHPVtypes6, 11, 16, 18, 31, 33, 35, 39, 40, 45, 54, 55, 56, and 58 (kindly provided by E.-M. de Villiers, A. Irincz, T. Matsukura, G. Orth, K. Shah, and H. zurHausen) were radioactively labeled and usedas probes for hybridization. The nylon filters were prehybrid-ized andhybridized in a solution containing 5 x SSC (0.75 M NaCl plus 0.075 M sodium citrate), 5x Denhardt solution (0.1% bovine serum albumin, 0.1% Ficoll, 0.1%

polyvi-nylpyrrolidone), 1mMEDTA,0.1%sodium dodecyl sulfate

(SDS), sonicated salmon sperm DNA(100

p,g/ml),

and 50% formamideat 42°C. The filters were then washed at 20°C in 2x SSC-0.1% SDS for 30 min, two times for 15 min each time with 2x SSC-0.1% SDS at65°C, and finally, two times for15 min each time with 0.5x SSC-0.1% SDS at 65°C. The filterswereexposedtoCronex 4 film(DuPont) for 1to3 days

by using intensifyingscreens. RESULTS

Cytology. The cytological evaluation of the cell samples revealed that of 558 cell samples, 539 (96.6%) showed normal cytology and 19 (3.4%) had pathological signs. Of

these, 12 (2.2%) had cytological patterns of condyloma, 3

(0.5%)had patterns of dysplasia, and 4 (0.7%)hadsigns of inflammation.

One-stepPCR withthe outerprimer pair.To determinethe

prevalence of HPV infection in the cell samples, we first usedaone-step PCRprocedure with the MY11-MY09 con-sensusprimers(Fig. 1 and 2A). Among cervical scrapes from womenwith normalcytology, 30 of 539 (5.6%) were shown to contain HPV DNA. Of the women with pathological

signs,5 of 19(26.3%)werepositive for HPV DNA (Table 1). In total, samples from 590 women were tested by the MY11-MY09 one-step PCR. We detected HPV DNA in 35

(5.9%)of the cervical scrapes (Table 2). Theproportion of

HPV-positivewomen increased with age, from 1.4% HPV

positive among the women aged 19 years to 9.2% HPV

positiveamongthewomenaged 25 years(Table 2).The SiHa cell line(which containsone totwocopiesof HPV type16) was usedto determine thesensitivity of the one-step PCR.

BydilutingSiHa cellDNA,whichwaspreparedin thesame wayasthecervical scrapes were,wedetected100copies of

the HPV type 16 genomebyethidium bromidestaining. Two-step PCR. We then assembled a nested PCR with

general primerstobe abletoincrease thesensitivityof HPV DNAdetection(Fig.1and2B).The two-step PCRwasused foranalysisof allsamplesexceptfor ninesamplesthatwere notavailable foranalysis, thatwere negative by the MY11-MY09 one-step PCR.Among cervical scrapes fromwomen with normalcytology, 102 of 530 (19.2%)werepositivefor HPVDNAafterthe two-step PCR.Amongthewomenwith

pathological signs, specimensfrom 16 of 19(84.2%)of them had HPV DNA (Table 1). In addition to the 35 HPV

DNA-positive specimens from the MY11-MY09 one-step

PCR, we detected 83 HPV-containing specimens by the two-step PCR,givingatotal HPVprevalenceof 118 of 581

(20.3%) (Table 3). The proportion ofHPV-positive women increased with age, also when the two-step PCRwasused, from17.4% amongwomenaged 19 yearsto31.9%amongthe women aged 25 years (Table 3). The two-step PCR was showntodetect1 to10copiesof theHPVtype 16 genomeby usingthe SiHa cell lineasdescribed above.

Hybridization. Afteramplification,223 of theMY11-MY09 one-step PCRamplimerswerehybridizedtothe

oligonucle-otideprobesMY1019 andMY18(15).All samplesthatwere

negative after ethidium bromide staining (209 of 223) were

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6722 6764 6904

MYll GP5 GP6

_ -b -04

7170

MY09

El

FIG. 1. Locations of the MY11-MYO9consensusprimers(15)and theGP5-GP6general primers (23)in the Li regionof the HPVgenome.

alsonegative after hybridization. Of the ethidium

bromide-positive samples, 9 of 14were also hybridization positive. Detection by ethidium bromide staining revealed 83 speci-mensthatwere negative bytheMY11-MY09 one-step PCR

A- 1 2

-.142bp

FIG. 2. DNA fromcervicalscrapes wasanalyzedby the

MY11-MY09 one-stepPCR(A) and the two-stepPCR (B). Lanes 1to 6,

DNAfrom cervicalscrapes. +,cloned HPVtype16 DNAamplified

as a positive control; -, negative control containing all PCR

reagents except DNA; m, DNAmolecular mass standard. In the

one-stepPCR,allsamplesweresubjectedto40cyclesof

amplifica-tion withtheMY11-MY09 primer pair (15).Inthetwo-stepPCR, all samples were subjected to 20 cycles of amplification with the

MY11-MY09 primer pair; thiswasfollowedby30cyclesof

ampli-fication with theGP5-GP6primer pair (23).

but that were positive by the two-step PCR. Ofthese, 36 specimens were hybridized to the oligonucleotide probes. None of thesampleswas positiveafterhybridization.

One-step PCR with the inner primer pair. We also

per-formedaone-step PCR with the innerprimer pairGP5-GP6. Weanalyzed 62 of the 83specimensearlier scored asHPV positive by the two-step PCR but HPV negative by the MY11-MY09 one-step PCR. After ethidium bromide stain-ing, 35 specimenswere foundto be HPVpositive. We also analyzed 90HPV-negative specimens bythe two-step PCR with the GP5-GP6primer pair. Allof these specimenswere negative. The number of HPV-positive specimens in the GP5-GP6one-step PCRcorresponds toabout 14% of HPV-positive specimens in the total population. The GP5-GP6 one-step PCR was shown to detect 10 copies of the HPV

type 16 genome by using the SiHa cell line as described

above.

HPVtyping.The HPVtypesweredeterminedbyslot blot hybridizationof the HPV DNA-positive amplification prod-ucts to 14 different genital HPV types. Themost prevalent types were HPV type 6, 12 of590 (2.0%) specimens, and HPVtype 16,16 of 590(2.7%) specimens (Table 4).We also

TABLE 1. PrevalenceofHPVinfection comparedwith cytologicalevaluation ofPapsmearsdetermined

byMY11-MY09one- ortwo-stepPCR'

Cytologyand PCR No.(%)HPV:

method(no.ofpatients) Positive Negative

Normalcytology

One-stepPCR(539) 30(5.6) 509(94.4)

Two-step PCR(530) 102(19.2) 428(80.8) Pathological signsb

One-stepPCR(19) 5(26.3) 14(73.7)

Two-stepPCR(19) 16(84.2) 3(15.8)

aAll the HPV DNA-positive specimens from the MY11-MYO9 one-step

PCRareincludedastwo-stepPCRpositives.

bCytological patterns of condyloma (n = 12), dysplasia (n = 3), or

inflammation(n =4).

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TABLE 2. MY11-MYO9 one-stepamplification for determination

of theprevalence of HPVinfection in comparison withage

Patientage No. (%) HPV:

(yr [no.ofpatients]) Positive Negative

19(69) 1 (1.4) 68(98.6)

21(158) 6(3.8) 152 (96.2)

23(221) 15(6.8) 206(93.2)

25(142) 13(9.2) 129(90.8)

Total(590) 35(5.9) 555(94.1)

detected HPVtypes11, 18, 31, 33, 35, 39, 40, 45, 55, and 56. Sixty HPV-positive samples could not be typed. Infection with more than one typewasfound in 16 specimens (Table 4).

DISCUSSION

Several approaches have been tested to determine the prevalence of HPV in the population. Southern blot, dot blot, and filter in situ hybridization have been used for the detection of HPV DNA in women attending clinics for routine gynecologic screening (5, 13) or large

population-based studies(10). Morerecently, PCR has beenapplied for the demonstration of HPV DNA in clinical specimens. Several selections oftype-specific primers for HPV detec-tionareavailable(7, 17, 21, 30).Primers for thedetection of a broad spectrum of HPV types, consensus or general primers, have been presented (8, 9, 15, 23). Degenerate primers for HPV DNA detection, arranged in a nested fashion, have alsobeen developed (29).

We chose to use the MY11-MY09consensus primer pair (15)for detection of HPVDNA, toallowcomparison ofour

results with thoseof other studies. TheMY11-MY09 primer pair is localized within the Li region of the HPVgenome.

The Li regioncanbe lostatlatestagesofneoplasia (22),but this is hardly relevant in HPV infections in very young women.

By using the MY11-MY09 consensus primer pair in a

one-stepPCRdirectly oncytological scrapesfroma nonse-lected, young female population, we demonstrated HPV

DNA in5.9%ofthewomen.Among thewomenwithnormal cytology, 5.6%were shownto beHPV DNApositive.

To check the sensitivity of the one-stepPCR directlyon cytological scrapes,weassembledatwo-stepPCR. We took advantageof thefact that the GP5-GP6 general primers (23) werepositionedwithin theMY11-MY09 consensusprimers

and used thesetwo primerpairs to setup a two-step PCR with theprimers arranged in a nestedfashion. Anew PCR system should beevaluated inaclinicalsettingbefore itcan

TABLE 3. Two-stepPCR amplification for determination of the prevalenceof HPVinfection incomparisonwithagea

Patientage No. (%)HPV:

(yr [no.ofpatients]) Positive Negative

19(69) 12(17.4) 57(82.6)

21(158) 24(15.2) 134(84.8)

23(218) 39(17.9) 179(82.1)

25(135) 43(31.9) 92(68.1)

Total(581) 118(20.3) 463(79.7)

aAll the HPV DNA-positive specimens from the MY11-MYO9 one-step

PCRareincludedastwo-stepPCRpositives.

TABLE 4. Determination of the HPV type in the HPV-containing specimens by slot blothybridizationa

HPV type No.positive

6... 12

11... 2

16... 16

18... 7

31... 8

33... 8

35... 4

39... 3

40... 4

45... 4

54... 0

55... 1

56... 4

58... 0

Unknown... 60

a Sixteenspecimenswere demonstratedtocontaintwo or moretypes: 4

contained HPVtypes6 and16;twocontainedHPV types 16 and33;andthe

10remaining specimenscontained HPV types 6 and 31, HPV types 6 and 39,

HPVtypes 11 and16,HPV types 16 and18,HPV types 16 and 56, HPVtypes

18and45,HPVtypes31 and33,HPVtypes 33 and 40, HPV types 35 and 56,

andHPVtypes6, 39, and 45, respectively.

be regardedasanapplicable system(20). The nested primer-based PCRwas evaluated on samples from the same non-selectedfemalepopulation thatwereanalyzed by the MY11-MY09 one-step PCR. The total detection rate of HPVDNA increased from 5.9 to20.3%. Among the women with normal cytology, the detection rate of HPV increased from 5.6 to

19.2%.Itwasclear from theMY11-MY09 one-step PCR that HPVprevalence increases withage(Table2). The frequency of HPV-positive women also increased with age in the

samples analyzed bythe two-step PCR(Table 3).

Demonstration by the two-step PCR of 83 HPV-positive specimens thatwerenegative bytheMY11-MY09 one-step PCR may indicate variationsin the HPV copy number in the lesions. The two-step PCR was more sensitive than the one-stepPCR; i.e.,fewercopies of the HPV genome could be detected. Theinnerprimer pair GP5-GP6was also used foramplificationof some of thespecimens,which increased thesensitivityof the one-step PCR. However, the two-step PCRwasthemostsensitivetechnique.TheGP5-GP6primer pairspansaregionof about 140bp compared with450bp for

the MY11-MY09 primer pair. The shorter amplification product may increase the efficiency of the PCR.

Further-more, the GP5-GP6 primersdo notcontain anydegenerate bases, which may result in increased amplification

effi-ciency.

The HPV type 6 genomewas detected in 10 of 35

HPV-positive sampleswhen the MY11-MY09 one-step PCRwas used, but only 2additional HPV type 6-containing cervical scrapeswerefoundbythe two-step PCR(datanotshown).

This could reflect the fact that HPV type 6was present at

high copy numbers in the lesions, while other types may havehad alower genome copy number. It is alsopossible

thatsometypes(unknowntypes)couldnotbeamplifiedtoa detectable level by using ethidiumbromide-stained gels in the one-step PCR. Inthe two-step system, theamplification

products can be visualized on an agarose gel when the second amplification round is performed with the inner

primer pair. Theconditions foramplification can also vary

considerably between different specimens, since the PCR was performed directly on the DNA prepared from cell

scrapings.However, allsamplesthatwereanalyzedwith the

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HPV-specific primers were positive for the

P-globin

geneby a one-stepPCR.

HPV types 6and 16 werethe most commonly found HPV types amongthewomenin this study. In total,wedetected 12 different types. We were not able to determine the HPV type amonga largeproportion of the two-step PCR-positive specimens incomparisonwith theproportion we determined amongthe one-step PCR-positive specimens. A higher pro-portion of unknown HPV types was amplified with the nestedprimer-based PCR system, andit is also possible that thetypingwaseasiertoperform on the larger amplimer (450 bp) from the MY11-MY09 one-step PCR.

The increase in the unknown HPV types among the positive specimens after the two-step PCR may indicate nonspecific amplification. However, the ethidium bromide-stained amplification product was always of the predicted size. Furthermore, women with antichlamydial antibodies

displayed a statistically significant higher presence of HPV than other women did (P < 0.001; data not shown). This correlateswith the increase in the number of HPV-positive women with age determined by the two-step PCR. We also analyzed DNA prepared from the HPV-negative cell line A549originating from a human lung carcinoma. No amplifi-cation product of the predicted HPV amplimer size was

detected(datanotshown).Thenatureof theamplimers from theunknown HPV types can beverifiedby sequencing.

By use ofthe two-step general PCR, we determined the HPVprevalencetobe 19.2% among young (age range, 19 to 25 years) Swedish women with normal cytology in a popu-lation-based study. Some 20.3% of all women were HPV

positive, and the prevalence of HPV 16 was 2.7%. In Holland, HPV DNA was found by using the GP6-GP6 general primer pair in 14 to 25% of cervical scrapes from womenwith normalcytologyattending gynecological clinics

forawide range ofgynecological complaints (agerange, 16 to60years) (26, 27).Amongfemale university students from

Berkeley, Calif., who underwent a routine gynecological

examination, 31% had normalcytology and 46% were HPV

positive when the consensus primer pair MY11-MY09was used(1). Theirmean age was22.9 years.

The one-step PCR consensusprimer system

from

theLi

region (15) was, in our hands, not sufficiently robust to detect all the HPV DNA-positive cytological scrapes that werepositive by use of a two-step general PCRfromthe

Li

region. By

using

the two-stepPCR,wefound that even in a population-based study of young women, HPV infection of theuterine cervix is common.

ACKNOWLEDGMENTS

We aregrateful to T.

Angstrom

of the Department of Cytology, UniversityofUmea, forcytological analysis.

This work was supported by a grant from the Labor Market Insurance Company, grant 1547-B90 from the Swedish Cancer Society, and grants from the Research Foundation of the Depart-mentofOncology, University ofUmeg.

REFERENCES

1. Bauer, H. M., Y. Ting, C. E. Greer, J. C. Chambers, C. J. Tashiro, J. Chimera, A.Reingold,and M.Manos. 1991. Genital humanpapillomavirus infection infemale university students as determined byaPCR-based method. JAMA 265:472-477. 2. Boshart, M., L. Gissmann, H. Ikenberg, A. Kleinheinz, W.

Scheurlen,and H.zurHausen. 1984. A new type of papilloma-virus DNA and its presence in genital cancer biopsies and in cell linesderived from cervical cancer. EMBO J. 3:1151-1157. 3. Chow,L.T.,M.Nasseri, S. M. Wolinsky, and T. Broker. 1987.

Humanpapillomavirus types [sic] 6 and 11 mRNAs from genital

condylomata. J. Virol. 61:2581-2588.

4. de Villiers, E.-M. 1989.Heterogeneity of the human papilloma-virus group. J. Virol. 63:4898-4903.

5. de Villiers, E.-M., D. Wagner, A. Schneider, H. Wesch, H. Miklaw, J. Wahrendorf, U. Papendick, and H. zur Hausen. 1987. Human papillomavirus infections in women with and without abnormal cervical cytology. Lancet ii:703-706.

6. Durst, M., L. Gissmann, H. Ikenberg, and H. zur Hausen. 1983. A papillomavirus DNA from a cervical carcinoma and its prevalence in cancer biopsy samples from different geographic regions.Proc. Natl. Acad. Sci.USA 80:3812-3815.

7. Evander, M., E. Boden, L. Bjersing, E. Rylander, and G. Wadell. 1991.Oligonucleotide primers forDNAamplificationof theearly regions 1, 6,and 7 from humanpapillomavirustypes 6, 11, 16, 18, 31, and 33. Arch. Virol. 116:221-233.

8. Evander, M., and G. Wadell. 1991. A general primer pair for amplification and detection of genital human papillomavirus types. J. Virol. Methods 31:239-250.

9. Gregoire, L., M. Arella, J. Campione-Piccardo, and W. D. Lancaster. 1989. Amplificationof human papillomavirus DNA sequences by using conserved primers. J. Clin. Microbiol. 27:2660-2665.

10. Kjaer, S. K., E.-M. de Villiers, B. J. Haugaard, R. B. Christen-sen,C.Teisen, K. A. M0ller,P.Poll, H. Jensen, B. F. Vester-gaard, E. Lynge, and0.M.Jensen. 1988. Human papillomavi-rus, herpes simplex virus and cervical cancer incidence in Greenland and Denmark. A population-based cross-sectional study.Int.J. Cancer41:518-524.

11. Koutsky, L. A., D. A. Galloway, and K. K. Holmes. 1988. Epidemiology of genital human papillomavirusinfection. Epi-demiol. Rev. 10:122-163.

12. Lbrincz, A. T., W. D. Lancaster, and G. F. Temple. 1986. Cloning and characterization of the DNA of a new human papillomavirus from a woman with dysplasia of the uterine cervix. J. Virol. 58:225-229.

13. Lirincz, A. T., G. F. Temple, J. A. Patterson, A. B. Jenson, R. J. Kurman, and W. D. Lancaster. 1986. Correlation of cellular atypia and human papillomavirus deoxyribonucleic acid sequences in exfoliated cells of the uterine cervix. Obstet. Gynecol.68:508-512.

14. MacNab, J. S., J. Walkinshaw, J. Cordiner, and J. B.Clements. 1986. Human papillomavirus in clinically and histologically normaltissue ofpatients with genitalcancer. N.Engl.J. Med. 315:1052-1058.

15. Manos, M. M., Y.Ting,D. K.Wright, A. J.Lewis,T. R.Broker, and S. M.Wolinsky. 1989. Use ofpolymerase chain reaction amplificationfor the detection ofgenitalhumanpapillomavirus. CancerCells7:209-214.

16. Meisels, A., C. Morin, and M. Casas-Cordero. 1982. Human papillomavirusinfection of the uterine cervix. Int. J. Gynecol. Pathol.1:75-94.

17. Melchers, W., A. van den Brule, J. Walboomers, M. de Bruin, M.Burger, P. Herbrink, C.Mejer,J. Lindeman, and W. Quint. 1989. Increased detection rate of human papillomavirus in cervicalscrapesbythepolymerase chain reaction as compared to modified FISH and Southern blot analysis. J. Med. Virol. 27:329-335.

18. Pfister, H.1984. Biology andbiochemistry of papillomaviruses. Rev.Physiol. Biochem. Pharmacol. 99:111-181.

19. Saiki, R. K., S. Scharf, F. Faloona, K. B. Mullis, G. T. Horn, H.A.Erlich,ahdN. Arnheim.1985. Enzymaticamplificationof beta-globin genomic sequences andrestriction siteanalysisfor diagnosisof sickle cell anemia. Science 230:1350-1354. 20. Schiffman, M. H., H. M. Bauer, A. L.Lorincz,M. M. Manos,

J. C. Byrne, A. G. Glass, D. M. Cadell, and P. M. Howley. 1991. Comparison of Southern blot hybridization and polymerase chain reaction methods for the detection of human papilloma-virus DNA. J. Clin. Microbiol. 29:573-577.

21. Shibata, D. K., N. Arnheim, and J. Martin. 1988. Detection of humanpapillomavirus in paraffin-embedded tissueusing poly-merase chainreaction. J. Exp. Med. 167:225-230.

22. Shirasawa,H.,Y.Tomita, K. Kubota, T. Kasai, S. Sekiya, H. Takamizawa, and B. Simizu. 1988.Transcriptionaldifferences of

on April 12, 2020 by guest

http://jcm.asm.org/

(6)

thehumanpapillomavirustype 16 genome between precancer-ous lesions andcervical carcinomas. J. Virol. 62:1022-1027. 23. Snijders, P. J. F., A. J. F. van den Brule, H. J. F.

SchriJnemak-ers, G. Snow, C.J. L. M.Meijer, and J. M. M. Walboomers. 1990. The use of general primers in the polymerase chain reaction permits the detection of a broad spectrum of human papillomavirusgenotypes. J. Gen. Virol. 71:173-181.

24. Stanley, M. 1990. Genital papillomaviruses, polymerase chain reaction andcervicalcancer. Genitourin. Med. 66:415-417. 25. van denBrule, A. J. C., E. C. J. Claas, M. du Maine, W. J. G.

Melchers, T. Helmerhorst, W. G. V. Quint, J. Lindeman, C.J. L. M. Meijer, and J. M. M. Walboomers. 1989. Use of anticontaminationprimers in the polymerase chain reaction for the detection of human papillomavirus genotypes in cervical scrapes andbiopsies.J. Med. Virol.29:20-27.

26. van den Brule, A. J. C., C. J. L. M. Meijer, V. Bakels, P. Kenemans, and J. M. M.Walboomers. 1990.Rapid detection of humanpapillomaviruses in cervical scrapes by combined gen-eralprimer-mediated andtype-specific polymerasechain

reac-tion. J. Clin. Microbiol. 28:2739-2743.

27. van den Brule, A. J. C., P. J. F.Sniders,R. L.J.Gordijn, 0. P. Bleker, C. J. L. M. Meijer, and J. M. M. Walboomers. 1990. Generalprimer-mediated polymerase chainreactionpermits the detectionofsequenced andstill unsequenced human papilloma-virus genotypes in cervical scrapes and carcinomas. Int. J. Cancer.45:644-649.

28. Ward, P., G. N. Parry, R. Yule, D. V.Coleman, and A. D. B. Malcolm. 1989. Human papillomavirus subtype 16a. Lancet ii:170.

29. Williamson, A. L., and E. P. Rybicki. 1991.Detectionofgenital humanpapillomavirusby polymerasechainreaction amplifica-tionwithdegeneratenestedprimers.J. Med.Virol. 33:165-171. 30. Young, L. S.,I. S. Bevan, M. A. Johnson, P. I. Blomfield, T. Bromidge, N. J. Maitland, and C. B. J. Woodman. 1989. The polymerase chain reaction: a new epidemiological tool for investigatingcervical humanpapillomavirus infection.Br.Med. J. 298:14-18.