and opisthorchis viverrini using real-time pcr and high

Research ArticleRapid Detection and Differentiation ofClonorchis sinensis and Opisthorchis viverrini Using Real-TimePCR and High Resolution Melting Analysis

Xian-Quan Cai12 Hai-Qiong Yu3 Rong Li2 Qiao-Yun Yue2 Guo-Hua Liu1

Jian-Shan Bai4 Yan Deng4 De-Yi Qiu2 and Xing-Quan Zhu1

1 State Key Laboratory of Veterinary Etiological Biology Key Laboratory of Veterinary Parasitology of Gansu ProvinceLanzhou Veterinary Research Institute Chinese Academy of Agricultural Sciences Lanzhou Gansu Province 730046 China

2 Technical Center Zhongshan Entry-Exit Inspection and Quarantine Bureau Zhongshan Guangdong Province 528403 China3 Technical Center Guangdong Entry-Exit Inspection and Quarantine Bureau Guangzhou Guangdong Province 510630 China4Guangzhou Airport Entry-Exit Inspection and Quarantine Bureau Guangzhou Guangdong Province 510470 China

Correspondence should be addressed to Xing-Quan Zhu zhuxingquancaascn

Received 4 March 2014 Revised 3 August 2014 Accepted 3 August 2014 Published 19 October 2014

Academic Editor Rafael Toledo

Copyright copy 2014 Xian-Quan Cai et alThis 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

Clonorchis sinensis and Opisthorchis viverrini are both important fish-borne pathogens causing serious public health problem inAsia The present study developed an assay integrating real-time PCR and high resolution melting (HRM) analysis for the specificdetection and rapid identification of C sinensis and O viverrini Primers targeting COX1 gene were highly specific for these liverflukes as evidenced by the negative amplification of closely related trematodes Assays using genomic DNA extracted from the twoflukes yielded specific amplification and their identity was confirmed by sequencing having the accuracy of 100 in reference toconventional methods The assay was proved to be highly sensitive with a detection limit below 1 pg of purified genomic DNA 5EPG or 1 metacercaria of C sinensis Moreover C sinensis and O viverrini were able to be differentiated by their HRM profilesThe method can reduce labor of microscopic examination and the contamination of agarose electrophoresis Moreover it candifferentiate these two flukes which are difficult to be distinguished using other methods The established method provides analternative tool for rapid simple and duplex detection of C sinensis and O viverrini

1 Introduction

Opisthorchis viverrini and Clonorchis sinensis are patholog-ically important members of the family OpisthorchiidaeChronic infections with these liver flukes closely associatewith the development of the bile duct cancer (cholan-giocarcinoma) and the liver cancer (hepatocarcinoma) inhumans [1] In a recent survey in Chinese endemic areasthe overall prevalence of C sinensis infection was 058in 356629 residents from 688 sampled pilot sites in China[2] Opisthorchiasis caused by O viverrini is considered animportant food-borne parasitic disease in Southeast Asiaincluding Thailand Lao PDR Vietnam and Cambodiaapproximately 673 million people are at risk of the infection

[3] Humans get infected by ingestion of undercooked orpickled freshwater fish containing metacercariae of O viver-rini and C sinensis which are classified by the InternationalAgency for Research on Cancer as a group 1 biologicalcarcinogen in 2009 [4]

C sinensis and O viverrini have similar life cycles loca-tions pathogenicity and theirmorphologies in particular theforms of their eggs and metacercariae differ only slightly [5]However the ability to differentiate the species of liver andminute intestinal flukes is important from both a clinicaland epidemiological perspective The frequency and typesof pathology and clinical diseases among C sinensis and Oviverrini seem to differ for example cholelithiasis is one ofthe most serious complications of clonorchiasis but a rare

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 893981 6 pageshttpdxdoiorg1011552014893981

2 The Scientific World Journal

complication of opisthorchiasis [6] Although both flukesare implicated as predisposing factors for cholangiocarci-noma this is more frequent with O viverrini From anepidemiological perspective C sinensis will make controlmore challenging

A number of methods have been developed to detectthese two species such as direct microscopy ELISA [7]conventional PCR [8] and loop-mediated isothermal ampli-fication (LAMP) [9] There are however many drawbacksof these methods For example microscopic examinationis cumbersome and time consuming requiring experiencedlaboratory technicians and often it is difficult to distin-guish eggs from those of closely related heterophyids whichhave similar egg morphologies Eggs can therefore onlybe characterised as ldquoOpisthorchisClonorchis-likerdquo eggs [10]Crude or recombinant antigens have been evaluated forimmunodiagnosis however the specificity and sensitivityof these methods are still in great need of improvement[11 12] Both conventional PCR and LAMP have high riskof contamination Moreover even with expensive probelabeled real-time PCR the two liver flukes are difficult to bedistinguished [13]

Recently high resolution melting (HRM) for fast high-throughput analysis of many pathogens has been developedfor example variation scanning [14] species determination[15] genotyping [16] and even the identification of recent andnonrecent HIV infections [17] The real-time PCR platformwith HRM is a single-step closed tube reduces turnaroundtime of the assay reported here to almost 1 h eliminatesthe risk of contamination and saves expense These featuresmake it advantageous for use in laboratories Here wedeveloped a real-time PCR assay coupled with HRM analysisfor rapid identification and differentiation of C sinensis andO viverrini

2 Materials and Methods

21 Samples Adult worms of C sinensis were collected fromnaturally infected cats in Guangdong Province Adult wormsof O viverrini were kindly provided by Professor Sung-Jong Hong Department of Medical Environmental BiologyChung-Ang University College of Medicine Republic ofKorea which were originated from the Lao PDR Severalclosely related trematodes infecting humans were included asldquoheterologousrdquo control samples for assessing the specificity ofthe real-time PCR assay namely Fasciola hepatica Fasciolagigantica Schistosoma mansoni and Schistosoma japonicumAll parasitematerials were preserved in 70 ethanol and keptat minus20∘Cuntil the extraction of genomicDNAMetacercariaeof C sinensis were collected from the fish muscles essentiallyaccording to a previously reported method [9] Fecal sampleswere obtained from the residents in Guangdong ProvinceChina The fecal samples were examined three times bythe Kato-Katz thick smear using nylon screens and plastictemplates

22 Genomic DNA Extraction Total genomic DNA wasextracted from adult worms using the commercial QiAamp

DNA extraction kit (Qiagen) In the case of adult wormsonly a single specimen was used in each DNA extractionBriefly the sample was enzymatically digested in 180120583L ofa lysis solution (ATL buffer-Qiagen) then 20 120583L proteinase-K (50 gmL) was added and incubated at 56∘C for 2-3 h withbrief vortexing every 30min For eggs the incubation timewas set 1-2 h longer The mixture after adding 200120583L bufferAL (Qiagen) containing guanidine hydrochloride and 4120583LRNase A (100mgmL) andmixing by pulse-vortexing for 15 swas further incubated at 70∘C for 10min Thereafter 200120583Lof ethanol (96ndash100) was added and mixed by vortexingfor 15ndash20 s The contents were then loaded onto a QIAampSpin Column for DNA binding and spin down for 1minThecolumnwith the DNA bound was washed several times usingsolutions (AW1 AW2 buffersmdashQiagen) provided accordingto manufacturerrsquos instruction Finally the genomic DNA waseluted in 50 120583L elution buffer (AE) and stored at minus20∘Cuntil use The DNA from fecal samples was extracted usingQIAampDNAStoolMini Kit according to themanufacturerrsquosinstructions and the DNA from metacercariae in fish tissuewas extracted according to a previous description [9 13]Theconcentration of DNA samples was estimated using ThermoNanodrop 1000

23 Primers Real-Time PCR and HRM Analysis A pairof primers targeting the COX1 gene of C sinensis and Oviverrini (COX1e-F 51015840-GGTAGGGTGGTTTGAGC-31015840 andCOX1e-R 51015840-TCATAGTAACCGAGCTAAA-31015840) was synthe-sized by Takara Biotechnology (Dalian China) Real-timePCR amplification was performed in Lightcycler 480 (RocheUSA) The total reaction volume was 20 120583L which consistedof 1x Fast-Plus EvaGreen qPCR Master Mix (Bio-rad USA)reaction buffer containing dNTPs MgCl

2 fast-activating

chemically modified hot start enzyme Cheetah Taq 03 120583Mof each primer and 1 120583L genomic DNA as template

The PCR was carried out with initiation at 95∘C for 60 sthen 45 cycles of denaturation at 95∘C for 10 s annealingat 59∘C for 10 s and extension at 72∘C for 20 s When PCRamplification was completed HRM analysis was performedby lowering the temperature to 60∘C for 5min followedby increasing the temperature ramping from 60∘ to 95∘at 011∘Cs 25 acquisitions∘C In this process the PCRamplicons were allowed to denature and reanneal in fluo-rescence with changes in temperature (119889119865119889119879) The HRMprofile was then analyzed using HRM analysis software asreported previously [15]The normalizedmelting curves wereanalyzed and clustered samples into different groups withsimilar melting profiles Hence the difference in the shapeof the ldquonormalized melting curverdquo will help cluster samplesinto different species and the samples can be detected andgrouped at the same time

3 Results

31 Specificity of the Detection Assay and Confirmationof Amplicon Identity The specificity of the primers wasdetermined by performing PCR using pure genomic DNAfrom F hepatica F gigantica S mansoni and S japonicum

The Scientific World Journal 3

A BLAST search of the chosen primer and probe sequencesresulted in a hit of the target sequence in C sinensis and Oviverrini suggesting the specificity of the primers Moreoverno fluorescence signal was detected from the ldquoheterologouscontrol samplesrdquo as mentioned above In all 57 C sinensissamples including 36worms 11 fish samples 10 fecal samplesand 10 O viverrini worms get positive results which wasconfirmed by microscopic method while 31 uninfected fishor fecal samples appear negative (Figure 1(a)) To ensure theaccuracy of the method all amplifications were sequencedand proved highly homologous to corresponding sequences

32 Detection Limit and Correlation between Cp and DilutedDNA In order to evaluate the detection limit and correlationof the real-time PCR assay the 10-fold serial dilutions ofa genomic DNA were prepared The DNA was extractedusing QiAamp DNA extraction kit (Qiagen) and quantifiedby spectrophotometry The 10-fold diluted DNA was per-formed and good result acquired The formula of Cp valueand template concentration (10 ng120583L to 1 pg120583L) is 119910 =minus18603 log(119909) + 22815(1198772 = 09978) when tested with Csinensis (Figure 2(a)) while it is 119910 = minus15718 log(119909) + 22203(119877

2

= 09938) when tested with O viverrini (Figure 2(b))It seems that the sensitivity ofO viverrini approaches the

sensitivity of C sinensis which are shown in Figures 2(c) and2(d) The detection limit of the assay was 1 pg for O viverrinigenomic DNA when considering 40 cycles as the cutoff Todetermine the detection limit in fecal samples and fish tissue1 2 5 and 10 C sinensis eggs or metacercariae were spikedinto samples which were proved negative by microscopicmethod Each mixed sample was separately used for DNAextraction as described earlier Finally we concluded thatthe detection limit was 5 EPG for fecal samples and 1 MPG(metacercariae per gramfish filet) for fish samples which wasconsistent with that of a previous study [13]

33 HRM Analysis The sequence difference between theforward and reverse primers will bring different meltingtemperature (119879

119898) values and normalized melting curves

Constant HRM profiles with distinct 119879119898

peaks were per-sistently obtained for all positive samples As shown inFigure 1(b) there were two kinds of characteristic profilesThe amplification product from all 57 C sinensis samples hada 119879119898of 7805 plusmn 007∘C while the amplification product from

10 O viverrini samples had an average 8089 plusmn 006∘C Asshown in Figure 1(c) there were two obvious groups onerepresentedC sinensis and the other representedO viverriniTo ensure the accuracy of themethod the amplified productswere sequenced in both directionsThe results showed that allsequences were uniform with the HRM analysis result

TheHRM analysis with different template concentrations(10 ng120583L to 1 pg120583L) of the template appeared to be reliablewhile the profile is unsatisfactory when the template concen-trations were le1 pg The melting peaks were obtained fromeach dilution (10 ng120583L to 1 pg120583L) and it shows that 119879

119898of C

sinensis was 7897 plusmn 011 while 119879119898of O viverrini was 8092

plusmn 008∘C (Figure 2(e)) All the dilutions were divided into

5 10 15 20 25 30 35 40 45

0744774414744217442874435744427444974456744637447074477744

Amplification curves

Cycles

C sinensis and O viverrini

Fluo

resc

ence

(465

ndash510

)

(a)

Temperature (∘C)70 72 74 76 78 80 8482 86 88 90 92 94

034733476347934712347153471834721347243472734730347

Melting peaks7905 plusmn 007∘C

8089 plusmn 006∘C

1 2minus(dd

T) fl

uore

scen

ce (4

65

ndash510

)

(b)

C sinensis O viverrini

75 76 77 78 79 80 81 82 83 84

Normalized melting curves

Temperature (∘C)

Rela

tive s

igna

l (

)

0000100002000030000400005000060000700008000090000100000

(c)

Figure 1 Real-time PCR and HRM analysis for all samples (a)Real-time PCR amplification for all samples All the heterologousand blank control had no amplification curve before 40 cycles (b)Melting peaks of liver flukes 1 C sinensis 119879

119898

7905 plusmn 007∘C 2 Oviverrini 119879

119898

8089 plusmn 006∘C (c) Normalized melting curves for twoliver flukes

two obvious clusters one represented differently diluted Csinensis and the other represented O viverrini (Figure 2(f))

4 Discussion

Diagnosis of trematode infection is very important foreffective treatment and controlling the spread of infections


15

20

25

30

35

40

minus10 minus9 minus8 minus7 minus6 minus5 minus4 minus3 minus2 minus1 0 1 2

y = minus15718x + 22203

R2 = 09978

Cros

sing

poin

t

Log concentration (ng120583L)

(a)

15

20

25

30

35

40


y = minus18603x + 22815

R2 = 09938

Cros

sing

poin

t


(b)


Cycles5 10 15 20 25 30 35 40 45

10ng 10ng01ng 10pg1pg01pg

minus0392660813608206082760834608416084860855608626086960876608

Fluo

resc

ence

(465

ndash510

)

(c)


Cycles5 10 15 20 25 30 35 40 45

10ng 10ng 01ng10pg1pg 01pg

370110701177012470131701387014570152701597016670173701

Fluo

resc

ence

(465

ndash510

)

(d)

Temperature (∘C)70 72 74 76 78 80 8482 86 88 90 92 94

Melting peaks7897 plusmn 011∘C 8092 plusmn 008∘C

1 2

minus022522754775727597751227514775172751977522275247752727529775

minus(dd

T) fl

uore

scen

ce (4

65

ndash510

)

(e)


757473 76 77 78 79 80 81 82 83 84 85


Temperature (∘C)

Rela

tive s

igna

l (

)

0000100002000030000400005000060000700008000090000100000

(f)

Figure 2 Real-time PCR and HRM analysis of serial diluted genomic DNA (a) A linear regression of the data providing a formula of119910 = minus18603 log(119909) + 22815 (1198772 = 09978) template dilutions 10 ng120583L to 1 pg120583L of C sinensis (b) A linear regression of the data providinga formula of 119910 = minus15718 log(119909) + 22203 (1198772 = 09938) template dilutions 10 ng120583L to 1 pg120583L of O viverrini (c) Amplification curves ofdiluted genomic DNA of C sinensis 1ndash6 dilutions 10 ng120583L to 1 pg120583L (d) Amplification curves of diluted genomic DNA of O viverrini1ndash6 dilutions 10 ng120583L to 1 pg120583L (e) Melting peaks of amplicon of diluted genomic DNA from two liver flukes (10 ng120583L to 1 pg120583L) 1 Csinensis 7897 plusmn 011∘C 2O viverrini 8092 plusmn 008∘C (f) Normalized melting curves of diluted genomic DNA from two liver flukes (10 ng120583Lto 1 pg120583L)

Since more and more travelers visited endemic countriesan accurate differential diagnostic method for humanclonorchiasis and opisthorchiasis should be developed [1 2]Here we developed an unlabelled real-time PCR with HRMassay targeting the COX1 for the two important liver flukesThe differentiation may not be that important in areas whereonly one species of the liver flukes is prevalent but it isnecessary in endemic areas where C sinensis and O viverriniare present Accurate laboratory tests will help diagnosesuspected patients

The method established in the present study has anaccuracy of 100 in reference to microscopic methodsMoreover the detection limit of the method was below 1 pgof purified genomic DNA 5 EPG or 1 metacercaria whichwas equal to probed real-time PCR [13] and superior toconventional PCR [8] Real-time PCR using SYBR Green hasalso been developed targeting many pathogens but it canonly detect gross differences between amplicons generatedby real-time PCR with nonsaturating dyes which wouldinhibit the PCR reaction EvaGreen has been proven to


be superior to SYBR Green with high reaction efficiencies[18] The instrumentation for HRM analysis has also beenimproved with a high rate of data acquisition ideal opticstight temperature control and adequate analysis software theaccuracy of the dissociation versus temperature (ie melting)curve is as sensitive as 001∘C so single base can also bedifferentiated [19]

O viverrini C sinensis and Opisthorchis felineus arethe three most medically important species in the familyOpisthorchiidae [3] Now we still could not validate thedetection for the three liver flukes in the same time becauseunfortunately both the samples and the genetic informationof O felineus are absent However once the sequence differ-ence between three species was proved enough a multiplexreal-time PCR for three liver flukes will be developed in thenear future

A previous molecular method targeting the secondinternal transcribed spacer (ITS2) of rDNA showed goodsensitivity but O viverrini and C sinensis were not able to bedifferentiated because of high homology [20] Here we havedesigned more than 20 pairs of primers targeting differentgenes and the result shows that COX1 is superior to 16SrRNA and ITS rDNA when used for HRM analysis Anotheradvantage of mitochondrial DNA (mtDNA) rather thannuclear rDNA is that the mitochondrial genome is present inhundreds or thousands of copies per cell A further researchshows that the length of amplicons smaller than 100 bpis preferable because shorter amplicon size increases thedifference in signal All these conclusions are in accordancewith previous research

5 Conclusion

The present study established a method based on real-timePCR and HRM analysis for the rapid identification anddifferentiation of C sinensis and O viverrini simultaneouslyIt offers a potential means for detection of these flukesin the intermediate hosts Moreover it would also help indetermining the worm burden in fish or infected individualsin endemic areas and would therefore be a useful tool forepidemiological surveys

Conflict of Interests

The authors declare that there is no conflict of interests in thispaper

Acknowledgments

Project support was provided by the Science Fund forCreative Research Groups of Gansu Province (Grant no1210RJIA006) the National Science and Technology SupportProgram (Grant no 2012BAK11B05) and the Science amp Tech-nology Program of Zhongshan (Grant no 20123A299)

References

[1] B Fried A Reddy and D Mayer ldquoHelminths in humancarcinogenesisrdquoCancer Letters vol 305 no 2 pp 239ndash249 2011

[2] L Xue-Ming C Ying-Dan Y Ouyang Z Hong-Man L Ruiand M Wei ldquoOverview of human clonorchiasis sinensis inChinardquo Southeast Asian Journal of Tropical Medicine and PublicHealth vol 42 no 2 pp 248ndash254 2011

[3] B Sripa S Kaewkes P M Intapan W Maleewong andP J Brindley ldquoFood-borne trematodiases in Southeast Asiaepidemiology pathology clinical manifestation and controlrdquoAdvances in Parasitology vol 72 pp 305ndash350 2010

[4] S Hong and Y Fang ldquoClonorchis sinensis and clonorchiasis anupdaterdquo Parasitology International vol 61 no 1 pp 17ndash24 2012

[5] N D Young B E Campbell R S Hall et al ldquoUnlockingthe transcriptomes of two carcinogenic parasites Clonorchissinensis and Opisthorchis viverrinirdquo PLoS Neglected TropicalDiseases vol 4 no 6 article e719 2010

[6] L A Marcos A Terashima and E Gotuzzo ldquoUpdate on hepa-tobiliary flukes fascioliasis opisthorchiasis and clonorchiasisrdquoCurrent Opinion in Infectious Diseases vol 21 no 5 pp 523ndash530 2008

[7] S Sirisinha D Sahassananda D Bunnag and H J RimldquoImmunological analysis of Opisthorchis and Clonorchis anti-gensrdquo Journal of Helminthology vol 64 no 2 pp 133ndash138 1990

[8] R J Traub JMacaranasMMungthin et al ldquoA new PCR-basedapproach indicates the range of Clonorchis sinensis now extendsto Central Thailandrdquo PLoS Neglected Tropical Diseases vol 3no 1 article e367 2009

[9] X Q Cai M J Xu Y HWang et al ldquoSensitive and rapid detec-tion of Clonorchis sinensis infection in fish by loop-mediatedisothermal amplification (LAMP)rdquo Parasitology Research vol106 no 6 pp 1379ndash1383 2010

[10] R K Schuster ldquoOpisthorchiidosismdasha reviewrdquo InfectiousDisordersmdashDrug Targets vol 10 no 5 pp 402ndash415 2010

[11] I Nagano F Pei Z Wu et al ldquoMolecular expression of a cys-teine proteinase of Clonorchis sinensis and its application to anenzyme-linked immunosorbent assay for immunodiagnosis ofclonorchiasisrdquo Clinical and Diagnostic Laboratory Immunologyvol 11 no 2 pp 411ndash416 2004

[12] V Eursitthichai V Viyanant S Tesana P Sithithaworn NKosa and R Grams ldquoOpisthorchis viverrini evaluation of 28kDa glutathione S-transferase as diagnostic tool in humanopisthorchiasisrdquo Acta Tropica vol 114 no 2 pp 76ndash80 2010

[13] X-Q Cai H-Q Yu J-S Bai et al ldquoDevelopment of a TaqManbased real-time PCR assay for detection of Clonorchis sinensisDNA in human stool samples and fishesrdquo Parasitology Interna-tional vol 61 no 1 pp 183ndash186 2012

[14] P H Nissen S E Christensen S A Ladefoged K BrixenL Heickendorff and L Mosekilde ldquoIdentification of rare andfrequent variants of the CASR gene by high-resolutionmeltingrdquoClinica Chimica Acta vol 413 no 5-6 pp 605ndash611 2012

[15] M R Zianni M R Nikbakhtzadeh B T Jackson J Panescuand W A Foster ldquoRapid discrimination between Anophelesgambiae ss and Anopheles arabiensis by High-Resolution Melt(HRM) analysisrdquo Journal of Biomolecular Techniques vol 24 no1 pp 1ndash7 2013

[16] E Tajiri-Utagawa M Hara K Takahashi M Watanabe andT Wakita ldquoDevelopment of a rapid high-throughput methodfor high-resolution melting analysis for routine detection andgenotyping of norovirusesrdquo Journal of ClinicalMicrobiology vol47 no 2 pp 435ndash440 2009

[17] M M Cousins D Swan C A Magaret D R Hoover and SH Eshleman ldquonalysis of HIV using a High Resolution Melting(HRM) diversity assay automation of HRM data analysis


enhances the utility of the assay for analysis of HIV incidencerdquoPLoS ONE vol 7 no 12 Article ID e51359 2012

[18] A C Eischeid ldquoSYTO dyes and EvaGreen outperform SYBRGreen in real-timePCRrdquoBMCResearchNotes vol 4 article 2632011

[19] D Jin Y Luo Z Zhang et al ldquoRapid molecular identificationof Listeria species by use of real-time PCR and high-resolutionmelting analysisrdquo FEMSMicrobiology Letters vol 330 no 1 pp72ndash80 2012

[20] E-M Kim J J Verweij A Jalili et al ldquoDetection of Clonorchissinensis in stool samples using real-time PCRrdquo Annals ofTropical Medicine and Parasitology vol 103 no 6 pp 513ndash5182009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


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Virolog y

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Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


complication of opisthorchiasis [6] Although both flukesare implicated as predisposing factors for cholangiocarci-noma this is more frequent with O viverrini From anepidemiological perspective C sinensis will make controlmore challenging

A number of methods have been developed to detectthese two species such as direct microscopy ELISA [7]conventional PCR [8] and loop-mediated isothermal ampli-fication (LAMP) [9] There are however many drawbacksof these methods For example microscopic examinationis cumbersome and time consuming requiring experiencedlaboratory technicians and often it is difficult to distin-guish eggs from those of closely related heterophyids whichhave similar egg morphologies Eggs can therefore onlybe characterised as ldquoOpisthorchisClonorchis-likerdquo eggs [10]Crude or recombinant antigens have been evaluated forimmunodiagnosis however the specificity and sensitivityof these methods are still in great need of improvement[11 12] Both conventional PCR and LAMP have high riskof contamination Moreover even with expensive probelabeled real-time PCR the two liver flukes are difficult to bedistinguished [13]

Recently high resolution melting (HRM) for fast high-throughput analysis of many pathogens has been developedfor example variation scanning [14] species determination[15] genotyping [16] and even the identification of recent andnonrecent HIV infections [17] The real-time PCR platformwith HRM is a single-step closed tube reduces turnaroundtime of the assay reported here to almost 1 h eliminatesthe risk of contamination and saves expense These featuresmake it advantageous for use in laboratories Here wedeveloped a real-time PCR assay coupled with HRM analysisfor rapid identification and differentiation of C sinensis andO viverrini

2 Materials and Methods

21 Samples Adult worms of C sinensis were collected fromnaturally infected cats in Guangdong Province Adult wormsof O viverrini were kindly provided by Professor Sung-Jong Hong Department of Medical Environmental BiologyChung-Ang University College of Medicine Republic ofKorea which were originated from the Lao PDR Severalclosely related trematodes infecting humans were included asldquoheterologousrdquo control samples for assessing the specificity ofthe real-time PCR assay namely Fasciola hepatica Fasciolagigantica Schistosoma mansoni and Schistosoma japonicumAll parasitematerials were preserved in 70 ethanol and keptat minus20∘Cuntil the extraction of genomicDNAMetacercariaeof C sinensis were collected from the fish muscles essentiallyaccording to a previously reported method [9] Fecal sampleswere obtained from the residents in Guangdong ProvinceChina The fecal samples were examined three times bythe Kato-Katz thick smear using nylon screens and plastictemplates

22 Genomic DNA Extraction Total genomic DNA wasextracted from adult worms using the commercial QiAamp

DNA extraction kit (Qiagen) In the case of adult wormsonly a single specimen was used in each DNA extractionBriefly the sample was enzymatically digested in 180120583L ofa lysis solution (ATL buffer-Qiagen) then 20 120583L proteinase-K (50 gmL) was added and incubated at 56∘C for 2-3 h withbrief vortexing every 30min For eggs the incubation timewas set 1-2 h longer The mixture after adding 200120583L bufferAL (Qiagen) containing guanidine hydrochloride and 4120583LRNase A (100mgmL) andmixing by pulse-vortexing for 15 swas further incubated at 70∘C for 10min Thereafter 200120583Lof ethanol (96ndash100) was added and mixed by vortexingfor 15ndash20 s The contents were then loaded onto a QIAampSpin Column for DNA binding and spin down for 1minThecolumnwith the DNA bound was washed several times usingsolutions (AW1 AW2 buffersmdashQiagen) provided accordingto manufacturerrsquos instruction Finally the genomic DNA waseluted in 50 120583L elution buffer (AE) and stored at minus20∘Cuntil use The DNA from fecal samples was extracted usingQIAampDNAStoolMini Kit according to themanufacturerrsquosinstructions and the DNA from metacercariae in fish tissuewas extracted according to a previous description [9 13]Theconcentration of DNA samples was estimated using ThermoNanodrop 1000

23 Primers Real-Time PCR and HRM Analysis A pairof primers targeting the COX1 gene of C sinensis and Oviverrini (COX1e-F 51015840-GGTAGGGTGGTTTGAGC-31015840 andCOX1e-R 51015840-TCATAGTAACCGAGCTAAA-31015840) was synthe-sized by Takara Biotechnology (Dalian China) Real-timePCR amplification was performed in Lightcycler 480 (RocheUSA) The total reaction volume was 20 120583L which consistedof 1x Fast-Plus EvaGreen qPCR Master Mix (Bio-rad USA)reaction buffer containing dNTPs MgCl

2 fast-activating

chemically modified hot start enzyme Cheetah Taq 03 120583Mof each primer and 1 120583L genomic DNA as template

The PCR was carried out with initiation at 95∘C for 60 sthen 45 cycles of denaturation at 95∘C for 10 s annealingat 59∘C for 10 s and extension at 72∘C for 20 s When PCRamplification was completed HRM analysis was performedby lowering the temperature to 60∘C for 5min followedby increasing the temperature ramping from 60∘ to 95∘at 011∘Cs 25 acquisitions∘C In this process the PCRamplicons were allowed to denature and reanneal in fluo-rescence with changes in temperature (119889119865119889119879) The HRMprofile was then analyzed using HRM analysis software asreported previously [15]The normalizedmelting curves wereanalyzed and clustered samples into different groups withsimilar melting profiles Hence the difference in the shapeof the ldquonormalized melting curverdquo will help cluster samplesinto different species and the samples can be detected andgrouped at the same time

3 Results

31 Specificity of the Detection Assay and Confirmationof Amplicon Identity The specificity of the primers wasdetermined by performing PCR using pure genomic DNAfrom F hepatica F gigantica S mansoni and S japonicum




2









119898of C



5 10 15 20 25 30 35 40 45

0744774414744217442874435744427444974456744637447074477744


Cycles


Fluo

resc

ence

(465

ndash510

)

(a)

Temperature (∘C)70 72 74 76 78 80 8482 86 88 90 92 94

034733476347934712347153471834721347243472734730347


8089 plusmn 006∘C

1 2minus(dd

T) fl

uore

scen

ce (4

65

ndash510

)

(b)


75 76 77 78 79 80 81 82 83 84


Temperature (∘C)

Rela

tive s

igna

l (

)

0000100002000030000400005000060000700008000090000100000

(c)


119898


119898



4 Discussion



15

20

25

30

35

40


y = minus15718x + 22203

R2 = 09978

Cros

sing

poin

t


(a)

15

20

25

30

35

40


y = minus18603x + 22815

R2 = 09938

Cros

sing

poin

t


(b)


Cycles5 10 15 20 25 30 35 40 45


minus0392660813608206082760834608416084860855608626086960876608

Fluo

resc

ence

(465

ndash510

)

(c)


Cycles5 10 15 20 25 30 35 40 45


370110701177012470131701387014570152701597016670173701

Fluo

resc

ence

(465

ndash510

)

(d)

Temperature (∘C)70 72 74 76 78 80 8482 86 88 90 92 94


1 2

minus022522754775727597751227514775172751977522275247752727529775

minus(dd

T) fl

uore

scen

ce (4

65

ndash510

)

(e)


757473 76 77 78 79 80 81 82 83 84 85


Temperature (∘C)

Rela

tive s

igna

l (

)

0000100002000030000400005000060000700008000090000100000

(f)








5 Conclusion




Acknowledgments


References






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




2









119898of C



5 10 15 20 25 30 35 40 45

0744774414744217442874435744427444974456744637447074477744


Cycles


Fluo

resc

ence

(465

ndash510

)

(a)

Temperature (∘C)70 72 74 76 78 80 8482 86 88 90 92 94

034733476347934712347153471834721347243472734730347


8089 plusmn 006∘C

1 2minus(dd

T) fl

uore

scen

ce (4

65

ndash510

)

(b)


75 76 77 78 79 80 81 82 83 84


Temperature (∘C)

Rela

tive s

igna

l (

)

0000100002000030000400005000060000700008000090000100000

(c)


119898


119898



4 Discussion



15

20

25

30

35

40


y = minus15718x + 22203

R2 = 09978

Cros

sing

poin

t


(a)

15

20

25

30

35

40


y = minus18603x + 22815

R2 = 09938

Cros

sing

poin

t


(b)


Cycles5 10 15 20 25 30 35 40 45


minus0392660813608206082760834608416084860855608626086960876608

Fluo

resc

ence

(465

ndash510

)

(c)


Cycles5 10 15 20 25 30 35 40 45


370110701177012470131701387014570152701597016670173701

Fluo

resc

ence

(465

ndash510

)

(d)

Temperature (∘C)70 72 74 76 78 80 8482 86 88 90 92 94


1 2

minus022522754775727597751227514775172751977522275247752727529775

minus(dd

T) fl

uore

scen

ce (4

65

ndash510

)

(e)


757473 76 77 78 79 80 81 82 83 84 85


Temperature (∘C)

Rela

tive s

igna

l (

)

0000100002000030000400005000060000700008000090000100000

(f)








5 Conclusion




Acknowledgments


References






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


15

20

25

30

35

40


y = minus15718x + 22203

R2 = 09978

Cros

sing

poin

t


(a)

15

20

25

30

35

40


y = minus18603x + 22815

R2 = 09938

Cros

sing

poin

t


(b)


Cycles5 10 15 20 25 30 35 40 45


minus0392660813608206082760834608416084860855608626086960876608

Fluo

resc

ence

(465

ndash510

)

(c)


Cycles5 10 15 20 25 30 35 40 45


370110701177012470131701387014570152701597016670173701

Fluo

resc

ence

(465

ndash510

)

(d)

Temperature (∘C)70 72 74 76 78 80 8482 86 88 90 92 94


1 2

minus022522754775727597751227514775172751977522275247752727529775

minus(dd

T) fl

uore

scen

ce (4

65

ndash510

)

(e)


757473 76 77 78 79 80 81 82 83 84 85


Temperature (∘C)

Rela

tive s

igna

l (

)

0000100002000030000400005000060000700008000090000100000

(f)








5 Conclusion




Acknowledgments


References






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





5 Conclusion




Acknowledgments


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

and opisthorchis viverrini using real-time pcr and high

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