Loop-mediated isothermal amplification test for Trypanosoma vivax ...
Loop-mediated isothermal amplification test for Trypanosoma vivax ... Loop-mediated isothermal amplification test for Trypanosoma vivax ...
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 PRESSZ.K. Njiru et al. / Veterinary Parasitology xxx (2011) xxx–xxx 3Table 1The analytical sensitivity of T. <strong>vivax</strong> LAMP assay compared with PCR <strong>test</strong>s using templates from 10-fold serial dilution of T. <strong>vivax</strong> isolate EATRO 1186.Type of <strong>test</strong> 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. <strong>vivax</strong> + + + + + + + – – 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. <strong>vivax</strong> EATRO 1186DNA was used to compare and determine the analyticalsensitivity of T. <strong>vivax</strong> LAMP and the PCR <strong>test</strong>s. The templatewas 1-l <strong>for</strong> each serial dilution. The specificity ofthe LAMP <strong>test</strong> 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. <strong>for</strong>est, 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. <strong>vivax</strong> DNAas previously described (Njiru et al., 2008) and 2-l ofthe template was used. 25-l LAMP reactions (Section 2.3)were carried out <strong>for</strong> 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 <strong>for</strong> each dilution and increasedthe assay analytical sensitivity by 10 3 -fold to an equivalentof 1 trypanosome/ml (1 pg). The post <strong>amplification</strong>analysis of LAMP product showed reproducible melt curveswith a melting temperature (T m )of∼90 ◦ C <strong>for</strong> all T. <strong>vivax</strong>isolates while restriction enzyme NdeI gave the predictedsizes of ∼80 bp and 140 bp respectively. T. <strong>vivax</strong> LAMP <strong>test</strong>was 10 and 10 3 -fold more sensitive than satellite repeatPCR (Masiga et al., 1992) and diagnostic antigen PCR <strong>test</strong>(Masake et al., 1997) respectively (Table 1). The positivereactions showed ladder like pattern after electrophoresisin agarose gel indicating the <strong>for</strong>mation of stem-loops andturned green on addition of 1/10 dilution of SYBR ® GreenI. On the analysis of the T. <strong>vivax</strong> samples, the LAMP <strong>test</strong>detected 20/23, satellite repeat 15/23 and the diagnosticantigen PCR 7/23 (Table 2). The analysis of 357 archivedbovine samples revealed a T. <strong>vivax</strong> prevalence of 7.6%through LAMP <strong>test</strong>, 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. <strong>vivax</strong> 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. <strong>vivax</strong>samples 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. <strong>vivax</strong> satellite DNA based LAMP assay designed here israpid and shows superior analytical sensitivity to classicalPCR <strong>test</strong>s (Table 2). Moreover <strong>amplification</strong> is achievedwithin 35 min <strong>for</strong> T. <strong>vivax</strong> 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 <strong>for</strong> <strong>amplification</strong>.Although the use of a normal water bath is alternativeTable 2The analysis of field samples from Kenya using T. <strong>vivax</strong> PCR and LAMP assay.Sample type No of samples PCR <strong>test</strong>sDA-PCR SA-PCR SA-LAMP<strong>Trypanosoma</strong> <strong>vivax</strong> isolates 23 7 (30.3%) 15 (65.2%) 20 (90%)Tsetse fly (T. <strong>vivax</strong> 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 <strong>for</strong> trypanosomes (Njiru et al., 2005).c Samples stored at KARI-TRC cryo-bank and confirmed positive <strong>for</strong> trypanosomes at the time of storage.Please cite this article in press as: Njiru, Z.K., et al., <strong>Loop</strong>-<strong>mediated</strong> <strong>isothermal</strong> <strong>amplification</strong> <strong>test</strong> <strong>for</strong> <strong>Trypanosoma</strong> <strong>vivax</strong>based on satellite repeat DNA. Vet. Parasitol. (2011), doi:10.1016/j.vetpar.2011.03.021
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