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