Abstract
Karnal bunt in wheat is caused by the fungus Tilletia indica. It is a quarantine disease, and therefore its timely and specific detection is important. Current detection protocols involve DNA amplification by polymerase chain reaction (PCR). However, this technique has encountered specificity issues due to the high DNA homology of T. indica with other Tilletia species, in particular T. walkeri. Here, we report the specific and rapid detection of T. indica DNA using the loop-mediated isothermal amplification (LAMP) at 62 °C. Alignment of the mitochondrial DNA of T. indica and T. walkeri has revealed four major unique regions in T. indica. Six LAMP primers designed in one of these unique regions were able to amplify T. indica DNA. The amplification could be completed in 30 min and was nearly as sensitive as conventional PCR. The amplification was found to be highly specific to T. indica DNA during the screening of 17 isolates of T. indica, T. walkeri, T. horrida, T. ehrhartae and T. caries. The use of the fluorescent chemical calcein has enabled endpoint detection with the naked eye. This method, with its specificity, rapidity and simple visualization, is well suited for the detection of this important disease in wheat.
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References
Castlebury LA, Carris LM (1999) Tilletia walkeri, a new species on Lolium multiflorum and L-perenne (vol 91, pg 121, 1999). Mycologia 91(2):416-+
Chen S, Wang F, Beaulieu JC, Stein RE, Ge B (2011) Rapid detection of viable salmonellae in produce by coupling Propidium Monoazide with loop-mediated isothermal amplification. Appl Environ Microbiol 77(12):4008–4016
Fan F, Du P, Kan B, Yan M (2015) The development and evaluation of a loop-mediated isothermal amplification method for the rapid detection of salmonella enterica serovar Typhi. PLoS One 10(4)
Feng J, Tang S, Liu L, Kuang X, Wang X, Hu S, You S (2015) Development of a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of common genetically modified organisms (GMOs). Int J Food Sci Nutr 66(2):186–196
Ferreira M, Tooley PW, Hatziloukas E, Castro C, Schaad NW (1996) Isolation of a species-specific mitochondrial DNA sequence for identification of Tilletia Indica, the Karnal bunt of wheat fungus. Appl Environ Microbiol 62(1):87–93
Frederick RD, Snyder KE, Tooley PW, Berthier-Schaad Y, Peterson GL, Bonde MR, Schaad NW, Knorr DA (2000) Identification and differentiation of Tilletia Indica and T. walkeri using the polymerase chain reaction. Phytopathology 90(9):951–960
Levy L, Castlebury LA, Carris LM, Meyer RJ, Pimentel G (2001) Internal transcribed spacer sequence-based phylogeny and polymerase chain reaction-restriction fragment length polymorphism differentiation of Tilletia Walkeri and T-indica. Phytopathology 91(10):935–940
Li QC, Fang JH, Liu X, Xi X, Li MJ, Gong YF, Zhang MZ (2013) Loop-mediated isothermal amplification (LAMP) method for rapid detection of cry1Ab gene in transgenic rice (Oryza sativa L.). Eur Food Res Technol 236(4):589–598
McDonald JG, Wong E, White GP (2000) Differentiation of Tilletia species by rep-PCR genomic fingerprinting. Plant Dis 84(10):1121–1125
Mishra A, Singh US, Goel R, Kumar A (2002) PCR based molecular technique for identification and discrimination of quarantined and non-quarantined Tilletia sps. Indian J Exp Biol 40(10):1137–1142
Mori Y, Notomi T (2009) Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases. J Infect Chemother 15(2):62–69
Nagamine K, Watanabe K, Ohtsuka K, Hase T, Notomi T (2001) Loop-mediated isothermal amplification reaction using a nondenatured template. Clin Chem 47(9):1742–1743
Nagamine K, Hase T, Notomi T (2002) Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes 16(3):223–229
Nakao R, Stromdahl EY, Magona JW, Faburay B, Namangala B, Malele I, Inoue N, Geysen D, Kajino K, Jongejan F, et al. (2010) Development of Loop-Mediated Isothermal Amplification (LAMP) Assays for Rapid Detection of Ehrlichia ruminantium Bmc Microbiology:10
Niessen L, Luo J, Denschlag C, Vogel RF (2013) The application of loop-mediated isothermal amplification (LAMP) in food testing for bacterial pathogens and fungal contaminants. Food Microbiol 36(2):191–206
Nixon G, Garson JA, Grant P, Nastouli E, Foy CA, Huggett JF (2014) Comparative study of sensitivity, linearity, and resistance to inhibition of digital and Nondigital polymerase chain reaction and loop mediated isothermal amplification assays for quantification of human cytomegalovirus. Anal Chem 86(9):4387–4394
Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28(12):E63
Notomi T, Mori Y, Tomita N, Kanda H (2015) Loop-mediated isothermal amplification (LAMP): principle, features, and future prospects. J Microbiol 53(1):1–5
Ou SC, Giambrone JJ, Macklin KS (2012) Comparison of a TaqMan real-time polymerase chain reaction assay with a loop-mediated isothermal amplification assay for detection of Gallid herpesvirus 1. J Vet Diagn Investig 24(1):138–141
Pack TD, Deng X (2008) Stable reagents and kits useful in loop-mediated isothermal amplification (LAMP). US Patent US 20080182312:A1
Pimentel G, Carris LM, Levy L, Meyer RJ (1998) Genetic variability among isolates of Tilletia Barclayana, T-indica and allied species. Mycologia 90(6):1017–1027
Smith OP, Peterson GL, Beck RJ, Schaad NW, Bonde MR (1996) Development of a PCR-based method for identification of Tilletia Indica, causal agent of Karnal bunt of wheat. Phytopathology 86(1):115–122
Tan MK, Murray GM (2006) A molecular protocol using quenched FRET probes for the quarantine surveillance of Tilletia Indica, the causal agent of Karnal bunt of wheat. Mycol Res 110:203–210
Tan M, Ghalayini A, Indu S, Yi J, Shivas R, Priest M, Wright D (2009) A one-tube fluorescent assay for the quarantine detection and identification of Tilletia Indica and other grass bunts in wheat. Australas Plant Pathol 38(2):101–109
Tomita N, Mori Y, Kanda H, Notomi T (2008) Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat Protoc 3(5):877–882
Yang BC, Wang FX, Zhang SQ, Song N, Li JX, Yang ZQ, Wen YJ, Wu H (2013) Comparative evaluation of conventional polymerase chain reaction (PCR), with loop-mediated isothermal amplification and SYBR green I-based real-time PCR for the quantitation of porcine circovirus-1 DNA in contaminated samples destined for vaccine production. J Virol Methods 191(1):1–8
Zhou D, Guo J, Xu L, Gao S, Lin Q, Wu Q, Wu L, and Que Y. 2014. Establishment and application of a loop-mediated isothermal amplification (LAMP) system for detection of cry1Ac transgenic sugarcane. Scientific Reports 4:4912
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The authors would like to thank CSIRO Manufacturing and Biosecurity Flagships for funding the research.
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Gao, Y., Tan, M.K. & Zhu, Y.G. Rapid and specific detection of Tilletia indica using loop-mediated isothermal DNA amplification. Australasian Plant Pathol. 45, 361–367 (2016). https://doi.org/10.1007/s13313-016-0422-7
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DOI: https://doi.org/10.1007/s13313-016-0422-7