Loop-mediated isothermal amplification test for Trypanosoma vivax ...

G ModelVETPAR-5767; No. of Pages 5ARTICLE IN PRESSVeterinary Parasitology xxx (2011) xxx–xxxContents lists available at ScienceDirectVeterinary Parasitologyjournal homepage: www.elsevier.com/locate/vetparShort communicationLoop-mediated isothermal amplification test for Trypanosoma vivaxbased on satellite repeat DNAZ.K. Njiru a,∗ , J.O. Ouma b , R. Bateta b , S.E. Njeru b , K. Ndungu b , P.K. Gitonga b ,S. Guya b , R. Traub aa School of Veterinary Sciences, University of Queensland, Gatton, QLD 4343, Australiab Trypanosomiasis Research Centre, Kenya Agricultural Research Institute, P.O. Box 362-00902, Kikuyu, KenyaarticleinfoabstractArticle history:Received 15 January 2011Received in revised form 10 March 2011Accepted 11 March 2011Keywords:Loop-mediated isothermal amplificationTrypanosoma vivaxNaganaTrypanosomiasisPCRTrypanosoma vivax is major cause of animal trypanosomiasis and responsible for enormouseconomic burden in Africa and South America animal industry. T. vivax infections mostly runlow parasitaemia with no apparent clinical symptoms, making diagnosis a challenge. Thiswork reports the design and evaluation of a loop-mediated isothermal amplification (LAMP)test for detecting T. vivax DNA based on the nuclear satellite repeat sequence. The assay israpid with results obtained within 35 min. The analytical sensitivity is ∼1 trypanosome/mlwhile that of the classical PCR tests ranged from 10 to 10 3 trypanosomes/ml. The T. vivaxLAMP test reported here is simple, robust and has future potential in diagnosis of animaltrypanosomiasis in the field.© 2011 Elsevier B.V. All rights reserved.1. IntroductionTrypanosoma vivax is a widely distributed parasite anda major pathogen of ruminants in sub-Saharan Africa andSouth America. In Africa, it is predominantly transmittedcyclically by tsetse flies and to a lesser extent by bitingflies while in South America it is mechanically transmittedby blood-sucking flies such as tabanids and stableflies (Wells, 1984; Meléndez et al., 1993). T. vivax is economicallyimportant because it causes mortality and highmorbidity in ruminant livestock. Different patterns of diseaseoccur in Africa and South America; in South Americathe disease occurs in cattle as epizootics and with rare outbreaks(Osório et al., 2008) while in Africa the disease runsa more severe and acute form (Stephen, 1986). A few EastAfrican isolates of T. vivax cause an acute hemorrhagic syn-∗ Corresponding author at: School of Veterinary Sciences, Universityof Queensland, Inner Ring Road, Blgd 8114, RM 231, Gatton, QLD 4343,Australia. Tel.: +61 7 5460 1973.E-mail addresses: zablon@iprimus.com.au, z.njiru@uq.edu.au(Z.K. Njiru).drome with a mortality rate of 6–35% (Assoku and Gardiner,1989; Magona et al., 2008).The definition of accurate geographical endemicity ofT. vivax has been limited by the ease in which variety ofinsect vectors (some yet to be identified) transmits theparasite (Masake et al., 1997). As such the most definitiveway of verifying the presence of the parasite is throughits detection in the host and to a lesser extent in vectors.Detection of T. vivax in animals is a challenging tasksince infection runs typically a low parasitaemia limitingthe use of parasitological techniques such as microscopyin the field diagnosis (Nantulya, 1990). These drawbacksled to development of antibody based techniques such asantibody indirect fluorescent staining technique (Platt andAdams, 1976) and enzyme-linked immunosorbent assays(ELISA) (Luckins, 1977; Nantulya, 1990; Ferenc et al., 1990).The persistence of antibodies in the host after treatmentimplied that these antibody detecting tests could not differentiatebetween past and current infection. To overcomethe limitation imposed by the antibody assays, an antigenbased ELISA was developed (Nantulya et al., 1992) but itsdiagnostic parameters in the field were not encouraging0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.vetpar.2011.03.021Please cite this article in press as: Njiru, Z.K., et al., Loop-mediated isothermal amplification test for Trypanosoma vivaxbased on satellite repeat DNA. Vet. Parasitol. (2011), doi:10.1016/j.vetpar.2011.03.021

G ModelVETPAR-5767; No. of Pages 5ARTICLE IN PRESS2 Z.K. Njiru et al. / Veterinary Parasitology xxx (2011) xxx–xxx(Desquesnes, 1996). Molecular based test (PCR) promisedbetter sensitivity and a range of them were developed(Masiga et al., 1992; Masake et al., 1997; Morlais et al.,2001) but the demanding nature of PCR technique has limitedits use in routine diagnosis. Nevertheless due to lowparasitaemia and difficulties in propagating T. vivax in mice,PCR has remained the most appropriate method for laboratorybased diagnosis (Jones and Dávila, 2001). To datea definitive field based assay for T. vivax has been elusive,therefore it is essential to continue evaluating newdiagnostic technologies and especially those that offer platformsfor developing point of use tests.Loop-mediated isothermal amplification (LAMP) is anovel gene amplification method that amplifies DNA underisothermal conditions (Notomi et al., 2000). The techniqueuses four to six primers that recognize six to eight regionsof the target DNA and relies on Bst DNA polymerase, anenzyme that synthesizes DNA through strand displacementactivity. The LAMP method has several advantages over PCRin that: (i) LAMP amplification can be achieved using simpleheating device that maintains temperature at isothermal(60–65 ◦ C), (ii) amplification can be achieved using partiallyor non-processed template (Kaneko et al., 2007; Njiruet al., 2008) therefore DNA extraction may not be necessary,(iii) reactions are rapid and require shorter time (Nagamineet al., 2002), (iv) sensitivity is equal or higher than that ofPCR and (v) the technology allows the use of varied productdetection formats. These characteristics make LAMP strategyideal for T. vivax diagnosis in resource poor endemicregions. In this study, we have designed a rapid T. vivaxLAMP test based on the satellite repeat DNA. The nuclearsatellite repeat sequence is a desired target because it ismulticopy gene and widely conserved among T. vivax isolatesin Africa and S. America. The test was evaluated andcompared with PCR tests using a panel of T. vivax isolatesand archived field samples.2. Materials and methods2.1. Preparation of templateA total of 23 T. vivax samples collected from Kenya,Uganda, Tanzania and Nigeria from bovine and tsetseflies between 1951 and 1991 were used in this study.The samples included three hemorrhagic T. vivax samplesfrom Kenya. The DNA was prepared using the QiagenDNeasy blood and tissue kit and following the manufacturerinstructions. The resulting DNA was stored at −20 ◦ C.2.2. Polymerase chain reactionThe PCR test reactions for satellite repeat DNA (Masigaet al., 1992) and diagnostic antigen gene (Masake et al.,1997) were done using the published conditions. A thirdPCR for satellite repeat DNA was performed using outerforward (F3) and backward (B3) LAMP primers. The newPCR test was performed using a 25 l reaction consistingof 1× PCR buffer, 200 M of each dNTP, 1.5 mM of MgCl 2 ,12.5 pmol of each F3 and B3 primers, 1 U Taq DNA Polymerase(Fisher Biotec, WA, Australia) and 1-l of DNAtemplate. The reactions were done in duplicates using a96-Well GeneAmp ® PCR System 9700 (Applied Biosystems,Australia) at: 1 cycle 95 ◦ C for 10 min, followed by 40 cyclesof 94 ◦ C for 45 s, 63 ◦ C for 30 s and 72 ◦ C for 30 s and a finalextension at 72 ◦ C for 10 min. After amplifications, 10-lofthe PCR products were resolved for 70 min in 2.0% agarosegel stained with SYBR ® safe DNA gel stain (Invitrogen, Victoria,Australia). The gel images were documented usingGel- Doc-XR system (Bio-Rad Lab).2.3. LAMP primers and reactionT. vivax satellite repeat DNA (Accession number J03989)was used for designing six LAMP primers. The primerswere forward and backward primers: F3 = TGTTCTGGTGG-CCTGTTGC and B3 = GGCCGGAGCGAGAGGTGC, forwardand backward inner primers: FIP = GTGGAGCGTG-CCAACGTGGCACCCGCTCCCAGACCATA and BIP = TGTCT-AGCGTGACGCGATGGAAGAGGGAGTGGGGAAGG and loopforward and backward primers: CACATGGAGCATCAGGACand LB: CCGTGCACTGTCCCGCAC. The LAMP tests werecarried out in 25-l reactions consisting of 5 pmol ofthe outer primers, 20 pmol of loop primers, 40 pmol ofthe inner primers, 4 mM of extra MgSO 4 , 1 M betaine(Sigma–Aldrich, St. Louis, MO, USA) and 2.5 mM deoxynucleotidetriphosphates mix (dNTP). The 1× ThermoPolreaction buffer contained 20 mM Tris–HCl (pH 8.8), 10 mMKCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 and 0.1% TritonX-100. The Bst DNA polymerase (Large fragment; NewEngland Biolabs, MA, USA) was 1 l (8 units) while SYTO-9fluorescence dye at 1.5 M (Molecular Probes, OR USA)was used for each real time reaction. The template was1-l (∼1 ng) of T. vivax isolate EATRO 1186. The mixturewas incubated at 63 ◦ C in the Rotor-Gene 6000 (Qiagen,Victoria, Australia) and data acquired on HRM channel(460–510 nm) followed by reaction inactivation at 80 ◦ Cfor 4 min. Later the assay was trialed using a normal waterbath. Briefly, water was heated to ∼63–64 ◦ C and the LAMPreaction tubes suspended in water using a floater for 1 h,after which the temperature was raised to approximately80 ◦ C to stop the reactions. To select an appropriaterestriction enzyme for product analysis, the targetsequence was analyzed using restriction enzyme mapperin DNAman software (Lynnon Corporation, Quebec,Canada).2.4. Detection and confirmation of LAMP productThe LAMP reactions were monitored in real timethrough fluorescence of double stranded DNA (dsDNA) inRotor-Gene 6000. After amplification, the products werefurther analyzed through electrophoresis in 2.0% agarosegels stained with SYBR ® safe DNA gel stain and throughaddition of 1/10 dilution of SYBR ® Green I. Two methodswere used to confirm that the T. vivax LAMP amplifiedthe correct target: (i) the melt curves were acquired using1 ◦ C step, with a hold of 30 s, from 63 ◦ Cto96 ◦ C and (ii)1–2 l of the amplification product was incubated withrestriction enzyme NdeI (New England Biolabs, MA, USA)at 37 ◦ C for 4 h followed by electrophoresis in 3% agarosegel.Please cite this article in press as: Njiru, Z.K., et al., Loop-mediated isothermal amplification test for Trypanosoma vivaxbased on satellite repeat DNA. Vet. Parasitol. (2011), doi:10.1016/j.vetpar.2011.03.021

G ModelVETPAR-5767; No. of Pages 5ARTICLE IN PRESSZ.K. Njiru et al. / Veterinary Parasitology xxx (2011) xxx–xxx 3Table 1The analytical sensitivity of T. vivax LAMP assay compared with PCR tests using templates from 10-fold serial dilution of T. vivax isolate EATRO 1186.Type of test Target gene Specificity 10-fold dilution a ReferenceNeat 10 −1 10 −2 10 −3 10 −4 10 −5 10 −6 10 −7 10 −8LAMP Satellite DNA T. vivax + + + + + + + – – This studyPCR Satellite DNA ” + + + + + + – – – This study (F3/B3primers)PCR Satellite DNA ” + + + + + + – – – Masiga et al. (1992)PCR Diagnostic antigen ” + + + + – – – – – Masake et al. (1997)Neat = approximately 100 ng.a 10 −1 (∼1.0 × 10 5 tryps/ml), 10 −2 (∼1.0 × 10 4 tryps/ml) and 10 −8 (∼0.01 tryps/ml).2.5. LAMP and PCR analytical sensitivitiesTen-fold serial dilution of ∼10 ng of T. vivax EATRO 1186DNA was used to compare and determine the analyticalsensitivity of T. vivax LAMP and the PCR tests. The templatewas 1-l for each serial dilution. The specificity ofthe LAMP test was assessed with DNA prepared from bitingflies (stomoxys), Glossina pallidipes, bovine and otherpathogenic trypanosomes; T.b. brucei, T.b. gambiense, T.b.evansi, T. congolense savannah, T.c. kilifi, T.c. forest, T. simiae,T.s. tsavo and T. godfreyi.2.6. Analysis of archived bovine samplesA total of 376 archived DNA samples prepared fromthree tsetse flies, 16 bovine buffy coats collected fromLambwe valley, Kenya and 357 from blood samples collectedin Nguruman, Kenya (Njiru et al., 2005) were used(Table 2). The DNA was prepared using Qiagen DNeasyBlood & Tissue Kit. In addition, supernatant was preparedfrom bovine blood spiked with ∼1–100 pg of T. vivax DNAas previously described (Njiru et al., 2008) and 2-l ofthe template was used. 25-l LAMP reactions (Section 2.3)were carried out for the field samples and comprised ofruns of 1 and 3-l of the purified template.3. ResultsThe inclusion of loop primers reduced the reaction timeby an average of 25 min for each dilution and increasedthe assay analytical sensitivity by 10 3 -fold to an equivalentof 1 trypanosome/ml (1 pg). The post amplificationanalysis of LAMP product showed reproducible melt curveswith a melting temperature (T m )of∼90 ◦ C for all T. vivaxisolates while restriction enzyme NdeI gave the predictedsizes of ∼80 bp and 140 bp respectively. T. vivax LAMP testwas 10 and 10 3 -fold more sensitive than satellite repeatPCR (Masiga et al., 1992) and diagnostic antigen PCR test(Masake et al., 1997) respectively (Table 1). The positivereactions showed ladder like pattern after electrophoresisin agarose gel indicating the formation of stem-loops andturned green on addition of 1/10 dilution of SYBR ® GreenI. On the analysis of the T. vivax samples, the LAMP testdetected 20/23, satellite repeat 15/23 and the diagnosticantigen PCR 7/23 (Table 2). The analysis of 357 archivedbovine samples revealed a T. vivax prevalence of 7.6%through LAMP test, 5.3% by satellite repeat PCR and 1.7%with diagnostic antigen PCR (Table 2). The use of 3-l oftemplate did not improve the detection rate. We recordeda 100% agreement in detection of T. vivax DNA using realtime machine and the water bath. In addition, a similaragreement was recorded using the gel electrophoresis andSYBR ® Green I fluorescence dye. The LAMP assay was specificand no cross reactivity was recorded with non-targetDNA, however the assay failed to amplify three T. vivaxsamples from Kenya.4. DiscussionLAMP technology represents an innovative strategy thathas the potential to offer alternative detection method ofpathogen DNA within a range of infectious diseases. TheT. vivax satellite DNA based LAMP assay designed here israpid and shows superior analytical sensitivity to classicalPCR tests (Table 2). Moreover amplification is achievedwithin 35 min for T. vivax DNA concentration ranging from10 ng to 1 pg (10 6 –1 trypanosomes/ml) using a real timePCR machine. This reaction time was recorded to increaseto 45 min when a normal water bath is used for amplification.Although the use of a normal water bath is alternativeTable 2The analysis of field samples from Kenya using T. vivax PCR and LAMP assay.Sample type No of samples PCR testsDA-PCR SA-PCR SA-LAMPTrypanosoma vivax isolates 23 7 (30.3%) 15 (65.2%) 20 (90%)Tsetse fly (T. vivax positive) c 3 – 3 3Buffy coat a (Trypanosome positive) 16 3 7 8Bovine samples b 357 6 (1.7%) 19 (5.3%) 27 (7.6%)DA = diagnostic antigen gene; SA = satellite repeat DNA.a Samples were collected from Lambwe valley and were confirmed trypanosome positive by microscopy.b Samples collected from Nguruman valley and found to be microscopically positive for trypanosomes (Njiru et al., 2005).c Samples stored at KARI-TRC cryo-bank and confirmed positive for trypanosomes at the time of storage.Please cite this article in press as: Njiru, Z.K., et al., Loop-mediated isothermal amplification test for Trypanosoma vivaxbased on satellite repeat DNA. Vet. Parasitol. (2011), doi:10.1016/j.vetpar.2011.03.021

G ModelVETPAR-5767; No. of Pages 5ARTICLE IN PRESS4 Z.K. Njiru et al. / Veterinary Parasitology xxx (2011) xxx–xxxto a thermal cycler, the need for power to heat water is stilla drawback. As such, alternative sources of heat such asexothermal chemicals and battery operated units need tobe considered. The recorded analytical sensitivity of 1 trypanosome/mlis a good detection level for T. vivax, notingits low parasitaemia in hosts. However, experience showsthat these laboratory based sensitivities may not necessarilybe reproducible under field conditions (Njiru et al.,2008). Thus, rigorous T. vivax LAMP field evaluations willbe the next major step.The potential usefulness of T. vivax LAMP as a point ofuse test is demonstrated by the ability of the new assayto amplify target DNA from mixed infections and supernatantprepared from spiked bovine blood, meaning thatDNA extraction may not be necessary. We recorded noinhibition of LAMP reaction or formation of non-specificproduct with the use of up to 3-l of supernatant per 25-l. The possibility of using partially processed templatesbypasses extraction, a contamination prone step, shortensthe LAMP test and improves detection limit since less DNAis lost. Thus T. vivax LAMP assay reported in this work hasa clear advantage over PCR in that the test is robust, simpleand requires less instrumentation to make a diagnosis.Further work is required to define the supernatant preparationprotocols and analyze various extraction and storagebuffers that may help improve supernatant viability.It is crucial to ensure that T. vivax LAMP amplifies thecorrect product since the LAMP strategy does not offer thechance of confirming the end product unlike in PCR whereDNA marker can be used to size the amplicon. In this workthe resulting LAMP product was confirmed through restrictionenzyme digestion with NdeI which gave the predictedtwo products of ∼80 bp and 140 bp sizes respectively. Inaddition, the acquisition and analysis of the melt curves(post LAMP amplification) not only showed reproduciblemelt curves but revealed consistent T m of ∼90 ◦ C indicatingsimilar sequence. A combination of these results andthree negative controls per each test run increases our confidencein using the non-specific SYBR ® Green I to progressthe test for further analysis. However absolute detection ofthe correct LAMP product can be achieved through the useof probes that bind to specific sequence within the LAMPproduct followed by detection of the resulting probe-LAMPproduct complex through a simple lateral flow dipstick(LFD) format (Njiru, 2011). This method has downside inthat it is expensive compared to non-specific dyes, howeverthe need for absolute diagnosis and the advantages ofusing supernatant as template merits further research inconverting LAMP/LFD into a single amplification and detectionsystem, a device that our research group is workingon.The diagnosis of T. vivax is the most challenging amongthe animal pathogenic trypanosomes. Therefore the developmentof LAMP test based on a target used to develop themost sensitive PCR test (Masiga et al., 1992) may potentiallyimprove the field diagnosis of this parasite. This perceptionis supported by the recorded superior sensitivity of LAMPtest over PCR tests in the analysis of T. vivax and bovine samplescollected from the endemic area (Table 2). The T. vivaxLAMP was specific and no cross reactivity was recordedwith other trypanosomes, however it failed to detect threesamples that were microscopically positive at the time ofpreservation. Two ideas could be advanced for this observation:(i) that the pathogen DNA concentration in thetemplate was below the LAMP detection level and/or (ii)the possibility that the three samples contained T. vivax thathad different satellite repeat DNA sequence. Previous studieshave identified isolates from East Africa (albeit few) thatare not detected by satellite DNA based PCR test (Adamset al., 2010). It is worth noting that the overall percentageof these isolates is so low and only a handful of themexist in the laboratories. Studies to sequence the satelliteDNA of the three isolates will be undertaken to reveal theirsequence composition.An initial comprehensive analysis of the bovine fieldsamples showed a trypanosome prevalence of 86/357(24.1%) consisting of 71 single infections (27 Trypanozoon,33 Nannomonas and 11 T. vivax) and 15 double infections (7Tbr/Tcs, 4Tbr/Tv and 4 Tcs/Tv) (Njiru et al., 2005). Analysisof the same samples with T. vivax LAMP not only confirmedthe T. vivax positive samples but picked an extra eight (8) T.vivax infected animals that were previously negative withPCR. Since DNA degradation may have occurred over theyears due to sample storage, the prevalence of this parasitecould have even be higher than earlier reported. Such undiagnosedcases continue to act as a source of new infectionin the field. Since mixed infection is a common occurrencein the field, research on development of a universal LAMPtest for pathogenic trypanosomes need to be considered.5. ConclusionIt is a routine procedure for animal blood to be collectedfrom the field and taken to the laboratory for analysis.Moreover, the animals that are treated during the field visitsare those that are microscopically positive and/or thosethat show convincing clinical symptoms as per the opinionof the treating veterinary officer. Once the laboratoryanalysis are complete, it is not always possible to go backand treat the sick animals due to expenses involved andthe migrating nature of the cattle owners. The LAMP technologydescribed here shows potential for field diagnosisand may be a future viable alternative to laboratory diagnosis.However before T. vivax LAMP can be deployed underfield conditions as a point of use test, a number of issueswill need to be addressed, namely; (i) template preparationprotocols, (ii) development of lyophilized reagents that arestable in the endemic areas, (iii) source of power for amplificationand (iv) development of accurate visual detectionformats. Until these objectives are realized, the T. vivaxLAMP test will be a valuable tool in mid-tier laboratorieswhere it may compliment PCR test.AcknowledgementsThis work was funded through the University ofQueensland postgraduate grant to Zablon Njiru and thesamples analyzed were contributed by Trypanosomiasisresearch Center, Kenya. The views expressed by the authorsdo not necessarily reflect the views of their respective institutes.Please cite this article in press as: Njiru, Z.K., et al., Loop-mediated isothermal amplification test for Trypanosoma vivaxbased on satellite repeat DNA. Vet. Parasitol. (2011), doi:10.1016/j.vetpar.2011.03.021

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