review article application of pcr-elisa in molecular diagnosis · review article application of...

Review ArticleApplication of PCR-ELISA in Molecular Diagnosis

Mei Jean Sue Swee Keong Yeap Abdul Rahman Omar and Sheau Wei Tan

Laboratory of Vaccines and Immunotherapeutics Institute of Bioscience Universiti Putra Malaysia (UPM)43400 Serdang Selangor Malaysia

Correspondence should be addressed to Sheau Wei Tan tansheauputraupmedumy

Received 21 February 2014 Accepted 12 May 2014 Published 27 May 2014

Academic Editor Nikolaos Siafakas

Copyright copy 2014 Mei Jean Sue et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA) is an immunodetection method that can quantifyPCR product directly after immobilization of biotinylated DNA on amicroplateThis method which detects nucleic acid instead ofprotein is a muchmore sensitive method compared to conventional PCRmethod with shorter analytical time and lower detectionlimit Its high specificity and sensitivity together with its semiquantitative ability give it a huge potential to serve as a powerfuldetection tool in various industries such as medical veterinary and agricultural industries With the recent advances in PCR-ELISA it is envisaged that the assay is more widely recognized for its fast and sensitive detection limit which could improve overalldiagnostic time and quality

1 Introduction

In the early 90s there was a sudden interest in DNAstudies when Friedrich Miescher first identified and isolatedDNA and when James D Watson and Francis Crick firstdiscovered the double helix structure of DNA in 1953 Fromthen on various molecular techniques and knowledge wereintroduced such as gel electrophoresis DNA double helixstructure and the invention of polymerase chain reaction(PCR) by Kary Mullis in 1983 one of the most innovativeand still widely used techniques in the field of life sciencesAlthough PCR is a powerful tool its applications cannot befully expressed without a powerful detection tool

Gel electrophoresis is one of the commonly usedmethodsfor the detection of an amplified PCR product but thismethod has a low detection limit and only allows the userto detect the presence or absence of a particular gene Manydetectionmethods and equipment have since been developedand amongst those commonly used is real-time PCR Inthe late 1980s there was a sudden boom of interest in thestudy of immunodetection of DNA Various methods ofimmunodetection were published and amongst them is astudy by Coutlee et al [1] where they studied the immun-odetection of DNA using biotinylated RNA probes Fromthen on numerous studies on immunodetection of DNA

using enzyme linked immunosorbent assay techniques werepublished which subsequently lead to the introduction ofpolymerase chain reaction-enzyme linked immunosorbentassay (PCR-ELISA) This method combines both PCR andELISA into a single analytical technique and its applicationis very much similar to ELISA except that this method allowsthe detection of nucleic acid instead of protein [2]

How does PCR-ELISA work PCR-ELISA is an immuno-logical method to quantify the PCR product directly afterimmobilization of biotinylated DNA on a microplate Thewhole method involves 3 steps amplification immobiliza-tion and detection At the very beginning of the method thegene of interest will be amplified through PCR in the presenceof digoxigenin-11-dUTP (DIG-dUTP) DIG-labelled PCRproducts will then bind to specific oligonucleotide probeslabelled with biotin at their 51015840 end The next step involvesimmobilizing the gene of interest to the microplate Thisis achievable with the presence of streptavidin coated onmicroplates and biotin on the 51015840 end of the formed hybridStrong affinity of avidin-biotin interaction forms the avidin-biotin complex thus binding only PCR products with thespecific gene of interest to the microplate All other non-specific products will be washed off After immobilizationdetection of biotinylated DNA is required as the formationof these complexes cannot be detected through naked eyes

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 653014 6 pageshttpdxdoiorg1011552014653014

2 BioMed Research International

Amplification

5998400

3998400

DNA template

PCR amplification usingDIG-labelled dUTP

DIG

Detection

Immobilization

Microplate

Strep

Biotin

Biotin-labelledprobe

DIG-labelledmRNA

Anti-DIG peroxidase

ABTS ABTS

Substrate-enzymereaction to create

colour change

DIG DIG

DIG DIG DIG

DIGDIGDIG

DIG DIG DIG

Figure 1 Illustration of the 3-step PCR-ELISA method (i) amplification of the gene of interest using PCR in the presence of DIG-dUTPwhich is then bound to specific probes (ii) immobilization of the gene of interest to the microplate through strong affinity of avidin-biotininteraction followed by (iii) detection of biotinylatedDNAusing an anti-DIG-peroxidase conjugate with substrate ABTS to form a blue-greencolor reaction that is both visible and measured using a spectrophotometer

To do so the amplicons can be detected using an anti-DIG-peroxidase conjugate through the substrate 221015840-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) These will develop ablue-green color reaction that is both visible and measuredusing a spectrophotometer (Figure 1) [3] Another methodof PCR-ELISA detection includes the use of fluoresceinprobe where detection includes the use of antifluoresceinantibodies conjugated to horseradish peroxidase to detectthe hybridized fluorescein-labelled oligonucleotide probe[4]

2 Comparisons of PCR-ELISA with OtherPCR-Based Molecular Approaches

Since the introduction of this tool various studies havebeen carried out to compare the performance of PCR-ELISAwith other tools Many agreed that the detection of DIG-labelled products by microwell capture hybridization assaymakes PCR-ELISA a more sensitive tool than agarose gelelectrophoresis analysis because the specific hybridizationand enzymatic colourization increase the positive signal ofbiotin-labelled probe-bound PCR products

The PCR amplicons are analyzed using a colorimetricassay thus not only is there reduced risk on the use ofmutagen-staining materials and significant reduction of pos-sible DNA contamination but also it allows the method toserve as a semiquantitative tool [5] As this detection usesgene-specific probes for detection the specificity of the toolis very prominent [2 6] Not only can samples with that

particular gene be detected but also they can be quantifiedbased on colour intensity The presence of higher colourintensity indicates that more probe was bound to the specificgene sequence forming a hybrid complex that was laterboundby a peroxidase-conjugated anti-DIGantibody and thecolorimetric peroxidase substrate ABTS allowing detectionWhilst PCR-ELISA cannot provide an accurate estimation ofthe actual gene of interest that is present compared to real-time PCR (qPCR) it provides a quick summary of whethera particular substrate is high or low at a particular time ascolorimetric detection is in direct proportion to the numberof the intended gene of interest

Another main attraction of PCR-ELISA is that the assayallows large-scale screening to be done using only standardlaboratory equipmentmaking it suitable to be used in clinicallaboratories This should serve as another incentive forlaboratories with fewer resources as a survey done by Comley[7] showed that respondents do not turn to fully automatedequipment for ELISA despite its availability possibly due tothe high costs of purchasing and maintaining the equipmentLast but not least the overall analytical time of this assay isalso much shorter than conventional PCRmethod making ita promising tool for future uses especially when dealing witha large sample size

With new discoveries and new invention molecular biol-ogy tools need to be continuously improved and developedfor faster and more efficient results Each new technologythat was developed has its pros and cons compared to othertechnologies If the study is about an unknown gene thenconventional PCR using agarose gel detection would be the

BioMed Research International 3

Table 1 Comparisons between 3 different detection methods conventional PCR with agarose gel electrophoresis PCR-ELISA and qPCR

Comparison Conventional PCR PCR-ELISA qPCREquipment required Standard laboratory equipment Standard laboratory equipment Requires fluorescence detection instrumentReagent costs Low Moderate CostlyDetection limit 1ndash10 ng120583L 001 ng120583L 025 pg120583LQuantitative ability Not quantitative Semi-quantitative Quantitative

only available option as both qPCR and PCR-ELISA requirethe development of primers andor probes that is difficult toachieve on a new target gene

There were several articles comparing PCR-ELISA withqPCR for their high sensitivity ability Menotti et al [8] whodid a comparison between PCR-ELISA and qPCR assay forthe detection of Toxoplasma gondii a parasitic protozoanthat is responsible for causing life-threatening infections inimmunocompromised hosts found that the former methodyields negative results in 15 samples that were clinicallyproven to suffer from the disease while the latter presentedaccurate results throughout the study This was supportedby various other authors (Table 1) whereby qPCR provedto be a more sensitive tool Without the presence of anycompetitor so far in terms of sensitivity qPCR is still anessential tool in research studies for detection Howeverapart from this handicap PCR-ELISA was found to be morecost effective as real-time PCR requires the use of costlyfluorescent scoring system If the purpose of the study doesnot require such high sensitivity as compared to qPCR PCR-ELISA might just be the better option as it offers semi-quantitative ability and adequate sensitivity at lower costs[9]

Overall the main factors that need to be consideredwhile choosing the bestmethod for their experimental designwould be the pricing factor and level of sensitivity requiredSeveral studies compared the use of qPCR PCR-ELISAand conventional PCR for the detection of poultry virusinfectious bursal disease virus and the summary of the resultsis listed in Table 1 [2 10ndash14]

3 Applications of PCR-ELISA

With the aforementioned advantages of PCR-ELISA and itssemiquantitative ability a number of researchers propose theuse of PCR-ELISA in a diverse range of fields from basicdetection and diagnosis to quality control and quantitativemonitoring of infectious disease food allergen detectionplant pathogens and biomarkers with detection as its mainapplication

31 Detection and Diagnosis Due to its high sensitivity andspecificity various studies on the use of PCR-ELISA as adetection and diagnostic method were proven successful Asrapid diagnosis in the medical field can affect the life anddeath of the public the papers below are amongst some of therecent studies reportedwithin the last 5 years on the detectionof various diseases and pathogens in the medical diagnosis

identification of cancer cells [15ndash17] detection of the presenceof Hepatitis A B C and E types [14 18ndash21] species detectionand identification of dermatophyte species [22] invasivefungal infections in immunocompromised patients [23ndash25]detection of poliovirus enterovirus and norovirus [26 27]and determination of blood group antigens for hemolyticdisease of the newborn cases and polytransfused patients[28]

There are also a number of publications using PCR-ELISA in the food industry such as the detection of harmfulfood-borne pathogens such as Campylobacter sp Salmonella[6 29ndash32] Listeria monocytogenes [33 34] Escherichia coli[26 35] Brucella melitensis [36] andVibrio parahaemolyticus[2] Not limiting the use of the method in the medical andfood industries the study of PCR-ELISA extends even tothe veterinary industry Amongst the studies are Leishmaniaparasite detection [37 38] and the detection of various avianviruses in chickens [10 39 40] Other detection studies thathelp detect the presence of plant pathogens [41 42] includetomato spottedwilt [43] potato spindle tuber [12] prevalenceof each phylogenetic group among the infected grapevinevarieties [44] and plant viruses in woody plants [45] PCR-ELISA can also detect the presence of harmful waterbornepathogens in both water supply and industrial cooling towerwater [46 47]

As PCR-ELISA is a sensitive tool that allows detectionat very small concentration there are suggestions to developPCR-ELISA as an early detection system that allows pre-ventive measures to be taken before the condition of thepatient deteriorates or the situation worsens The use ofPCR-ELISA as an early warning system can be extendedfor the detection of latent symptoms of diseases or evengene expression studies through biomarkers Some of thestudies published include the development of PCR-ELISA asa replacement method for detection and validation of geneexpression studies [9] and detection of four 120573-thalassemiapoint mutations in Iranians using a PCR-ELISA genotypingsystem [5]

32 Quantitative Monitoring Many studies also suggest theuse of the assay for quantitative monitoring as a quick indica-tion in the presence or absence of a particular substrate and itsestimated concentration It is a very crucial tool especially forimmunocompromised patients who are sensitive and suscep-tible to their environment as it allows the determination ofappropriate level of antiviral management Amongst studieson the quantification monitoring using PCR-ELISA are eval-uation and monitoring of cytomegalovirus infection in bone


marrow transplant recipients [48] diagnostic value of thecombined determination of telomerase activity in inducedsputum pleural effusion and fibrobronchoscopic biopsy inlung cancer patients [49] and quantitative monitoring ofLeishmania parasite in livestock [37]

4 Recent Advances inPCR-ELISA Technologies

Due to its ability to test multiple samples in a single runwith shorter running time a number of PCR-ELISA trialruns are currently in progress for use in medical diagnosisWith the success of these trial runs it can be foreseenthat the assay would be used in various quality control andmedical diagnostic labs in the near future Even with its highsensitivity and specificity there are continuous attempts toimprove the applications of PCR-ELISA in recent years AsPCR protocols are already established the focal point forfurther improvements is on the immunoassay detection ofDIG-labelled PCRproducts such as the effects of streptavidinconcentration on the microplate addition of various PCRproducts to the microplate or other solid-phase interfaceswhich require thorough optimization [6]

One of recent attempts to improve PCR-ELISA is todevelopmultiplexing ability where several specific sequencesare detected simultaneously without any cross-reactivityThis method is designed as many felt that separate reactionsfor each species are both costly and time consuming com-pared to simultaneous detection The key development forthis procedure is to design probes that are highly specific foreach of the species of interest but with the absence of cross-reactions [11 13 18 50ndash55]

Kobets et al [37] developed an improved and optimizedPCR-ELISA method that eliminates the need for a separatestep of hybridization of the PCRproductwith labelled probesFocusing on the detection and quantification of Leishmaniaparasites these researchers use bothDIG- and biotin-labelledprimers to produce PCR products These products werethen attached to the streptavidin-coated plate before theproducts were detected using sandwich ELISA with anti-DIG antibodies Not only does this method eliminate thehybridization step but also it eliminates a number of stepsincluding all the washing procedures in between each stepAmongst the procedures that were also eliminated are thedenaturation step of PCR product prior to hybridization andthe need for designing and attaching specific probe to themicroplate Overall this design reduces the incubation andwashing time as well as reagent costs

Others developed a technique that eliminates the need todenature and neutralize samples prior to hybridization com-pared to the conventional PCR-ELISAmethodThis methodknown as asymmetric PCR-ELISA utilizes asymmetric PCRamplification to amplify only one DNA strand in a double-stranded DNA template using excess primer for the strandtargeted for amplification prior to detection Nolasco et al[41 42] found that asymmetric PCR-ELISA increases therelative concentration of the target DNA species making thismethod more sensitive than TaqMan detection method Themethod also utilizes less dNTPs and DIG labels which are

very costly by half and tenfold respectively without reducingits sensitivity

No studies have yet been attempted to compare these fewmethods to identify the advantages and disadvantages of eachmethod but it seems that the advances seem to be focusingon reducing the production costs and turn-round time whilstmaintaining the assayrsquos sensitivity and specificity

5 Summary

Overall PCR-ELISA method (i) is much more sensitiveby about 100-fold than conventional PCR method (ii) iswith short analytical testing time allowing faster resultoutput (iii) allows multiple sample testing with the useof gene-specific probes (iv) is able to do quantitative andqualitative analyses (v) reduces risk of contamination (vi)omits the use of mutagen-staining materials and (vii) isan easy-to-use method as it only requires the use of basiclab equipment By offering faster diagnostic time and highsensitivity there is high potential for PCR-ELISA to serveas a powerful detection tool in all sectors from medicalsector to both food and agriculture sectors Early detectionallows earlier management intervention allowing more livesto be saved overcoming food shortages problem indirectlyand preventing contaminated food products from reachingthe consumers Since a number of trial runs on the tool arealready ongoing one can hope that the diagnostic time andquality will be further improved in the near future

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This work was supported by ScienceFund Ministry of Sci-ence Technology and Innovation (MOSTI)Malaysia Projectno 02-01-04-SF1450

References

[1] F Coutlee L Bobo K Mayur R H Yolken and R P ViscidildquoImmunodetection of DNA with biotinylated RNA probes astudy of reactivity of a monoclonal antibody to DNA-RNAhybridsrdquoAnalytical Biochemistry vol 181 no 1 pp 96ndash105 1989

[2] A di Pinto V Terio P di Pinto V Colao and G TantilloldquoDetection of Vibrio parahaemolyticus in shellfish using poly-merase chain reaction-enzyme-linked immunosorbent assayrdquoLetters in AppliedMicrobiology vol 54 no 6 pp 494ndash498 2012

[3] M Pislak M Ocepek J Zabavnik-Piano and M PogacnikldquoPCR-ELISA as the method for improving the diagnosis ofparatuberculosisrdquo in ProceedIngs of the 7th International Collo-quium on Paratuberculosis pp 251ndash254 Bilbao Spain 2003

[4] L I Gomes L H D S Marques M J Enk M C de OliveiraP M Z Coelho and A Rabello ldquoDevelopment and evaluationof a sensitive PCR-ELISA system for detection of Schistosomainfection in fecesrdquo PLoS Neglected Tropical Diseases vol 4 no4 article e664 2010


[5] P Gill M Forouzandeh N Eshraghi M Ghalami M SafaandM R Noori-Daloii ldquoDetection of four 120573-thalassemia pointmutations in Iranians using a PCR-ELISA genotyping systemrdquoMolecular and Cellular Probes vol 22 no 2 pp 103ndash109 2008

[6] S L Mousavi J Salimiyan A K Rahgerdi J Amani SNazarian and H Ardestani ldquoA rapid and specific PCR-ELISAfor detecting Salmonella typhirdquo Iranian Journal of ClinicalInfectious Diseases vol 1 pp 113ndash119 2006

[7] J Comley ldquoELISA assays recent innovations take analyte detec-tion to new levelsrdquo Drug Discovery World 2012 httpwwwddw-onlinecomscreeningp191009-elisa -2020assays-recent-innovations-take-analyte-detection-to-new-levels-fall-12html

[8] JMenotti Y J F Garin PThulliez et al ldquoEvaluation of a new 51015840-nuclease real-time PCR assay targeting the Toxoplasma gondiiAF146527 genomic repeatrdquo Clinical Microbiology and Infectionvol 16 no 4 pp 363ndash368 2010

[9] F A Palermo P Cocci M Angeletti A Polzonetti-Magniand G Mosconi ldquoPCR-ELISA detection of estrogen receptor 120573mRNA expression and plasma vitellogenin induction in juve-nile sole (Solea solea) exposed to waterborne 4-nonylphenolrdquoChemosphere vol 86 no 9 pp 919ndash925 2012

[10] H H Aini A R OmarM Hair-Bejo and I Aini ldquoComparisonof SybrGreen I ELISA and conventional agarose gel-based PCRin the detection of infectious bursal disease virusrdquoMicrobiolog-ical Research vol 163 no 5 pp 556ndash563 2008

[11] L Cabrera J de Witte B Victor et al ldquoSpecific detection andidentification of African trypanosomes in bovine peripheralblood bymeans of a PCR-ELISA assayrdquoVeterinary Parasitologyvol 164 no 2ndash4 pp 111ndash117 2009

[12] A M Shamloul and A Hadidi ldquoSensitive detection ofpotato spindle tuber and temperate fruit tree viroids byreverse transcription-polymerase chain reaction-probe capturehybridizationrdquo Journal of Virological Methods vol 80 no 2 pp145ndash155 1999

[13] M Musiani S Venturoli G Gallinella and M Zerbini ldquoQuali-tative PCR-ELISA protocol for the detection and typing of viralgenomesrdquo Nature Protocols vol 2 no 10 pp 2502ndash2510 2007

[14] D J Seo H Tahk K B Lee et al ldquoDetecting hepatitis Evirus with a reverse transcription polymerase chain reactionenzyme-linked immunosorbent assayrdquo Food and EnvironmentalVirology vol 4 no 1 pp 14ndash20 2012

[15] D T Cosan A Soyocak A Basaran I Degirmenci H VGunes and FM Sahin ldquoEffects of various agents on DNA frag-mentation and telomerase enzyme activities in adenocarcinomacell linesrdquo Molecular Biology Reports vol 38 no 4 pp 2463ndash2469 2011

[16] N Raji M Sadeghizadeh K N Tafreshi and E JahanzadldquoDetection of human papillomavirus 18 in cervical cancersamples using PCR-ELISA (DIAPOPS)rdquo Iranian Journal ofMicrobiology vol 3 no 4 pp 177ndash182 2011

[17] O Lungu X W Sun T C Wright Jr A Ferenczy R MRichart and S Silverstein ldquoA polymerase chain reaction-enzyme-linked immunosorbent assay method for detectinghuman papillomavirus in cervical carcinomas and high-gradecervical cancer precursorsrdquo Obstetrics and Gynecology vol 85no 3 pp 337ndash342 1995

[18] H Tahk M H Lee K B Lee D S Cheon and C ChoildquoDevelopment of duplex RT-PCR-ELISA for the simultaneousdetection of hepatitis A virus and hepatitis E virusrdquo Journal ofVirological Methods vol 175 no 1 pp 137ndash140 2011

[19] H Weijia Z Jianfeng G Xingcheng and Y Weiwei ldquoEval-uation of PCR-ELISA for detection of HBV-DNA in semenrdquoChinese Journal of Andrology vol 24 no 12 pp 18ndash20 2010

[20] F B Wu H Q Ouyan X Y Tang and Z X Zhou ldquoDouble-antigen sandwich time-resolved immunofluorometric assayfor the detection of anti-hepatitis C virus total antibodieswith improved specificity and sensitivityrdquo Journal of MedicalMicrobiology vol 57 no 8 pp 947ndash953 2008

[21] H Tahk K B Lee M H Lee D J Seo D S Cheon and CChoi ldquoDevelopment of reverse transcriptase polymerase chainreaction enzyme-linked immunosorbent assay for the detectionof hepatitis A virus in vegetablesrdquo FoodControl vol 23 no 1 pp210ndash214 2012

[22] B Beifuss G Bezold P Gottlober et al ldquoDirect detection offive common dermatophyte species in clinical samples using arapid and sensitive 24-h PCR-ELISA technique open to protocoltransferrdquoMycoses vol 54 no 2 pp 137ndash145 2011

[23] I Hadrich C Mary F Makni et al ldquoComparison of PCR-ELISA and real-time PCR for invasive aspergillosis diagnosis inpatients with hematological malignanciesrdquo Medical Mycologyvol 49 no 5 pp 489ndash494 2011

[24] P Badiee P Kordbacheh A Alborzi et al ldquoStudy on invasivefungal infections in immunocompromised patients to presenta suitable early diagnostic procedurerdquo International Journal ofInfectious Diseases vol 13 no 1 pp 97ndash102 2009

[25] P Badiee P Kordbacheh A Alborzi M Zakernia and PHaddadi ldquoEarly detection of systemic Candidiasis in the wholeblood of patients with hematologic malignanciesrdquo JapaneseJournal of Infectious Diseases vol 62 no 1 pp 1ndash5 2009

[26] J Madic C Lecureuil F Dilasser et al ldquoScreening of food rawmaterials for the presence of Shiga toxin-producing EscherichiacoliO91H21rdquo Letters in Applied Microbiology vol 48 no 4 pp447ndash451 2009

[27] K Park K Lee K Baek et al ldquoApplication of a diagnosticmethod using reverse transcription-PCR ELISA for the diag-nosis of enteroviral infectionsrdquo Korean Journal of LaboratoryMedicine vol 29 no 6 pp 594ndash600 2009

[28] M St-Louis ldquoPCR-ELISA for high-throughput blood groupgenotypingrdquo Methods in Molecular Biology vol 496 pp 3ndash132009

[29] B E Gillespie A GMathew F ADraughon BM Jayarao andS P Oliver ldquoDetection of Salmonella enterica somatic groups C1and E1 by PCR-enzyme-linked Iimmunosorbent assayrdquo Journalof Food Protection vol 66 no 12 pp 2367ndash2370 2003

[30] Y Hong M E Berrang T Liu et al ldquoRapid detection ofCampylobacter coliC jejuni and Salmonella enterica onpoultrycarcasses by using PCR-enzyme-linked immunosorbent assayrdquoApplied and Environmental Microbiology vol 69 no 6 pp3492ndash3499 2003

[31] C Metzger-Boddien A Bostel and J Kehle ldquoAnDiaTecSalmonella sp PCR-ELISA for analysis of food samplesrdquo Journalof Food Protection vol 67 no 8 pp 1585ndash1590 2004

[32] S Perelle F Dilasser B Malorny J Grout J Hoorfar and PFach ldquoComparison of PCR-ELISA and LightCycler real-timePCR assays for detecting Salmonella spp in milk and meatsamplesrdquoMolecular and Cellular Probes vol 18 no 6 pp 409ndash420 2004

[33] H J Kim and J C Cho ldquoRapid and sensitive detection of Lis-teria monocytogenes using a PCR-enzyme-linked immunosor-bent assayrdquo Journal of Microbiology and Biotechnology vol 18no 11 pp 1858ndash1861 2008


[34] W Cao N Wang X Wang H Liu and Y Guo ldquoStudy onrapid detection techniques of PCR-ELISA for Listeria monocy-togenesrdquo Journal of Hygiene Research vol 38 no 6 pp 662ndash6662009

[35] P Daly T Collier and S Doyle ldquoPCR-ELISA detection ofEscherichia coli in milkrdquo Letters in AppliedMicrobiology vol 34no 3 pp 222ndash226 2002

[36] V K Gupta J Vohra V S Vihan and R Kumari ldquoPCR-ELISAfor diagnosis of brucellosis in goat milkrdquo Indian Journal ofAnimal Sciences vol 78 no 8 pp 797ndash800 2008

[37] T Kobets J Badalova I Grekov H Havelkova M Svobodovaand M Lipoldova ldquoLeishmania parasite detection and quan-tification using PCR-ELISArdquo Nature Protocols vol 5 no 6 pp1074ndash1080 2010

[38] A Alborzi B Pourabbas F Shahian J Mardaneh G RPouladfar andM Ziyaeyan ldquoDetection of Leishmania infantumkinetoplast DNA in the whole blood of asymptomatic indi-viduals by PCR-ELISA and comparison with other infectionmarkers in endemic areas southern IranrdquoTheAmerican Journalof Tropical Medicine and Hygiene vol 79 no 6 pp 839ndash8422008

[39] S F Phong M Hair-Bejo A R Omar and I Aini ldquoSequenceanalysis of Malaysian infectious bursal disease virus isolateand the use of reverse transcriptase nested polymerase chainreaction enzyme-linked immunosorbent assay for the detectionof VP2 hypervariable regionrdquo Avian Diseases vol 47 no 1 pp154ndash162 2003

[40] D Barlic-Maganja O Zorman-Rojs and J Grom ldquoDetection ofinfectious bursal disease virus in different lymphoid organs bysingle-step reverse transcription polymerase chain reaction andmicroplate hybridization assayrdquo Journal of VeterinaryDiagnosticInvestigation vol 14 no 3 pp 243ndash246 2002

[41] G Nolasco Z Sequeira C Soares A Mansinho A MBailey and C L Niblett ldquoAsymmetric PCR ELISA increasedsensitivity and reduced costs for the detection of plant virusesrdquoEuropean Journal of Plant Pathology vol 108 no 4 pp 293ndash2982002

[42] G Nolasco C Santos G Silva and F Fonseca ldquoDevelopmentof an asymmetric PCR-ELISA typing method for citrus tristezavirus based on the coat protein generdquo Journal of VirologicalMethods vol 155 no 2 pp 97ndash108 2009

[43] R Weekes I Barker and K R Wood ldquoAn RT-PCR test forthe detection of tomato spotted wilt tospovirus incorporatingimmunocapture and colorimetric estimationrdquo Journal of Phy-topathology vol 144 no 11-12 pp 575ndash580 1996

[44] P Gouveia M T Santos J E Eiras-Dias and G NolascoldquoFive phylogenetic groups identified in the coat protein gene ofgrapevine leafroll-associated virus 3 obtained from Portuguesegrapevine varietiesrdquoArchives of Virology vol 156 no 3 pp 413ndash420 2011

[45] A Rowhani L Biardi G Routh S D Daubert and D AGolino ldquoDevelopment of a sensitive colorimetric-PCR assay fordetection of viruses in woody plantsrdquo Plant Disease vol 82 no8 pp 880ndash884 1998

[46] M Soheili M R Nejadmoghaddam M Babashamsi JGhasemi and M Jeddi Tehrani ldquoDetection of Legionella pneu-mophila by PCR-ELISA method in industrial cooling towerwaterrdquo Pakistan Journal of Biological Sciences vol 10 no 22 pp4015ndash4021 2007

[47] J T Kuo C Y Cheng H H Huang C F Tsao and Y CChung ldquoA rapid method for the detection of representative

coliforms inwater samples polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA)rdquo Journal of Indus-trial Microbiology and Biotechnology vol 37 no 3 pp 237ndash2442010

[48] M Ziyaeyan F Sabahi A Alborzi et al ldquoQuantification ofhuman cytomegalovirus DNA by a new capture hybrid poly-merase chain reaction enzyme-linked immunosorbent assay inplasma andperipheral bloodmononuclear cells of bonemarrowtransplant recipientsrdquo Experimental and Clinical Transplanta-tion vol 6 no 4 pp 294ndash300 2008

[49] H Li F Hua C Zhao G Liu and Q Zhou ldquoDiagnoticvalue of the combined determination of telomerase activityin induced sputum pleural effusion and fiberobronchoscopicbiopsy samples in lung cancerrdquo Chinese Journal of Lung Cancervol 13 no 2 pp 128ndash131 2010

[50] A Avlami S Bekris G Ganteris et al ldquoDetection of metallo-120573-lactamase genes in clinical specimens by a commercialmultiplex PCR systemrdquo Journal of Microbiological Methods vol83 no 2 pp 185ndash187 2010

[51] W Puppe J A IWeigl GAron et al ldquoEvaluation of amultiplexreverse transcriptase PCR ELISA for the detection of ninerespiratory tract pathogensrdquo Journal of Clinical Virology vol 30no 2 pp 165ndash174 2004

[52] H Schutzle J Weigl W Puppe J Forster and R BernerldquoDiagnostic performance of a rapid antigen test for RSV incomparison with a 19-valent multiplex RT-PCR ELISA in chil-dren with acute respiratory tract infectionsrdquo European Journalof Pediatrics vol 167 no 7 pp 745ndash749 2008

[53] B Chaharaein A R Omar I Aini K Yusoff and S S HassanldquoDetection ofH5H7 andH9 subtypes of avian influenza virusesby multiplex reverse transcription-polymerase chain reactionrdquoMicrobiological Research vol 164 no 2 pp 174ndash179 2009

[54] N Santos S Honma M D C S T Timenetsky et al ldquoDevel-opment of amicrotiter plate hybridization-based PCR-enzyme-linked immunosorbent assay for identification of clinicallyrelevant human group A rotavirus G and P genotypesrdquo Journalof Clinical Microbiology vol 46 no 2 pp 462ndash469 2008

[55] R S W Tsang C M Tsai A M Henderson et al ldquoImmuno-chemical studies and genetic background of two Neisseriameningitidis isolates expressing unusual capsule polysaccharideantigens with specificities of both serogroup Y and W135rdquoCanadian Journal of Microbiology vol 54 no 3 pp 229ndash2342008

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Microbiology


Amplification

5998400

3998400

DNA template

PCR amplification usingDIG-labelled dUTP

DIG

Detection

Immobilization

Microplate

Strep

Biotin

Biotin-labelledprobe

DIG-labelledmRNA

Anti-DIG peroxidase

ABTS ABTS

Substrate-enzymereaction to create

colour change

DIG DIG

DIG DIG DIG

DIGDIGDIG

DIG DIG DIG

Figure 1 Illustration of the 3-step PCR-ELISA method (i) amplification of the gene of interest using PCR in the presence of DIG-dUTPwhich is then bound to specific probes (ii) immobilization of the gene of interest to the microplate through strong affinity of avidin-biotininteraction followed by (iii) detection of biotinylatedDNAusing an anti-DIG-peroxidase conjugate with substrate ABTS to form a blue-greencolor reaction that is both visible and measured using a spectrophotometer

To do so the amplicons can be detected using an anti-DIG-peroxidase conjugate through the substrate 221015840-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) These will develop ablue-green color reaction that is both visible and measuredusing a spectrophotometer (Figure 1) [3] Another methodof PCR-ELISA detection includes the use of fluoresceinprobe where detection includes the use of antifluoresceinantibodies conjugated to horseradish peroxidase to detectthe hybridized fluorescein-labelled oligonucleotide probe[4]

2 Comparisons of PCR-ELISA with OtherPCR-Based Molecular Approaches

Since the introduction of this tool various studies havebeen carried out to compare the performance of PCR-ELISAwith other tools Many agreed that the detection of DIG-labelled products by microwell capture hybridization assaymakes PCR-ELISA a more sensitive tool than agarose gelelectrophoresis analysis because the specific hybridizationand enzymatic colourization increase the positive signal ofbiotin-labelled probe-bound PCR products

The PCR amplicons are analyzed using a colorimetricassay thus not only is there reduced risk on the use ofmutagen-staining materials and significant reduction of pos-sible DNA contamination but also it allows the method toserve as a semiquantitative tool [5] As this detection usesgene-specific probes for detection the specificity of the toolis very prominent [2 6] Not only can samples with that

particular gene be detected but also they can be quantifiedbased on colour intensity The presence of higher colourintensity indicates that more probe was bound to the specificgene sequence forming a hybrid complex that was laterboundby a peroxidase-conjugated anti-DIGantibody and thecolorimetric peroxidase substrate ABTS allowing detectionWhilst PCR-ELISA cannot provide an accurate estimation ofthe actual gene of interest that is present compared to real-time PCR (qPCR) it provides a quick summary of whethera particular substrate is high or low at a particular time ascolorimetric detection is in direct proportion to the numberof the intended gene of interest

Another main attraction of PCR-ELISA is that the assayallows large-scale screening to be done using only standardlaboratory equipmentmaking it suitable to be used in clinicallaboratories This should serve as another incentive forlaboratories with fewer resources as a survey done by Comley[7] showed that respondents do not turn to fully automatedequipment for ELISA despite its availability possibly due tothe high costs of purchasing and maintaining the equipmentLast but not least the overall analytical time of this assay isalso much shorter than conventional PCRmethod making ita promising tool for future uses especially when dealing witha large sample size

With new discoveries and new invention molecular biol-ogy tools need to be continuously improved and developedfor faster and more efficient results Each new technologythat was developed has its pros and cons compared to othertechnologies If the study is about an unknown gene thenconventional PCR using agarose gel detection would be the























5 Summary




Acknowledgment


References


































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology























5 Summary




Acknowledgment


References


































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

review article application of pcr-elisa in molecular diagnosis · review article application of...

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