A loop-mediated isothermal amplification assay for the visual detection of duck circovirus

METHODOLOGY Open Access

A loop-mediated isothermal amplification assayfor the visual detection of duck circovirusLiji Xie Zhixun Xie Guangyuan Zhao Jiabo Liu Yaoshan Pang Xianwen Deng Zhiqin Xie Qing Fan and Sisi Luo

Abstract

Background Duck circovirus (DuCV) infection in farmed ducks is associated with growth problems or retardationsyndromes Rapid identification of DuCV infected ducks is essential to control DuCV effectively Therefore this studyaims to develop of an assay for DuCV to be highly specific sensitive and simple without any specializedequipment

Methods A set of six specific primers was designed to target the sequences of the Rep gene of DuCV and Aloop-mediated isothermal amplification (LAMP) assay were developed and the reaction conditions were optimizedfor rapid detection of DuCV

Results The LAMP assay reaction was conducted in a 62degC water bath condition for 50 min Then the amplificationproducts were visualized directly for color changes This LAMP assay is highly sensitive and able to detecttwenty copies of DuCV DNA The specificity of this LAMP assay was supported by no cross-reaction with otherduck pathogens

Conclusion This LAMP method for DuCV is highly specific and sensitive and can be used as a rapid and directdiagnostic assay for testing clinical samples

Keywords Duck circovirus Loop-mediated isothermal amplification Visual detection

BackgroundThe duck circovirus (DuCV) was reported initially inGermany in 2003 which was detected from two female6-week-old female Mulard ducks with feathering disor-ders poor body condition immunosuppression and lowweight [1] Subsequently DuCV infection was confirmedin Hungary [2] Taiwan [3] United States [4] and Main-land China [56] Histopathologic examination of thebursa of Fabricius (BF) demonstrated lymphocyte deple-tion necrosis and histiocytosis DuCV was detected inMulard ducks as well as other duck species of MuscovyMule Cherry Valley Pekin and Pockmark ducks [4-8]DuCV is a small (15ndash16 nm in diameter) round none-

nveloped single-stranded DNA virus with a circular gen-ome of approximately 19 kb The genome contains twomajor open reading frames (ORFs) designated ORF V1(Rep gene) and ORF C1 (Cap gene) [1]

Virus isolation is a fundamental diagnostic methodbut no in vivo culture system is yet available for the pro-pagation of DuCV [910] Other diagnostic techniquessuch as conventional polymerase chain reaction (PCR) [2]nested PCR [11] real-time PCR [210] ELISA [9] and insitu hybridization (ISH) have been developed Howeverthese techniques usually need more time and require spe-cialized equipment For example PCR requires agarosegel analysis for the detection of amplification productsand must be performed in specialized laboratories andISH requires several days to complete the assayRecently a new technique known as loop-mediated iso-

thermal amplification (LAMP) has been described [12] Itcan be used to amplify specific target DNA sequenceswith high sensitivity and advantageously it can be com-pleted within 30 to 60 min under isothermal conditionswithout the need of a thermal cycler andor specializedlaboratory [12] This technique eliminates the heat de-naturation step for the DNA synthesis used in conven-tional PCR and relies instead on auto-cycling stranddisplacement DNA synthesis which is achieved by a DNA

Correspondence xiezhixun126comDepartment of Biotechnology Guangxi Key Laboratory of Animal Vaccinesand Diagnostics Guangxi Veterinary Research Institute 51 Youai North Road530001 Nanning China

copy 2014 Xie et al licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (httpcreativecommonsorglicensesby20) which permits unrestricted use distribution andreproduction in any medium provided the original work is properly credited The Creative Commons Public DomainDedication waiver (httpcreativecommonsorgpublicdomainzero10) applies to the data made available in this articleunless otherwise stated

Xie et al Virology Journal 2014 1176httpwwwvirologyjcomcontent11176

polymerase with high strand displacement activity and aset of specially designed primers two inner primers andtwo outer primers Another important feature of LAMP isthe color change which is visible to the naked eye andresults can be obtained in 60 min at a constant settemperature The LAMP assay has been previously usedto successfully detect other viral pathogens [13-19] Theobjective of the present study is to develop and optimizea LAMP assay for the detection of DuCV

MethodsEthics statementThis study was approved by the Animal Ethics Commit-tee of the Guangxi Veterinary Research Institute The in-stitute did not issue a number or ID to this animalstudy because the studied ducks are not an endangeredor protected species Sample collection was conductedbased on the protocol issued by the Animal Ethics Com-mittee of the Guangxi Veterinary Research Institute

Virus strains and DNARNA extractionThe DuCV strains and the other duck pathogen strainsused in this study are listed in Table 1 The genomicDNARNA was extracted from 200 μL of the viruses listedin Table 1 using an EasyPure Viral DNARNA kit (Trans-gen Beijing China) according to the manufacturerrsquosprotocol The DNA and RNA were eluted with 50 μL elu-tion buffer The concentrations of total DNA and RNAwere measured by UV spectrophotometry (Beckman UV-800 Beckman Coulter USA) DNA and RNA sampleswere stored immediately at minus70degC until required

Primer design for the LAMP assayPrimer design for DuCV LAMP was based on the pub-lished Rep gene sequence of the DuCV strain 33753ndash52(GenBank accession no DQ100076) The DuCV strain33753ndash52 sequence was aligned with the available se-quences of twenty viral isolates to identify the conservedregionsA set of six specific primers for the DuCV LAMP assay

was designed using LAMP primer design software PrimerExplorer V4 (httpprimer explorerjpelamp400indexhtml) The LAMP primer set comprised two outer primers(forward primer F3 and backward primer B3) two innerprimers (forward inner primer FIP and backward inner pri-mer BIP) and two loop primers (forward loop F and back-ward loop B) The outer primers (F3 and B3) were used inthe initial steps of the LAMP reactions but later during theisothermal cycling only the inner primers were used forstrand displacement DNA synthesis The loop primerswere designed to accelerate the amplification reaction aspreviously described [17] Since each inner primer (FIP(F1c + F2) and BIP (B1c + B2)) consists of two sequencestargeting two specific sites the six primers consequently

recognized eight sites on the target sequence specific tothe Rep gene segment The details of the primers areshown in Table 2 All primers were purchased fromInvitrogen (Guangzhou China)

Optimization of the DuCV LAMP conditionsThe DuCV LAMP assay was performed in tubes con-taining 10times Thermopolreg Reaction Buffer (New England

Table 1 Pathogens strains and LAMP assay results

Pathogen strainotherduck pathogens

Source Virus information

DuCV (GX1006) GVRI Original positive tissuespecimen






Muscovy duck parvovirus(AV238)

CIVDC Duck embryo cultured virus

Avian influenza virussubtype H5 (Inactivated)

HVRI Chick embryo cultured virus

Avian influenza virussubtype H9

GVRI Chick embryo cultured virus

Duck plague virus GVRI Duck embryo cultured virus

Duck paramyxovirus GVRI Duck embryo cultured virus

Gosling parvovirus GVRI Duck embryo cultured virus

Duck hepatitis virus(AV2111)


Negative tissue (Duck spleenand liver)

GVRI

GVRI = Guangxi Veterinary Research Institute ChinaCIVDC = China Institute of Veterinary Drugs Control ChinaHVRI = Harbin Veterinary Research Institute ChinaPositive= + Negative = minus

Table 2 Oligonucleotide primers used for RT-LAMP assay

Primer name Sequence (5primendash3prime) Genome position

FIP = F1c + F2 TTCAGGAATCCCTGAAGGTG-ATCGTCGGMGAGGAVAAGG

F1c203ndash222F2167ndash185

BIP = B1c + B2 GCGMGAGCTGCCGCCCT-TCTTCVTCAGATCCCCGG

B1c236ndash252B2292ndash309

F3 AGTTBTGCACGCTCGACAAT 135ndash154

B3 GTCGACTCTTTGGMGCAATA 320ndash339

LoopF AGGYGTVCCVTTCGCGC 186ndash202

LoopB AGGAAGAGCCTGGCTCTC 268ndash285

Genome position according to the DuCV complete genome sequence(GenBank accession no DQ100076)Abbreviations are as follows M = A or C V = A or C or G B = T or G or C

Xie et al Virology Journal 2014 1176 Page 2 of 6httpwwwvirologyjcomcontent11176

Biolabs Beijing China) Bst DNA polymerase (large frag-ment New England Biolabs) dNTPs (Takara DalianChina) primers betaine (SigmandashAldrich) MgSO4 (SigmandashAldrich) calcein (International Laboratory USA) MnCl2(International Laboratory USA) template DNARNA andnuclease-free water Based on the previous studies dif-ferent combinations of various concentrations of eachcomponent (dNTPs (04 mmolL ~16 mmolL) betaine(08 mmolL ~14 mmolL) MgSO4 (2 mmolL ~9 mmolL) were tested for amplification efficiency The amplifica-tion reaction was performed in a thermal block between59degC to 65degC within 40 to 80 min to ascertain the optimalincubation temperature and time At the end of each in-cubation period the reaction was terminated by heatingat 80degC for 5 min All of the experiments were repeatedthree times

Analysis of DuCV LAMP productsFor a visual inspection of the LAMP assay productsfluorescence reagents (calcein and MnCl2 InternationalLaboratory) were added to the reaction mixture beforeamplification and a color change of the reaction mixturewas noted upon successful amplification Samples thatturned green were considered positive while samplesthat remained orange were considered negative Alterna-tively 3 μL of the DuCV LAMP products were analyzedby 15 agarose gel electrophoresis The presence of asmear or a pattern of multiple bands with different mo-lecular weights indicated a positive result [13]

Evaluation of DuCV LAMP assaysTo evaluate the specificity of the assay DNARNA sam-ples extracted from different virus strains includingDuCV Muscovy duck parvovirus avian influenza virussubtypes H5 and H9 duck plague virus duck paramyxo-virus Gosling parvovirus and duck hepatitis virus weretested by the DuCV LAMP assay (Table 1) The detec-tion limit of the LAMP to DuCV was assessed and com-pared with conventional PCR [5] using a series of ten-fold dilutions of the DuCV templateBriefly DNA extracted from the DuCV strain GX1006

was used in the PCR reaction to amplify the Rep geneThe amplified product of the Rep gene was cloned intothe pMD18-T cloning vector (TaKaRa Dalian China)according to the manufacturerrsquos directions The recom-binant plasmids were sequenced The sequence datawere analyzed using DNASTAR software and were com-pared with the corresponding sequence data in Gen-Bank The copy number was calculated according to thefollowing formula (copiesμL = 6 times 1023 times DNA concen-tration gμL)molecular weight gmol as described byXie et al [20] A series of ten-fold dilutions (1 times 107 to1 times 10deg copiesμL) were used to assess the sensitivity ofthe DuCV LAMP assay

Detection of clinical samples by DuCV LAMP assayA total of 181 clinical samples were collected from eachsampling point (Nanning Yulin Hengxian NingmingShanglin Dongxing and Liuzhou) on commercial duckfarms in Guangxi Province China (Table 3) Spleen andliver (01 g) tissues were homogenized in 500 μL sterilesaline centrifugation at 12000 g for 15 min and then200 μL of the supernatant were used to extract DNA byusing an EasyPure Viral DNARNA kit (Transgen BeijingChina) according to the manufacturerrsquos protocol The ex-tracted DNAs were tested by both the DuCV LAMP assayand real-time PCR as previously described by Fringuelliet al [2]

ResultsDuCV LAMP assayAfter optimization of the reaction conditions the DuCVLAMP assay was carried out in a 25 μL reaction mixturecontaining 25 μL 10times Thermopolreg Reaction Buffer (20 mMTrisndashHCl 10 mM (NH4)2SO4 10 mM KCl 2 mM MgSO401 Triton X-100) 8 U Bst DNA polymerase 1 mmolLbetaine 7 mmolL MgSO4 14 mmolL dNTPs 02 mmolL F3 primer 02 mmolL B3 primer 08 mmolL forwardloop (LF) primer 08 mmolL backward loop (LB) primer16 mmolL forward inner primer (FIP) 16 mmolL back-ward inner primer (BIP) 25 mmolL calcein 05 mmolLMnCl2 2 μL template DNA and dH2O to make the finalvolume up to 25 μL The initial color of the reaction solu-tion prior to amplification was orange The optimal reac-tion time and incubation temperature were found to be50 min at 62degC

Specificity and sensitivity of DuCV LAMP assayThe optimized DuCV LAMP assay was used to specific-ally amplify six Guangxi field DuCV strains Test results(Figure 1A and B) showed that this technique exhibitedno cross-reactivity with other avian viral pathogens testedincluding Muscovy duck parvovirus Avian influenza virus

Table 3 Detection results of clinical samples by DuCVLAMP assay

Location ofsamples

LAMP Real-time PCR

Positivesamplestotalsamples

Positiverate


Positiverate

Nanning 529 1724 529 1724

Yulin 477 519 477 519

Hengxian 07 0 07 0

Ningmeng 013 0 013 0

Shanglin 138 263 138 263

Dongxing 111 909 111 909

Liuzhou 06 0 06 0

Total 11181 608 11181 608


subtype H5 Avian influenza virus subtype H9 Duckplague virus Duck paramyxovirus Gosling parvovirusand Duck hepatitis virus Six DuCV field strains weretested by the DuCV LAMP assay and all final products ofthe LAMP assay yielded a positive green color (Figure 1Blanes 1 to 6) and showed a typical DNA ladder patternafter 15 agarose gel electrophoresis (Figure 1A lanes 1to 6) The products of the DuCV LAMP assay for theother avian viral pathogens remained negative orangecolor (no color change) (Figure 1B lanes 7 to 13) fur-thermore the other viral pathogens lacked the typicalDNA ladder pattern showing amplification did notoccur (Figure 1A lanes 7 to 13)The sensitivity of the DuCV LAMP assay was then de-

termined and compared with that of conventional PCR inparallel As shown in Figure 2A the detection limit of theDuCV LAMP assay is twenty copies (Figure 2A and B)

However the detection limit of conventional PCR was 2 times103 copies (Figure 3) This indicates that the sensitivity ofthe DuCV LAMP assay was 100-fold higher than that ofconventional PCR Our results showed that the DuCVLAMP assay is highly specific sensitive and superior tothe conventional PCR for the detection of DuCV

Detection of clinical samples by DuCV LAMP assayThe uCV LAMP assay was used to test 181 clinical sam-ples which were also tested by real-time PCR for a com-parison of the two assays Eleven duck samples (608)were tested positive and 170 (9392) were tested negativeby both methods (Table 3) The DuCV LAMP and real-time PCR yielded 100 of agreement in testing these sam-ples However DuCV LAMP was noted to be a quickereasier and more cost-efficient method compared to real-time PCR

Figure 1 Specificity of DuCV LAMP assay (A) Agarose gel electrophoresis of the LAMP products M 100 bp DNA ladder (B) Visualization ofthe LAMP products The numbers in (A) and (B) represent DuCV strains and the negative controls used in the specificity test 1 DuCV GX1006 2DuCV GX1008 3 DuCV GX1104 4 DuCV GX1105 5 DuCV GX1209 6 DuCV GX1208 7 Muscovy duck parvovirus AV238 8 Avian influenza virussubtype H5 9 Avian influenza virus subtype H9 10 duck plague virus 11 duck paramyxovirus 12 Gosling parvovirus 13 duck hepatitisvirus AV2111

Figure 2 Sensitivity of the DuCV LAMP assay (A) Agarose gel electrophoresis of the LAMP products M 100 bp DNA ladder (B) Visualizationof the LAMP products Labels 1 2 times 107 copiestube 2 2 times 106 copiestube 3 2 times 105copiestube 4 2 times 104 copiestube 5 2 times 103 copiestube6 2 times 102 copiestube 7 20 copiestube 8 2 copiestube 9 negative control


DiscussionDuCV can cause feathering disorders poor body conditionimmunosuppression and low weight in ducks [1] Conse-quently the development of a rapid simple and sensitivedetection method for DuCV is essentialIn this study a set of six primers targeting the Rep gene

segment of DuCV were designed and the reaction condi-tions for LAMP were optimized through repeated experi-ments The concentrations of each reaction reagent ieMgSO4 betaine dNTP Bst DNA polymerase calcein andMnCl2 can influence the amplification efficiency of theLAMP assay which subsequently affects the fluorescenceemission of the resulting solution Therefore it is neces-sary to optimize the reaction system to obtain the appro-priate reagent concentrations reaction temperature andamplification time The results of the specificity testsindicated that the DuCV LAMP assay can successfullydetected the DuCV field strains and showed no cross-reaction with other viral pathogens The establishedDuCV LAMP assay has several advantages in comparedwith conventional PCR First the detection limit of theDuCV LAMP assay is twenty copies which is 100-foldhigher than that of conventional PCR Secondly theamplification reaction of DuCV LAMP assay can be ac-complished in a conventional laboratory water bath for50 min and does not require a PCR machine or anyother specialized equipment Thirdly the results can beinspected immediately and the color change is visible tothe naked eye which makes a rapid and easy determin-ation of the test result without the need of electrophor-esis analysisNevertheless the main challenge in the development

of a DuCV LAMP assay is the hurge genetic heterogen-eity of this virus [41821] with FIP and BIP being themost important primers in the LAMP assay that need tobe conserved across the many strains Another signifi-cant challenge in the development of a DuCV LAMPassay is its high amplification rate which can result in

potential cross-contamination issues To investigate thisissue we added a dye (calcein with MnCl2) into the reac-tion system before the amplification in order to elimin-ate the chance of contamination Prior to amplificationcalcein combines with the manganese ions (Mn2+) andthe reaction solution turns orange When the positiveLAMP amplification reaction proceeds the generatedpyrophosphate ions remove the manganese ions (Mn2+)from calcein resulting in the emission of fluorescencefrom calcein The free calcein may then combine withmagnesium ions (Mg2+) in the reaction mixture leadingto stronger fluorescence emission [22] By the way afterthe positive LAMP amplification reaction proceeds thegenerated pyrophosphate ions combine with manganeseions (Mn2+) and generate manganous pyrophosphateBy centrifuge the manganous pyrophosphate precipi-tated at the bottom of the tube (white sediment at thebottom of the tube) And the negative LAMP did nothave the white sediment The generated manganouspyrophosphate is another way to determine the LAMPresult by the white sediment besides examination of thecolor by naked eyeA LAMP assay for Goose circovirus detection has been

previously reported [23] but the sequence of the Goosecircovirus and DuCV was different To our knowledgethis is the first study to explore the use of DuCV LAMPtechnology in a diagnostic test for DuCV The methodand analysis is simple specific sensitive and rapidfurthermore it can be used to detect DuCV in clinicalsamples

ConclusionsA simple rapid highly sensitive and specific DuCVLAMP assay for the detection of DuCV has been devel-oped and established in our present study This tech-nique has the potential to be applied in clinical or fieldconditions as it does not require the use of sophisticatedequipment

Competing interestsThe authors declare that they have no competing interests

Authorsrsquo contributionsLX and ZX designed the experiments GZ and JL prepared the RNADNAsamples LX designed the primers and optimized conditions of the LAMPassay LX XD ZX QF SL carried out the experiments shown in Figures 1 2 3and in Table 3 GZ performed the data analysis LX wrote the manuscript Allauthors reviewed and approved the final version of the manuscript

AcknowledgementsThis research project was founded by Guangxi Science and TechnologyBureau (1222003-2-4 and 10100014ndash5) and Guangxi Government SeniorScientist Foundation (2011B020)

Received 15 January 2014 Accepted 25 April 2014Published 29 April 2014

Figure 3 Sensitivity of conventional PCR M 100 bp DNA ladder1 2 times 107 copiestube 2 2 times 106 copiestube 3 2 times 105 copiestube4 2 times 104 copiestube 5 2 times 103 copiestube 6 2 times 102 copiestube7 20 copiestube 8 2 copiestube 9 negative control


References1 Hattermann K Schmmitt C Soike D Mankertz A Cloning and sequencing

of duck circovirus (DuCV) Arch Virol 2003 1482471ndash24802 Fringuelli E Scott AN Beckett A McKillen J Smyth JA Palya V Glavits R

Ivanics E Mankertz A Franciosini MP Todd D Diagnosis of duck circovirusinfections by conventional and real-time polymerase chain reactiontests Avian Pathol 2005 34495ndash500

3 Chen C Wang P Lee M Shien J Shieh H Ou S Chen C Chang PDevelopment of a polymerase chain reaction procedure for detection anddifferentiation of duck and goose circovirus Avian Dis 2006 5092ndash95

4 Banda A Galloway-Haskins RI Sandhu TS Schat KA Genetic analysis of aduck circovirus detected in commercial Pekin ducks in New YorkAvian Dis 2007 5190ndash95

5 Zhang X Jiang S Wu J Zhao Q Sun Y Kong Y Li X Yao M Chai T Aninvestigation of duck circovirus and co-infection in Cherry Valley ducksin Shandong Province China Vet Microbiol 2009 133(3)252ndash256

6 Xie L Xie Z Zhao G Liu J Pang Y Deng X Xie Z Fan Q CompleteGenome Sequence Analysis of a Duck Circovirus from GuangxiPockmark Ducks J Virol 2012 86(23)13136ndash13136

7 Soike D Albrecht K Hattermann K Schmitt C Mankertz A Novel circovirusin mulard ducks with developmental and feathering disorders Vet Rec2004 154792ndash793

8 Wan C Fu G Shi S Cheng L Chen H Peng C Lin S Huang YEpidemiological investigation and genome analysis of duck circovirus inSouthern China Virol Sin 2011 26(5)289ndash296

9 Liu SN Zhang XX Zou JF Xie ZJ Zhu YL Zhao Q Zhou EM Jiang SJDevelopment of an indirect ELISA for the detection of duck circovirusinfection in duck flocks Vet Microbiol 2010 145(1)41ndash46

10 Wan C Huang Y Cheng L Fu G Shi SH Chen H Peng C Lin F Lin J Thedevelopment of a rapid SYBR Green I-based quantitative PCR fordetection of Duck circovirus Virol J 2011 8465

11 Halami MY Nieper H Muumlller H Johne R Detection of a novel circovirus inmute swans (Cygnus olor) by using nested broad-spectrum PCRVirus Res 2008 132208ndash212

12 Notomi T Okayama H Masubuchi H Yonekawa T Watanabe K Amino NHase T Loopmediated isothermal amplification of DNA Nucleic Acids Res2000 28E63

13 Xie ZX Tang Y Fan Q Liu JB Pang YS Deng XW Xie ZQ Peng Y Xie LJKhan MI Rapid detection of Group I avian adenoviruses by aLoop-mediated isothermal amplification Avian Dis 2011 55575ndash579

14 Enomoto Y Yoshikawa T Ihira M Akimoto S Miyake F Usui C Suga SSuzuki K Kawana T Nishiyama Y Asano Y Rapid diagnosis of herpessimplex virus infection by a loopmediated isothermal amplificationmethod J Clin Microbiol 2005 43951ndash955

15 Peng Y Xie ZX Lie JB Peng YS Deng XW Xie ZQ Xie LJ Fan Q Feng JXKhan MI Visual detection of H3 subtype avian influenza viruses byreverse transcription loop-mediated isothermal amplification assayVirol Jour 2011 81ndash10

16 Nemoto M Imagawa H Tsujimura K Yamanaka T Kondo T Matsumura TDetection of equine rotavirus by reverse transcription loop-mediatedisothermal amplification (RT-LAMP) J Vet Med Sci 2010 72823ndash826

17 Rovira A Abrahante J Murtaugh M Munoz-Zanzi C Reverse transcriptionloop-mediated isothermal amplification for the detection of Porcinereproductive and respiratory syndrome virus J Vet Diagn Invest 200921350ndash354

18 Xie Z Fan Q Liu J Pang Y Deng X Xie Z Xie L Khan M Reversetranscription loop-mediated isothermal amplification assay for rapiddetection of Bovine Rotavirus BMC vety res 2012 8(1)133

19 Fan Q Xie Z Xie L Liu J Pang Y Deng X Xie Z Peng Y Wang X A reversetranscription loop-mediated isothermal amplification method for rapiddetection of bovine viral diarrhea virus J Virol Methods 2012 186(1ndash2)43ndash48

20 Xie Z Xie L Pang Y Lu Z Xie Z Sun J Deng X Liu J Tang X Khan MDevelopment of a real-time multiplex PCR assay for detection of viralpathogens of penaeid shrimp Arch Virol 2008 1532245ndash2251

21 Wang D Xie X Zhang D Ma G Wang X Zhang D Detection of duckcircovirus in China a proposal on genotype classification Vet Microbiol2011 147(3)410ndash415

22 Tomita N Mori Y Kanda H Notomi T Loop-mediated isothermalamplification (LAMP) of gene sequences and simple visual detection ofproducts Nat Protoc 2008 3877ndash882

23 Woźniakowski G Kozdruń W Samorek-Salamonowicz E Loop-mediatedisothermal amplification for the detection of goose circovirus Virol J2012 9(1)1ndash11

doi1011861743-422X-11-76Cite this article as Xie et al A loop-mediated isothermal amplificationassay for the visual detection of duck circovirus Virology Journal2014 1176

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Abstract

Background

Methods

Results

Conclusion

Background

Methods

Ethics statement

Virus strains and DNARNA extraction

Primer design for the LAMP assay

Optimization of the DuCV LAMP conditions

Analysis of DuCV LAMP products

Evaluation of DuCV LAMP assays

Detection of clinical samples by DuCV LAMP assay

Results

DuCV LAMP assay

Specificity and sensitivity of DuCV LAMP assay


Discussion

Conclusions

Competing interests

Authorsrsquo contributions

Acknowledgements

References

polymerase with high strand displacement activity and aset of specially designed primers two inner primers andtwo outer primers Another important feature of LAMP isthe color change which is visible to the naked eye andresults can be obtained in 60 min at a constant settemperature The LAMP assay has been previously usedto successfully detect other viral pathogens [13-19] Theobjective of the present study is to develop and optimizea LAMP assay for the detection of DuCV

MethodsEthics statementThis study was approved by the Animal Ethics Commit-tee of the Guangxi Veterinary Research Institute The in-stitute did not issue a number or ID to this animalstudy because the studied ducks are not an endangeredor protected species Sample collection was conductedbased on the protocol issued by the Animal Ethics Com-mittee of the Guangxi Veterinary Research Institute

Virus strains and DNARNA extractionThe DuCV strains and the other duck pathogen strainsused in this study are listed in Table 1 The genomicDNARNA was extracted from 200 μL of the viruses listedin Table 1 using an EasyPure Viral DNARNA kit (Trans-gen Beijing China) according to the manufacturerrsquosprotocol The DNA and RNA were eluted with 50 μL elu-tion buffer The concentrations of total DNA and RNAwere measured by UV spectrophotometry (Beckman UV-800 Beckman Coulter USA) DNA and RNA sampleswere stored immediately at minus70degC until required

Primer design for the LAMP assayPrimer design for DuCV LAMP was based on the pub-lished Rep gene sequence of the DuCV strain 33753ndash52(GenBank accession no DQ100076) The DuCV strain33753ndash52 sequence was aligned with the available se-quences of twenty viral isolates to identify the conservedregionsA set of six specific primers for the DuCV LAMP assay

was designed using LAMP primer design software PrimerExplorer V4 (httpprimer explorerjpelamp400indexhtml) The LAMP primer set comprised two outer primers(forward primer F3 and backward primer B3) two innerprimers (forward inner primer FIP and backward inner pri-mer BIP) and two loop primers (forward loop F and back-ward loop B) The outer primers (F3 and B3) were used inthe initial steps of the LAMP reactions but later during theisothermal cycling only the inner primers were used forstrand displacement DNA synthesis The loop primerswere designed to accelerate the amplification reaction aspreviously described [17] Since each inner primer (FIP(F1c + F2) and BIP (B1c + B2)) consists of two sequencestargeting two specific sites the six primers consequently

recognized eight sites on the target sequence specific tothe Rep gene segment The details of the primers areshown in Table 2 All primers were purchased fromInvitrogen (Guangzhou China)

Optimization of the DuCV LAMP conditionsThe DuCV LAMP assay was performed in tubes con-taining 10times Thermopolreg Reaction Buffer (New England

Table 1 Pathogens strains and LAMP assay results

Pathogen strainotherduck pathogens

Source Virus information







Muscovy duck parvovirus(AV238)


Avian influenza virussubtype H5 (Inactivated)

HVRI Chick embryo cultured virus

Avian influenza virussubtype H9

GVRI Chick embryo cultured virus

Duck plague virus GVRI Duck embryo cultured virus

Duck paramyxovirus GVRI Duck embryo cultured virus

Gosling parvovirus GVRI Duck embryo cultured virus

Duck hepatitis virus(AV2111)


Negative tissue (Duck spleenand liver)

GVRI

GVRI = Guangxi Veterinary Research Institute ChinaCIVDC = China Institute of Veterinary Drugs Control ChinaHVRI = Harbin Veterinary Research Institute ChinaPositive= + Negative = minus

Table 2 Oligonucleotide primers used for RT-LAMP assay

Primer name Sequence (5primendash3prime) Genome position

FIP = F1c + F2 TTCAGGAATCCCTGAAGGTG-ATCGTCGGMGAGGAVAAGG

F1c203ndash222F2167ndash185

BIP = B1c + B2 GCGMGAGCTGCCGCCCT-TCTTCVTCAGATCCCCGG

B1c236ndash252B2292ndash309

F3 AGTTBTGCACGCTCGACAAT 135ndash154

B3 GTCGACTCTTTGGMGCAATA 320ndash339

LoopF AGGYGTVCCVTTCGCGC 186ndash202

LoopB AGGAAGAGCCTGGCTCTC 268ndash285

Genome position according to the DuCV complete genome sequence(GenBank accession no DQ100076)Abbreviations are as follows M = A or C V = A or C or G B = T or G or C

Xie et al Virology Journal 2014 1176 of 6httpwwwvirologyjcomcontent11176









Location ofsamples

LAMP Real-time PCR


Positiverate


Positiverate

Nanning 529 1724 529 1724

Yulin 477 519 477 519

Hengxian 07 0 07 0


Shanglin 138 263 138 263

Dongxing 111 909 111 909

Liuzhou 06 0 06 0

Total 11181 608 11181 608























































Abstract

Background

Methods

Results

Conclusion

Background

Methods

Ethics statement







Results

DuCV LAMP assay



Discussion

Conclusions

Competing interests


Acknowledgements

References









Location ofsamples

LAMP Real-time PCR


Positiverate


Positiverate

Nanning 529 1724 529 1724

Yulin 477 519 477 519

Hengxian 07 0 07 0


Shanglin 138 263 138 263

Dongxing 111 909 111 909

Liuzhou 06 0 06 0

Total 11181 608 11181 608























































Abstract

Background

Methods

Results

Conclusion

Background

Methods

Ethics statement







Results

DuCV LAMP assay



Discussion

Conclusions

Competing interests


Acknowledgements

References






















































Abstract

Background

Methods

Results

Conclusion

Background

Methods

Ethics statement







Results

DuCV LAMP assay



Discussion

Conclusions

Competing interests


Acknowledgements

References

A loop-mediated isothermal amplification assay for the visual detection of duck circovirus

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Transcript of A loop-mediated isothermal amplification assay for the visual detection of duck circovirus