Griffin, Mairead

Keegan, Helen

Lambkin, Helen

Malkin, Alison

Ryan, Fergus

English

Arrow@TUDublin

Technological University Dublin ARROW@TU Dublin Articles School of Biological Sciences 2005 Validation of a Multiplex PCR Assay for the Simultaneous Detection of Human Papillomavirus and Chlamydia Trachomatis in Cervical PreservCyt Samples. Helen Keegan Technological University Dublin, helen.keegan@tudublin.ie Alison Malkin Technological University Dublin, alison.malkin@tudublin.ie Mairead Griffin St. James's Hospital Fergus Ryan Technological University Dublin, fergus.x.ryan@tudublin.ie Helen Lambkin Technological University Dublin, helen.lambkin@tudublin.ie Follow this and additional works at: https://arrow.tudublin.ie/scschbioart  Part of the Bioinformatics Commons, Biology Commons, and the Molecular Genetics Commons Recommended Citation Keegan, H. et al. (2005). Validation of a Multiplex PCR Assay for the Simultaneous Detection of Human Papillomavirus and Chlamydia Trachomatis in Cervical PreservCyt Samples. Clinical Chemistry. 51 (7): 1301-1302. doi:10.1373/clinchem.2004.045302 This Article is brought to you for free and open access by the School of Biological Sciences at ARROW@TU Dublin. It has been accepted for inclusion in Articles by an authorized administrator of ARROW@TU Dublin. For more information, please contact yvonne.desmond@tudublin.ie, arrow.admin@tudublin.ie, brian.widdis@tudublin.ie. This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 3.0 License assisted laser desorption/ionization(MALDI)-TOF MS, with its higherresolution, is the capability of SELDIto enrich low-abundance proteinsfrom complex matrices such asplasma, through, for example, thecoupling of specific antibodies to thechip surfaces (4 ). Such an approachis an important methodologic aspectin “second phase” proteomics, whichare characterized by repetitive inves-tigation of the same protein to vali-date the protein phenotype in largepopulation-based studies. This pro-vides a basis for diagnostic progressin personalized medicine (5 ). ForTTR, such an approach is relevantnot only in the diagnosis of TTR-related amyloidosis but also in otherdiseases.The true problem with on-chip im-munopurification is not the resolu-tion of the MS, which can be solvedby use of specific available interfaces,but the efficient coupling of the anti-body to the chip surface. When anon-chip immunoassay format is be-ing used, it is important that theprotein chip retains the antibody inan active state at high density. Re-sults are greatly affected by function-ality characteristics, such as the sta-bility, affinity, and specificity of theantibody. On the basis of studiesrelating to microarrays, only 5%–20%of commercially available antibodiesare suitable for one or the other mi-croarray format (6 ).References1. Theberge R, Connors LH, Skinner M, CostelloCE. Detection of transthyretin variants usingimmunoprecipitation and matrix-assisted laserdesorption/ionization bioreactive probes: a clin-ical application of mass spectrometry. J Am SocMass Spectrom 2000;11:172–5.2. Wang Z, Yip C, Ying Y, Wang J, Meng XY, LomasL, et al. Mass spectrometric analysis of proteinmarkers for ovarian cancer. Clin Chem 2004;50:1939–42.3. Schweigert FJ, Wirth K, Raila J. Characterizationof the microheterogeneity of transthyretin inplasma and urine using SELDI-TOF-MS immuno-assay. Proteome Sci 2004;2:5.4. Schweigert FJ. Characterization of protein micro-heterogeneity using mass spectrometry basedimmunoassays. Brief Funct Genomic Proteomic2005;4:7–15.5. Hanash S. Disease proteomics. Nature 2003;422:226–32.6. MacBeath G. Protein microarrays and proteom-ics. Nat Genet 2002;32:526–32.Florian J. Schweigert*Jens RailaDepartment of Physiology andPathophysiologyUniversity of PotsdamPotsdam-Rehbrucke, Germany*Address correspondence to this au-thor at: Institute of Nutritional Science,University of Potsdam, Scheunert-Allee114-116, Potsdam-Rehbrucke, D-14558Germany. E-mail fjschwei@rz.uni-potsdam.de.DOI: 10.1373/clinchem.2005.051714Validation of a Multiplex PCR Assayfor the Simultaneous Detection ofHuman Papillomavirus and Chlamydiatrachomatis in Cervical PreservCytSamplesTo the Editor:Chlamydia trachomatis is the mostcommon sexually transmitted bacte-rium worldwide (1 ) and a leadingcause of infertility in women (2 ).Human papillomaviruses (HPVs) arethe most important single agentcausing carcinoma of the uterine cer-vix (3 ). Combined molecular screen-ing for C. trachomatis and HPV couldbe justified given their propensity tocause asymptomatic infections, par-ticularly in high-risk groups. Fea-tures of HPV infection of cells of theuterine cervix are traditionally re-ported by the Pap smear method (4 ).The introduction of liquid-based cy-tology, such as the ThinPrep® PapTestTM, has had the effect of improv-ing the sensitivity of conventionalcytologic screening with the poten-tial for HPV testing of residual cellu-lar material in borderline or difficultcases (5–7). The US Food and DrugAdministration (FDA) has recentlycleared a hybrid capture-based sys-tem (HCII; Digene) for screeningwomen over 30 years of age as anadjunct to Pap testing (8 ). Research-ers have developed consensus prim-ers for the detection of HPV DNA byPCR (9, 10). We developed and eval-uated a multiplex PCR for the simul-taneous detection of HPV and C.trachomatis from PreservCytTM (Thin-Prep) solution.The multiplex PCR was performedon 100 cervical PreservCyt fluidspecimens collected from women at-tending their general practitionersfor routine cervical screening. Weused the MY09/11 primers (9 ) forHPV and plasmid primers for C. tra-chomatis (11 ), which generated frag-ments of 450 and 201 bp, respec-tively. Primers for the human-globin gene were included in themultiplex as an internal DNA ampli-fication control, generating a 110-bpproduct (12 ) (see Fig. 1 in the DataSupplement that accompanies theonline version of this Letter at http://www.clinchem.org/content/vol51/issue7/). The PCR mixture contained5 pmol of each of the forward andreverse primers of the MY09/11 andCTP1/2 primer sets and 10 pmol ofeach of the forward and reverseprimers of the PCO3/4 primer set,200 M deoxynucleoside triphos-phates, 10	 PCR buffer [containing10 mM Tris-HCl (pH 8.3), 50 mMKCl], 2 mM MgCl2, and 1 U of Plat-inum Taq DNA polymerase (Invitro-gen Ltd.) in a final volume of 20 L.The PCR was initiated by a 10-mindenaturation and enzyme activationstep at 95 °C and was completed by a10-min extension step at 72 °C. TheTable 1. Comparison of multiplex and single PCR for the detection of HPV and C.trachomatis in PreservCyt cervical samples.Sample cohortMultiplex positive, n Single PCR positive, nMY09/11a CTP1/2b MY09/11 GP5/6a CTP1/2 Hsp60bGPc clinic (n  100) 21 2 24 14 2 2GUM clinic (n  34) 10 34 10 10 34 34a Primers for the detection of HPV.b Primers for the detection of C. trachomatis.c Women who attended general practitioner (GP) clinics for routine cervical screening.Clinical Chemistry 51, No. 7, 2005 1301temperature cycles were as follows:40 cycles of 30 s at 95 °C, 1 min at57 °C, and 1 min at 72 °C.The detection limit was estimatedon serial dilutions, from 108 to 101copies/L, of cloned HPV MY09/11and the C. trachomatis CTP PCRproduct in the pCR® 2.1-TOPO® (In-vitrogen Ltd). To evaluate the sensi-tivity and specificity of the multiplexPCR for the detection of each organ-ism, we performed single PCRs onall samples: one PCR that detectsHPV by use of the GP5/6 prim-ers (10 ), generating a 150-bp prod-uct; and one that detects C. trachoma-tis by use of primers specific to thehsp60 gene of C. trachomatis (sense,5-GAT GGT GTT ACC GTT GCGA-3; antisense, 5-TAA TAA TCGTCT TTA ACA ACG T-3), generat-ing a truncated version (309 bp) of apreviously described product (13 ).PCRs were also performed with theMY09/11 and CTP1/2 primer setssingly.In the screened population, 21%(21 of 100) of samples were positivefor HPV by the multiplex PCR (Table1). The MY09/11 primers identified 3other samples as HPV positive thatwere not detected by the multiplexassay (Table 1). Either the MY09/11or the GP5/6 primers confirmedall 21 of the samples positive by themultiplex assay. Two samples werepositive for C. trachomatis in the mul-tiplex assay (Table 1). These wereconfirmed positive by the CTP1/2and Hsp60 primers in single PCRs,and no additional positive sampleswere detected. A positive samplewas defined as positive by either themultiplex or any of the single PCRsfor that organism. The sensitivityand specificity of the multiplex withrespect to single PCR for the detec-tion of HPV in the opportunisticallyscreened samples were 89% (95%confidence interval, 70%–97%) and100% (94%–100%; Table 1). The mul-tiplex assay was 100% specific andsensitive for the detection of C. tra-chomatis with respect to single PCR inthe screened population (Table 1).To estimate the diagnostic sensitiv-ity of the multiplex assay for thedetection of C. trachomatis, we per-formed multiplex PCR on 34 Preserv-Cyt fluid specimens from womenattending a genitourinary (GUM)outpatient clinic who had tested pos-itive for C. trachomatis by the LCx(Abbott Laboratories). The sensitiv-ity of the multiplex assay for thedetection of C. trachomatis in theGUM clinic population was 100%with respect to the commercial LCxassay (Table 1). Ten of the 34 sampleswere positive for HPV by the multi-plex assay. These results were con-firmed by single PCRs (Table 1). Themultiplex assay could detect as fewas 100 copies of the C. trachomatisplasmid and 100 copies of the HPVgenome per microliter of extractedDNA.This is the first study looking at thedetection of both HPV and C. tracho-matis from a single ThinPrep sampleby use of multiplex PCR. This simplemultiplex is rapid and could be usedto screen cervical ThinPrep samplesfor both HPV and C. trachomatis, par-ticularly in a high-risk population.References1. World Health Organization. State of the art ofnew vaccines research and development: Ini-tiative for Vaccine Research. Geneva: WHO,April 2003:21. http://www.who.int/vaccine_research/documents/en/stateofart_excler.pdf(accessed October 10, 2004).2. Mardh PA. Tubal factor infertility, with specialregard to chlamydial salpingitis. Curr Opin In-fect Dis 2004;17:49–52.3. Munoz N. Human papillomavirus and cancer:the epidemiological evidence. J Clin Virol 2000;19:1–5.4. Renshaw AA, Young AN, Birdsong G, Styer P,Davey D, Mody D, et al. Comparison of perfor-mance of conventional and ThinPrep gyneco-logic preparations in the College of AmericanPathologists Gynecologic Cytology Program.Arch Pathol Lab Med 2004;128:17–22.5. Hoelund B. Implementation of liquid-based cy-tology in the screening programme againstcervical cancer in the County of Funen, Den-mark, and status for the first year. Cytopathol-ogy 2003;14:269–74.6. Cheung A, Szeto NE, Leung B, Khoo U, Ng A.Liquid-based cytology and conventional cervicalsmears: a comparison study in an Asianscreening population. Cancer 2003;99:331–5.7. Schiffman M, Solomon D. Findings to date fromthe ASCUS-LSIL Triage Study (ALTS). ArchPathol Lab Med 2003;127:946–9.8. FDA News. FDA approves expanded use of HPVtest. Washington: FDA, March 31, 2003.http://www.fda.gov/bbs/topics/news/2003/new00890.html (accessed October 10, 2004).9. Manos MM, Ting Y, Wright D, Lewis A, Broker T,Wolinsky S. Use of polymerase chain reactionamplification for the detection of genital humanpapillomaviruses. Cancer Cells 1989;7:209–14.10. de Roda Husman AM, Walboomers JM, van denBrule AJ, Meijer CJ, Snijders PJ. The use ofgeneral primers GP5 and GP6 elongated attheir 3 ends with adjacent highly conservedsequences improves human papillomavirus de-tection by PCR. J Gen Virol 1995;76:1057–62.11. Griffais R, Thibon M. Detection of Chlamydiatrachomatis by the polymerase chain reaction.Res Microbiol 1989;140:139–40.12. Saiki RK, Scharf S, Faloona F, Mullis K, Horn G,Erlich H, et al. Enzymatic amplification of -glo-bin genomic sequences and restriction siteanalysis for diagnosis of sickle cell anaemia.Biotechnology 1992;24:476–80.13. Wood H, Reischl U, Peeling R. Rapid detectionand quantification of Chlamydia trachomatis inclinical specimens by LightCycler PCR. In: Reis-chl U, Wittwer C, Cockerill F, eds. Rapid cyclereal-time PCR—methods and applications.New York: Springer, 2002:115–32.Helen Keegan1Alison Malkin2Mairead Griffin2Fergus Ryan1Helen Lambkin1*1 Biomedical Research GroupSchool of Biological SciencesDublin Institute of TechnologyDublin, Ireland2 Department of Clinical CytologyCentral Pathology LaboratorySt. James’ HospitalDublin, Ireland*Address correspondence to this au-thor at: School of Biological Sciences,Dublin Institute of Technology, Kevin St.,Dublin 8, Ireland. Fax 353-1-402-4995; e-mail helen.lambkin@dit.ie.DOI: 10.1373/clinchem.2004.045302Detection of Heavy Chain Disease byCapillary Zone ElectrophoresisTo the Editor:Luraschi et al. (1 ) recently publishedthe first report describing the use ofcapillary zone electrophoresis (CZE)coupled with immunosubtraction todetect and characterize low concen-trations of free  heavy chains inserum. By contrast, we describe acase in which CZE failed to detectand characterize  heavy chain dis-ease.A 90-year-old woman who com-plained of weight loss presentedwith progressive enlargement of theleft parotid gland, splenomegaly,and palpable inguinal lymph node1302 Letters

Validation of a Multiplex PCR Assay for the Simultaneous Detection of Human Papillomavirus and Chlamydia Trachomatis in Cervical PreservCyt Samples.

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Validation of a Multiplex PCR Assay for the Simultaneous Detection of Human Papillomavirus and Chlamydia Trachomatis in Cervical PreservCyt Samples.

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