High-Throughput Genotyping of Oncogenic Human Papilloma Viruses with MALDI-TOF Mass Spectrometry
High-Throughput Genotyping of Oncogenic Human Papilloma Viruses with MALDI-TOF Mass Spectrometry Anna So¨derlund-Strand,1,2Joakim Dillner,1,2and Joyce Carlson3*
BACKGROUND:Human papilloma virus(HPV)is the major cause of cervical https://www.wendangku.net/doc/8513026901.html,e of HPV geno-typing in cervical screening programs and for moni-toring the effectiveness of HPV vaccination pro-grams requires access to economical,high-throughput technology.
METHODS:We used the Sequenom MassARRAY plat-form to develop a high-throughput mass spectrometric (MS)method for detecting14specific oncogenic HPV genotypes in multiplex PCR products.We compared results from532cervical cell samples to the compari-son method,reverse dot blot hybridization(RDBH).
RESULTS:The MS method detected all samples found positive by RDBH.In addition,the MS method identi-fied5cases of cervical disease(cervical intraepithelial neoplasia of grade I or higher)that RDBH analysis had missed.Discrepancies in specific genotypes were noted in20samples,all positive by MS,with an overall con-cordance of??0.945.
CONCLUSIONS:The MS high-throughput method,with a processing capacity of10?384samples within2 working days and at a consumables cost of about US$2 per sample,performed as well as or better than the comparison method.
?2007American Association for Clinical Chemistry
Infection with oncogenic types of human papilloma-virus(HPV)4is the principal cause of invasive cervical cancer and its precursor lesion,cervical intraepithelial neoplasia(CIN)(1,2);HPV DNA is found in almost all cervical cancers(3).HPV types16,18,31,33,35,39, 45,51,52,56,58,59,68,73,and82are found in cervical cancer,whereas HPV6,11,40,42,43,44,54,61,70,72, and81are not associated with cervical cancer(4).Genotyping of HPV infections is important,because different HPV genotypes confer distinctly different risks for development of cervical disease(5).The addi-tion of HPV testing to cytology in primary screening produces a higher sensitivity for the detection of CIN than cytology alone(6,7).A knowledge of the prevalence and associated risks for each specific HPV genotype also has been essential for the development of vaccines against HPV.High-throughput HPV genotyping will be essential not only for cervical-screening programs but also for monitoring the effec-tiveness of HPV vaccination programs,i.e.,for docu-menting decreasing prevalence of the HPV types for which vaccines are available and possibly for changing the occurrence of HPV types without vaccines via type replacement or cross-protection(8).
Although several primarily PCR-based methods have been used to type HPV(9),the massive scale of the monitoring and primary-screening efforts likely to be launched in the near future will require more effi-cient methods.In the present study,we developed a high-throughput multiplex analysis of14distinct HPV high-risk genotypes on the Sequenom MassARRAY matrix-assisted laser desorption/ionization time-of-flight(MALDI-TOF)mass spectrometry(MS)system. We compared its performance with that of another method for HPV genotyping,reverse dot blot hybrid-ization(RDBH)of PCR amplimers,as well as with his-topathologic diagnoses.
Materials and Methods
STUDY POPULATION
We obtained samples from271patients,ages23–81 years(mean,38years;median,36years),who were referred to the Department of Gynecology,Va¨ster?s Hospital,Va¨ster?s,Sweden,for colposcopy after an atypical cervical smear,as described(10).A sample of
1WHO HPV LabNet Global Reference Laboratory and Departments of2Medical Microbiology and3Clinical Chemistry,Lund University,Malmo¨University Hos-pital,Malmo¨,Sweden.
*Address correspondence to this author at:Department of Clinical Chemistry, Lund University,Malmo¨University Hospital,SE-20502Malmo¨,Sweden.Fax 46-40-336286;e-mail joyce.carlson@med.lu.se.Received May28,2007;accepted October10,2007.
Previously published online at DOI:10.1373/clinchem.2007.092627
4Nonstandard abbreviations:HPV,human papillomavirus;CIN,cervical intraepi-thelial neoplasia;RDBH,reverse dot blot hybridization;MS,mass spectrometry; and hME,homogeneous mass extend.
Clinical Chemistry54:1
86–92(2008)
Molecular Diagnostics and Genetics 86
cervical cells for cytologic and HPV testing and a col-
poscopy-guided tissue biopsy sample were obtained
from all consenting patients.Patients testing positive
by at least2of the criteria(initial cytology results ?CIN I,biopsy results?CIN I,or abnormal colpos-copy results)were treated with loop or laser conization
(n?199).All patients were invited to follow-up visits
at4–6months and16–18months after treatment,
whether they received treatment or not,and261new
samples of cervical cells were obtained for cytologic
and HPV testing,for a total of532samples.The study
was approved by the ethics review board of Va¨ster?s
Hospital.
SAMPLE PREPARATION
Cervical cells were suspended in1mL154mmol/L
NaCl and stored at–20°C.After thawing,the cells
were centrifuged at3000g for10min,and pellets
were resuspended in1mL10mmol/L Tris-HCl,pH
7.4.Before RDBH analysis,100-?L aliquots were freeze-thawed and boiled for10min to release the DNA.
After storage at–80°C for3–6years,40-?L ali-quots of the same532DNA extracts were dried in96-well plates at37°C overnight and resuspended in8?L sterile water;2?L of this suspension was used as tem-plate in the primary PCR reaction of the MS method.
To evaluate the sensitivities of the2methods,we
used standard10-fold dilution series of1–1000plas-
mids/reaction with type-specific inserts of each target
HPV type and1–1000copies/reaction of DNA ex-
tracted from SiHa cells(a cervical cancer cell line con-
taining1copy of HPV16DNA per cell;American Type
Culture Collection).COMPARISON METHOD
GP5?/6?consensus primers(11)and BGPCO5/ BGPCO3primers(12)were used to amplify HPV DNA and the human?-globin gene(HBB),respectively,in separate50-?L PCR reaction mixes,each containing 10?L DNA solution as template.The presence of any of the14target HPV types or HBB DNA in5-?L ali-quots of PCR product was detected by enzyme immu-noassay(13).Samples positive for HPV(n?260)in the enzyme immunoassay were further tested by RDBH(10,14)for the specific HPV types16,18,31, 33,35,39,45,51,52,56,58,59,66,and68.
MASS SPECTROMETRY
The MS method involves a multiplex primary PCR,in this case with several HPV primers,followed by a ho-mogeneous mass extend(hME)reaction with a single primer of distinct mass that is specific for each geno-type.Subsequently,unextended primers demonstrate the absence and specifically extended primers verify the presence and identity of each specific genotype.After laser deionization,the time of flight,which increases with the m/z,is detected for each hME primer.Unless otherwise specified,all procedures were performed with protocols and materials from Sequenom. PRIMER DESIGN
For the primary PCR,we used Oligo6.0software (Cambio)to modify the design of GP5?/6?consen-sus primers to provide optimal annealing to the14tar-get HPV types(Table1).We produced new forward primers FA(all14HPV types),FB(HPV16,39,45,51, 56,59,66),FC(HPV18,31,33,52,58),and FD(HPV 35,68)and reverse primers RG(HPV16,18,45,56,66, 68),RH(HPV51,39),RI(HPV31,52,59),and RJ
Human Papilloma Virus Genotyping with Mass Spectrometry
Clinical Chemistry54:1(2008)87
(HPV33,35,58).The number of mismatches was min-imized,and A-C mismatches were avoided.All primers have the5?10-base extension recommended by Seque-nom to improve annealing stability in multiplex PCR reactions(see Table1).PCR programs were optimized, and products were viewed after agarose gel electro-phoresis.The14unique HPV type–specific hME prim-ers are shown in Table2.
PRIMARY PCR
PCR reaction mixes(6?L)contained2?L DNA tem-plate and a final concentration of0.3?mol/L of each outer primer,200?mol/L of each deoxynucleoside triphosphate,10mmol/L Tris HCl,pH8.3,50mmol/L KCl,2.5mmol/L MgCl2,and0.2U AmpliTaq Gold DNA polymerase(Roche Molecular Systems).The mixes were robotically pipetted with disposable tips and amplified in384-well plates.Separate dilutions of plasmids with HPV type–specific inserts,1000 copies/?L of each HPV type,and a pool of all HPV plasmids with1000copies of each type/?L were used as positive controls.Negative controls were sterile wa-ter and10ng/?L of human DNA extracted from pooled human peripheral blood leukocytes.We per-formed all primary PCR amplifications with a Gene-Amp PCR System9700(Applied Biosystems)as fol-lows:denaturation at95°C for10min;5cycles of95°C for30s,42°C for30s,and72°C for45s;45cycles of 95°C for30s,64°C for30s,and72°C for45s;and a final step at72°C for10min.Primary PCR reaction mixes were dephosphorylated with shrimp alkaline phosphatase(Sequenom).
hME REACTION
The hME reaction mix was added to the dephos-phorylated primary PCR reaction mix and included1?mol/L of each hME primer(Table2),0.229?L termi-
nator mix(Sequenom)containing equal amounts of dATP,dCTP,ddGTP,and ddTTP,and1.25U Thermo Sequenase(Sequenom)in a final volume of10?l.The PCR program was94°C for2min,followed by99cy-cles of94°C for5s,42°C for10s,and72°C for5s. After desalting by the addition of6mg Clean Resin (item#08040,Sequenom)to each384-well plate,we applied15nL of each hME product to a384-spot SpectroChip(Sequenom).MS analysis was performed and interpreted with MassARRAY Typer software (Sequenom).
DISCREPANCY BETWEEN METHODS
Seventy-eight samples showed genotypic discrepancies between the RDBH and MS results.For these samples, we purified DNA from100-?L aliquots of the original cell suspension in10mmol/L Tris-HCl by proteinase K digestion and DNA precipitation(15).The dried pel-lets were dissolved in20?L sterile water.The presence of human DNA in these samples was demonstrated by real-time PCR analysis on a7900HT instrument (Applied Biosystems)with primers and probes for the
88Clinical Chemistry54:1(2008)
human F25gene [coagulation factor II (thrombin)](C 8726802;Applied Biosystems).Samples testing negative for human DNA (n ?26)were considered not evaluable and were excluded.All 52samples posi-tive for human DNA were reanalyzed at least once by the MS method.Fluctuating results for specific geno-types in a few samples were apparently due to viral dosage effects.Of the 52samples,39had 1or more HPV genotypes detected with the RDBH method than were detected with the MS method,8had 1or more HPV genotypes detected with the MS method than were detected with the RDBH method,and 5had 1discrepant HPV genotype detected with each method before the MS reanalysis.These 52samples with per-sisting discrepancies between the RDBH and MS re-sults were reanalyzed by RDBH with the GP5?/6?primers.
After reanalysis with MS and RDBH,discrepant results remained for 24samples,of which 10had been positive for a single HPV type.After a new PCR reaction,these 10samples were sequenced with the same mix of forward primers as in the primary PCR (Table 1).The presence of HPV found only in the MS method for 6samples was verified by DNA sequencing.In 4other cases [none with the diagnosis CIN I or higher (CIN I ?)],DNA sequencing was inconclusive.
These 4samples were considered not evaluable and were excluded,for a final total of 502samples.
STATISTICAL METHODS
Sensitivity,specificity,and positive predictive value were calculated for each method separately regarding the detection of HPV in comparison with histopatho-logic diagnosis.The concordance between the RDBH and MS methods for the detection of each of the 14HPV types in all 502evaluable samples was calculated by means of ?statistics (16).Results
Analysis of the dilution series of plasmids revealed the following detection limits per PCR reaction for the MS method:1copy for HPV types 16,52,58,59,and 66;10copies for HPV types 18,33,35,45,51,56,and 68;and 100copies for HPV types 31and 39.The detection limits per PCR reaction for the comparison method (RDBH)were as follows:1copy for HPV types 16,51,58,and 66;10copies for HPV types 18,31,33,39,45,and 56;100copies for HPV types 35,52,and 59;and 1000copies for HPV 68.With CIN II or higher (CIN II ?)in the histopathologic results as a clinical refer-ence,the sensitivity for the MS method was 91.4%[95%confidence interval (CI),84.3%–95.6%],the specificity was 46.0%(95%CI,37.5%–54.7%),and the positive predictive value was 58.9%,whereas the sensi-tivity of the comparison method was 89.7%(95%CI,
5
Human genes:F2,coagulation factor II (thrombin);and HBB ,(hemoglobin,beta).
Human Papilloma Virus Genotyping with Mass Spectrometry
Clinical Chemistry 54:1(2008)89
82.3%–94.3%),the specificity was48.9%(95%CI, 40.3%–57.5%),and the positive predictive value was 59.8%(Table3).
CONCORDANCE OF METHODS
The degree of overall concordance(?)between the MS method and the RDBH method was0.945.Both meth-ods detected multiple HPV types in the same40sam-ples and single HPV types in the same175samples.In2 samples,the MS method detected multiple types, whereas the RDBH method detected single types.In another3samples,the RDBH method detected multi-ple types,whereas the MS method detected single types.In another8samples,the MS method detected single types,whereas the RDBH method detected no HPV.
We compared the efficiencies of type-specific HPV detection by the2methods for all samples.Table4 shows an overview of the number of type-specific in-fections detected with the2methods.For all502sam-ples(with2?14?502results),28results(0.2%)in20 samples did not agree between the2methods.Fifteen of the20discrepant samples were derived from pa-tients with the diagnosis CIN I?.The HPV types missed by RDBH were among10patients with a his-topathologic or cytologic diagnosis of CIN I?and among5patients with no CIN,whereas the HPV types missed by MS were among9patients with CIN I?(Table4).In samples with multiple HPV types present,the2methods may have had slightly different abilities to detect certain HPV types(Table4).The MS method alone detected all cases of cancer(Tables3and4)and detected HPV68in4samples.The latter result is in contrast to RDBH,which consistently failed to identify this genotype(Table4).
Discussion
Compared with the comparison method and most other reported methods,our new MS-based method had both greater clinical sensitivity for detecting HPV in HPV-associated disease(high-grade CIN II?)and better or comparable analytical sensitivity for the num-ber of HPV copies that could be detected.The use of robotic pipetting and the MS method enables the anal-ysis of10?384samples within2working days,with computerized documentation of sample identity and position at all steps,distinct identification of positive or negative signals by the Sequenom software,and cur-rent costs for consumables of about US$2per sample. By comparison,an analysis of36samples by RDBH requires4days of labor,and the results depend on a subjective interpretation of dot blots.
Our evaluation was based on a large clinical sam-ple consecutively enrolled from a population-based screening program,suggesting that our results are gen-erally valid with respect to the method’s usefulness in a screening setting.An additional strength of the study is
90Clinical Chemistry54:1(2008)
that we used the detection of HBB or F2human DNA in each available aliquot to demonstrate the presence of amplifiable DNA.Because the clinical priority to be able to review cytology prevents centrifugation of cer-vical samples and lysis of all cells to provide a homoge-neous DNA solution,the risk for false-negative HPV results due to nonrepresentative aliquots increases with each successive use of a sample.
Although there were discrepancies in the genotyp-ing results,all but8discrepant samples(all MS positive and RDBH negative)had at least1concordant HPV type and therefore would not have been misclassified as HPV negative in a screening situation.The causes for discrepancy include sampling error,DNA degradation during storage,and potential interference or false-pos-itive reporting of weak RDBH dot blots.
Plasmid dilution series generally demonstrated lower detection limits for MS than for the comparison method,with the most notable difference being the greater ability of the MS method to detect HPV type68. According to a pooled international analysis of HPV type–specific prevalence in3085cervical cancer cases (17),HPV68is the14th most common type in cervical cancer,more common than HPV66.
The numbers of type-specific HPV infections de-tected with the2methods were almost equal,but the results of the genotyping differed slightly between methods.HPV33,52,and58(detected more fre-quently by MS)and HPV45(detected more by RDBH) are among the8most common types in cervical cancer (18).HPV types16and18account for about70%of cervical cancers worldwide(18).In samples positive for multiple HPV types,these genotypes were each de-tected weakly in2samples by RDBH,whereas MS de-tected other HPV types,suggesting possible cross-hybridization.For one of the samples positive for HPV18by RDBH,there was a weak HPV18peak(be-low cutoff)in the MS analysis,suggesting a low viral load in the sample.Eight additional results were also both MS negative and RDBH positive,and all origi-nated from samples that were concordant with respect to at least1HPV type.All samples that were negative for all14genotypes by MS were also negative by RDBH.
In a WHO international collaborative study for the assessment of the performance of various HPV-detection assays(19),a panel of HPV types was ana-lyzed in a number of laboratories,of which half failed to detect10000copies of HPV31plasmids.Some laboratories did not detect the same copy numbers of HPV35and HPV52.In the present study,the MS analysis detected100copies of HPV31,10copies of HPV35,and a single copy of HPV52.The primary PCR primers of the MS method were designed to match some target HPV types better than others.A few primers match their templates perfectly,whereas oth-ers contain a minimal number of non–C-A mis-matches.Although the detection limit for HPV31is slightly higher than for all the other types(except HPV 39)with the MS method,the MS method was generally more sensitive for HPV31,35,and52than the meth-ods in the WHO study.In general,the number of mis-matches is slightly lower with the MS primers than with GP5?/6?primers,but,more importantly,the mis-matches are preferentially G-T to optimize thermo-dynamic stability.
In summary,we have designed a highly auto-mated,potentially high-throughput MS method for the specific detection of1–100copies of14different oncogenic HPVs in samples of cervical cells.The per-formance compares well with the comparison method and with other methods reported in the literature. With access to robotic pipetting and the Sequenom system,the MS method allows large-scale HPV geno-typing at a low cost,and this method may be useful for cervical screening and to evaluate the effectiveness of HPV vaccination programs via monitoring the circula-tion of HPV types in vaccinated populations.
Grant/funding Support:This work was supported by grants from the Swedish Cancer Society,the WHO,and by the EU6th Framework Grant CCPRB(Cancer Con-trol Using Population-Based Registries and Biobanks), principal investigator Joakim Dillner.The SWEGENE genotyping facility was supported by the Knut and Alice Wallenberg Foundation.
Financial Disclosures:None declared. Acknowledgments:We thank Maria Sterner and Lise-lott Hall for technical assistance and Per Rymark for the enrollment of patients.
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如何写先进个人事迹
如何写先进个人事迹 篇一：如何写先进事迹材料 如何写先进事迹材料 一般有两种情况：一是先进个人，如先进工作者、优秀党员、劳动模范等；一是先进集体或先进单位，如先进党支部、先进车间或科室，抗洪抢险先进集体等。无论是先进个人还是先进集体，他们的先进事迹，内容各不相同，因此要整理材料，不可能固定一个模式。一般来说，可大体从以下方面进行整理。 (1)要拟定恰当的标题。先进事迹材料的标题，有两部分内容必不可少，一是要写明先进个人姓名和先进集体的名称，使人一眼便看出是哪个人或哪个集体、哪个单位的先进事迹。二是要概括标明先进事迹的主要内容或材料的用途。例如《王鬃同志端正党风的先进事迹》、《关于评选张鬃同志为全国新长征突击手的材料》、《关于评选鬃处党支部为省直机关先进党支部的材料》等。 (2)正文。正文的开头，要写明先进个人的简要情况，包括：姓名、性别、年龄、工作单位、职务、是否党团员等。此外，还要写明有关单位准备授予他(她)什么荣誉称号，或给予哪种形式的奖励。对先进集体、先进单位，要根据其先进事迹的主要内容，寥寥数语即应写明，不须用更多的文字。 然后，要写先进人物或先进集体的主要事迹。这部分内容是全篇材料
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