การตรวจหาตัวบ่งชี้ทางพันธุกรรมของเชื้อรา Aspergillus flavus ที่สร้าง อะฟลาทอกซิน B1 จากตัวอย่างข้าวโพดอาหารสัตว์ด้วยเทคนิค Loop-mediated isothermal amplification (LAMP)
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ก.ค. 11, 2018
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อะฟลาทอกซิน B1 Aspergillus flavus ข้าวโพด Loop-mediated isothermal amplification (LAMP) ยีนที่ควบคุมการสร้างอะฟลาทอกซิน B1
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บทคัดย่อ
การปนเปื้อนอะฟลาทอกซินในข้าวโพดอาหารสัตว์ซึ่งเป็นหนึ่งในผลผลิตทางการเกษตรนับเป็นปัญหาสำคัญที่เกิดขึ้นทั่วโลกโดยเฉพาะในเขตร้อนชื้นเช่นประเทศไทย การศึกษาครั้งนี้นำตัวอย่างข้าวโพดที่มีการติดเชื้อราตามธรรมชาติมาเพาะเลี้ยง และพิสูจน์เชื้อ Aspergillus flavus โดยการตรวจหายีน ITS, Beta-tubulin and calmodulin genes ด้วยปฏิกิริยาพอลีมอเรส (Polymerase chain reaction; PCR ) ร่วมกับพิสูจน์เชื้อ A. flavus ชนิดที่สามารถสร้างอะฟลาทอกซินB1 (AFB1) โดยการตรวจหายีนสี่ชนิดคือ Ver1, Omt1, Nor1, aflR ซึ่งเป็นยีนที่ควบคุมการสร้าง AFB1 ด้วยวิธี PCR และตรวจยืนยันการสร้าง AFB1 จากเชื้อที่แยกได้โดยตรงด้วยวิธี high performance liquid chromatography (HPLC) จากนั้นนำเทคนิค Loop-mediated isothermal amplification (LAMP) ซึ่งใช้ในการเพิ่มขยายและตรวจหายีนเป้าหมายมาทดสอบในเชื้อราที่แยกได้ โดยใช้ไพรเมอร์ที่จำเพาะต่อยีนทั้งสี่ชนิดดังกล่าว และใช้สารฟลูออเรสเซนต์ calcein เติมก่อนการเพิ่มปริมาณยีน พบว่าสภาวะที่เหมาะสมของปฏิกิริยา LAMP คือ การใช้อุณหภูมิคงที่ 65°C เป็นเวลา 1 ชั่วโมง เมื่อสิ้นสุดปฏิกิริยาสามารถอ่านผลได้ด้วยตาเปล่า มีความแตกต่างของสีอย่างชัดเจนระหว่างผลบวกและผลลบ โดยภายใต้แสงธรรมชาติผลบวกให้สีเหลืองและผลลบให้สีส้ม ส่วนภายใต้แสง UV ผลบวกจะเรืองแสงสีเขียว วิธีนี้มีความไวในการตรวจที่ความเข้มข้นดีเอ็นเอ 10 -6 (89 pg/µl) ส่วนวิธี PCR มีความไวในการตรวจที่ความเข้มข้นดีเอ็นเอ 10 -5 (0.0089 ng/µl) สามารถตรวจยืนยันผลผลิตจาก LAMP โดยการทำ agarose gel electrophoresis ที่ความเข้มข้นร้อยละ 1 จากนั้นนำเทคนิค LAMP นี้ไปใช้ในการตรวจข้าวโพดอาหารสัตว์ที่เก็บจากฟาร์มของเกษตรกรจำนวน 50 ตัวอย่าง พบผลบวก 18 ตัวอย่าง (36%) ผลลบ 32 ตัวอย่าง (64%) โดยมีความจำเพาะร้อยละ 100 เมื่อเปรียบเทียบกับวิธี multiplex PCR นอกจากนี้เมื่อวิเคราะห์หาความสัมพันธ์ระหว่างผลของ LAMP กับการตรวจวิเคราะห์ AFB1 ด้วยวิธี HPLC โดยใช้ค่าสัมประสิทธิ์สหสัมพันธ์เพียร์สันพบว่ามีความสัมพันธ์ทางบวกอย่างมีนัยสำคัญทางสถิติ (r = 0.83, P<0.05) แสดงว่าตัวอย่างข้าวโพดที่ให้ผลบวกด้วยเทคนิค LAMP มีความเสี่ยงสูงที่จะมีโอกาสปนเปื้อน AFB1 ดังนั้นเทคนิค LAMP ที่ใช้ในการศึกษาครั้งนี้สามารถตรวจหาตัวบ่งชี้ทางพันธุกรรมของเชื้อ A. flavus ที่สร้าง AFB1 จากตัวอย่างข้าวโพด โดยเป็นวิธีที่ง่ายและรวดเร็ว มีความไวและความจำเพาะสูง สามารถนำไปใช้เป็นวิธีการทดสอบความเป็นไปได้และความเสี่ยงของการปนเปื้อนอะฟลาทอกซินจากอาหารในปศุสัตว์ได้
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Mimapan, S., Wattanasuntorn, P. และ Saijit, P. (2018) “การตรวจหาตัวบ่งชี้ทางพันธุกรรมของเชื้อรา Aspergillus flavus ที่สร้าง อะฟลาทอกซิน B1 จากตัวอย่างข้าวโพดอาหารสัตว์ด้วยเทคนิค Loop-mediated isothermal amplification (LAMP)”, สัตวแพทย์มหานครสาร, 11(2), น. 141–158. available at: https://li01.tci-thaijo.org/index.php/jmvm/article/view/133921 (สืบค้น: 18 ธันวาคม 2025).
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Abarca, M.L., Bragulat, M.R., Bruguera, M.T. and Cabanes, F.J. 1988. Comparison of some screening methods for aflatoxigenic moulds. Mycopathologia. 104: 75–79.
Abdel-Hadi, A., Carter, D. and Magan, N. 2010. Temporal monitoring of the Nor1 (aflD) gene of Aspergillus flavus in relation to aflatoxin B1 production during storage of peanuts under different water activity levels. J. Appl. Microbiol. 109(6): 1914-1922.
Abd El-Salam, M.H., Hippen, A.R., El-Shafie, K., Assem, F.M., Abbas, H., Abd El-Aziz, M., Sharaf, O. and El-Aassar, M. 2011. Preparation and properties of probiotic concentrated yoghurt (labneh) fortified with conjugated linoleic acid. J. Food Sci. Tech. 46(10): 2103-2110.
AflaTest Instruction Manual. 1999. Aflatest Fluorometer Procedure for Corn, Milo, Grains and Feeds (0-300 ppb, 0.2 gram sample equivalent). Vicam, L.P. 313 Pleasant Street Watertown, MA 02472 U.S.A: 34-35.
Bennett, J.W. and Christensen, S.B. 1983. New perspectives on aflatoxin biosynthesis. Adv. Appl. Microbiol. 29: 53-92.
Bennett, J.W. and Papa, K.E. 1988. The aflatoxigenic Aspergillus spp. Adv. Plant Pathol. 6: 263-280.
Criseo, G., Bagnara, A. and Bisignano, G. 2001. Differentiation of aflatoxin producing and non-producing strains of Aspergillus flavus group. Department of Microbiological, Genetic and Molecular Sciences, Messina, Italy. Lett. Appl. Microbiol. 33: 291-295.
Espada, Y., Domingo, M., Gomez, J. and Calvo, M.A. 1992. Pathological lesions following an experimental intoxication with aflatoxin B1 in broiler chickens. Res. Vet. Sci. 53: 275-279.
Faber, P., Geisen, R. and Holzapfel, W.H. 1997. Detection of aflatoxinogenic fungi in figs by a PCR reaction. Int. J. Food Microbial. 36: 215–220.
Fink-Gremmels, J. 2008. Mycotoxins in cattle feeds and carry-over to dairy milk: A review. Food Addit. Contam. 25(2): 172-180.
Gieson, R. 1998. PCR methods for the detection of mycotoxin producing fungi. In Bridge, P.D., Arora, D.K., Reddy, C.A. and Elander, R.P. (eds). Applications of PCR in mycology. Wallingford - CAB international. P. 243-266.
Ghosh, M., Bhadra, S., Adegoke, A., Bandyopadhyay, M. and Mukherjee, A. 2015. MWCNT uptake in Allium cepa root cells induces cytotoxic and genotoxic responses and results in DNA hyper-methylation. Mutat. Res. 774: 49–58.
Glass, N.L. and Donaldson, G.C. 1995. Development of primer sets designed for use with the PCR to amplify conserved genes from Filamentous Ascomycetes. Appl. Environ. Microbiol. 61(4): 1323–1330.
Gqaleni, N., Smith, J.E. and Lacey, J. 1996. Co-production of aflatoxins and cyclopiazonic acid in isolates of Aspergillus flavus. Food Addit. Contam. 13(6): 677-685.
Hamed, K., Abbas, R.M., Zablotowicz, M.A., Weaver, B.W., Horn, W.X. and Shie, W.T. 2004. Comparison of cultural and analytical methods for determination of aflatoxin production by Mississippi Delta Aspergillus isolates. Can. J. Microbiol. 50: 193-199.
Harvey, R.B., Kubena, L.F. and Huff, W. E. 1989. Effects of aflatoxin, deoxynivalenol, and their combinations in the diets of growing pigs. Am. J. Vet. Res. 50: 602-608.
Haugland, R.A., Brinkman, N. and Vesper, S.J. 2002. Evaluation of rapid DNA extraction methods for the quantitative detection of fungi using real-time PCR analysis. J. Microbiol. Methods. 50: 319–323.
Hong, S.G., Cramer, R.A., Lawrence, C.B. and Pryor, B.M. 2005. Alt a 1 allergen homologs from Alternaria and related texa: analysis of phylogenetic content and secondary structure. Fungal Genet. Biol. 42: 119-129.
Horn, B.W. and Dorner, J.W. 1999. Regional difference in production of aflatoxin B1 and cyclopiazonic acid by soil isolates of Aspergillus flavus along a transect within the United States. Appl. Environ. Microbiol. 65(4): 1444-1449.
Hussein, H.S. and Brasel, J.M. 2001. Toxicity, metabolism, and impact of mycotoxins on humans and animals. Toxicology. 167: 101-134.
Ikadai, H., Tanaka, H., Shibahara, N., Matsuu, A., Uechi, M., Itoh, N., Oshiro, S., Kudo, N., Igarachi, I. and Oyamada, T. 2004. Molecular evidence of infections with Babesia gibsoni parasites in Japan and evaluation of the diagnostic potential of a loop-mediated isothermal amplification method. J. Clin. Microbiol. 42: 2465-2469.
Iwamoto, R., Yamazaki, S., Asakura, M., Takashima, S., Hasuwa, H., Miyado, K., Adachi, S., Kitakaze, M., Hashimoto, K., Raab, G., Nanba, D., Higashiyama, S., Hori, M., Klagsbrun, M, and Mekada, E. 2003. Heparin-binding EGF-like growth factor and ErbB signaling is essential for heart function. Proc. Natl. Acad. Sci. USA. 100: 3221–3226
Jiufeng, S., Najafzadeh, M.J., Vicente, V., Xi, L. and de Hoog, G.S. 2010. Rapid detection of pathogenic fungi using loop-mediated isothermal amplification, exemplified by Fonsecaea Agents of chromoblastomycosis. J. Microbiol. Methods. 80: 19-24.
Klich, M.A. and Pitt, J.I. 1988. Differentiation of Aspergillus flavus and Aspergillus parasiticus and other closely related species. 91(1): 99-108.
Kuboki, N., Inouc, N., Sakurai, T., Di Cello, F., Grab, D.J., Suzuki, H., Sugimoto, C., Igarashi, I. 2003. Loop-mediated isothermal amplification for detection of African trypanosomes. J. Clin. Microbiol. 41: 5517-5524.
Levin, R.E. 2012. PCR detection of aflatoxin producing fungi and its limitations. Int. J. Food Microbiol. 156: 1-6.
Lindemann, M.D., Blodgett, D.J., Kornegay, E.T. and Schurig, G.G. 1993. Potential ameliorators of aflatoxicosis in weaning growing swine. .J. Anim. Sci. 71: 171-178.
Masclaux, F., Gueho, E., Hoog, G.S.D. and Christien, R. 1995. Phylogenetic relationships of human pathogenic Clasdosporium (Xylohypha) species inferred from partial LS rRNA sequences. J. Med. Vet. Mycol. 33: 327-338.
Medina, A., Rodriguez, A., and Magan, N. 2014. Effect of climate change on Aspergillus flavus and aflatoxin B1 production. Front. Microbiol. 5: 348.
Mori, Y., Nagamine, K., Tomita, N. and Notomi, T. 2001. Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem. Biophys. Res. Commun. 289: 150-154.
Nagamine, K., Hase, T. and Notomi, T. 2002. Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol. Cell Probe. 16: 223–229.
Niessen, L., Luo, J., Denschlag, C. and Vogel, R.F. 2013. The application of loop-mediated isothermal amplification (LAMP) in food testing for bacterial pathogens and fungal contaminants. Food Microbiol. 36(2): 191–206.
Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K, Amino, N. and Hase, T. 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 15:28 (12): E63.
Peterson S.W., Vega, F.E., Posada, F. and Nagai, C. 2005. Penicillium coffeae, a new endophytic species isolated from a coffee plant and its phylogenetic relationship to P. fellutanum, P. thiersii and P. brocaeon parsimony analysis of multilocus DNA sequences. Mycologia. 97: 659–666.
Priyanka, S.R., Ramana, M.V., Balakrishna, K., Murali, H.S. and Batra, H.V. 2012. A novel non radioactive PCR- DNA Probes for detection of Aflatoxins producing Aspergillus species from major crops grown in India. Adv. Microbiol. 2: 577-586.
Rahimi, P., Sharifnabi, B. and Bahar, M. 2008. Detection of aflatoxin in Aspergillus species isolated from pistachio in Iran. Phytopathology. 156: 15–20.
Rodrigues, P., Soares, C., Kozakiewicz, Z., Paterson, R.R.M., Lima, N. and Venancio, A. 2007. Identification and characterization of Aspergillus flavus and aflatoxins. Commun. Curr. Res. Educ. Top. Trends Appl. Microbiol. A Mendez–Vilas (ed). 527-534.
Shapira, R., Paster, N., Eyal, O., Menasherov, M., Mett, A. and Salomon, R. 1996. Detection of aflatoxigenic molds in grains by PCR. Appl. Environ Microbiol. 62: 3270-3273.
Squire, R.A. 1981. Ranking animal carcinogens-a proposed regulatory approach. Science. 214: 877-880.
Varga, J., Frisvad, J.C. and Samson, R.A. 2011. The new aflatoxin producing species, and overview of Aspergillus section Flavi. Stud. Mycol. 69(1): 57-80.
Wacoo, A.P., Wendiro, D., Vuzi, P.C. and Hawumba, J.F. 2014. Methods for detection of aflatoxins in agricultural food crops. J. Appl. Chem. 2014(Article ID 706291): 1-15.
Yin, H., Chu, A., Li, W., Wang, B., Shelton, F. and Otero, F. 2009. Lipid G-protein-coupled receptor ligand identification using β-arrestin PathHunterTM assay. J. Biol. Chem. 284: 12328–12338.
Yu, J., Whitelaw, C.A., Nierman, W.C., Bhatnagar, D. and Cleveland, T.E. 2004. Aspergillus flavus expressed sequence tags for identification of genes with putative roles in aflatoxin contamination of crops. FEMS Microbiol. Lett. 237: 333-340.
White, T.J., Bruns, T., Lee, S. and Taylor, J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protocols: A Guide to Methods and Applications. p. 315-322.
Zhang, Y, Fu., P., Li, J., Jiang, F., Li, J. and Wu, W. 2011. Development of loop-mediated isothermal amplification method for visualization detection of the highly virulent strains of porcine reproductive and respiratory syndrome virus (PRRSV) in China. Afr. J. Biotechnol. 10(6): 13278-13283.
Zhou, S., Han, S., Shi, J., Wu, J., Yuan, X., Cong, X., Xu, S., Wu, X., Li, J. and Wang, J. 2011. Loop-mediated isothermal amplification for detection of porcine circovirus type 2. Virol. J. 8: 497-501.
Zhuang, L., Gong, J., Li, Q., Zhu, C., Yu, Y., Dou, X., Liu, X., Xu, B. and Wang, C. 2014. Detection of Salmonella spp. by a loop-mediated isothermal amplification (LAMP) method targeting bcfD gene. Lett. Appl. Microbiol. 59(6): 658-664.
Abdel-Hadi, A., Carter, D. and Magan, N. 2010. Temporal monitoring of the Nor1 (aflD) gene of Aspergillus flavus in relation to aflatoxin B1 production during storage of peanuts under different water activity levels. J. Appl. Microbiol. 109(6): 1914-1922.
Abd El-Salam, M.H., Hippen, A.R., El-Shafie, K., Assem, F.M., Abbas, H., Abd El-Aziz, M., Sharaf, O. and El-Aassar, M. 2011. Preparation and properties of probiotic concentrated yoghurt (labneh) fortified with conjugated linoleic acid. J. Food Sci. Tech. 46(10): 2103-2110.
AflaTest Instruction Manual. 1999. Aflatest Fluorometer Procedure for Corn, Milo, Grains and Feeds (0-300 ppb, 0.2 gram sample equivalent). Vicam, L.P. 313 Pleasant Street Watertown, MA 02472 U.S.A: 34-35.
Bennett, J.W. and Christensen, S.B. 1983. New perspectives on aflatoxin biosynthesis. Adv. Appl. Microbiol. 29: 53-92.
Bennett, J.W. and Papa, K.E. 1988. The aflatoxigenic Aspergillus spp. Adv. Plant Pathol. 6: 263-280.
Criseo, G., Bagnara, A. and Bisignano, G. 2001. Differentiation of aflatoxin producing and non-producing strains of Aspergillus flavus group. Department of Microbiological, Genetic and Molecular Sciences, Messina, Italy. Lett. Appl. Microbiol. 33: 291-295.
Espada, Y., Domingo, M., Gomez, J. and Calvo, M.A. 1992. Pathological lesions following an experimental intoxication with aflatoxin B1 in broiler chickens. Res. Vet. Sci. 53: 275-279.
Faber, P., Geisen, R. and Holzapfel, W.H. 1997. Detection of aflatoxinogenic fungi in figs by a PCR reaction. Int. J. Food Microbial. 36: 215–220.
Fink-Gremmels, J. 2008. Mycotoxins in cattle feeds and carry-over to dairy milk: A review. Food Addit. Contam. 25(2): 172-180.
Gieson, R. 1998. PCR methods for the detection of mycotoxin producing fungi. In Bridge, P.D., Arora, D.K., Reddy, C.A. and Elander, R.P. (eds). Applications of PCR in mycology. Wallingford - CAB international. P. 243-266.
Ghosh, M., Bhadra, S., Adegoke, A., Bandyopadhyay, M. and Mukherjee, A. 2015. MWCNT uptake in Allium cepa root cells induces cytotoxic and genotoxic responses and results in DNA hyper-methylation. Mutat. Res. 774: 49–58.
Glass, N.L. and Donaldson, G.C. 1995. Development of primer sets designed for use with the PCR to amplify conserved genes from Filamentous Ascomycetes. Appl. Environ. Microbiol. 61(4): 1323–1330.
Gqaleni, N., Smith, J.E. and Lacey, J. 1996. Co-production of aflatoxins and cyclopiazonic acid in isolates of Aspergillus flavus. Food Addit. Contam. 13(6): 677-685.
Hamed, K., Abbas, R.M., Zablotowicz, M.A., Weaver, B.W., Horn, W.X. and Shie, W.T. 2004. Comparison of cultural and analytical methods for determination of aflatoxin production by Mississippi Delta Aspergillus isolates. Can. J. Microbiol. 50: 193-199.
Harvey, R.B., Kubena, L.F. and Huff, W. E. 1989. Effects of aflatoxin, deoxynivalenol, and their combinations in the diets of growing pigs. Am. J. Vet. Res. 50: 602-608.
Haugland, R.A., Brinkman, N. and Vesper, S.J. 2002. Evaluation of rapid DNA extraction methods for the quantitative detection of fungi using real-time PCR analysis. J. Microbiol. Methods. 50: 319–323.
Hong, S.G., Cramer, R.A., Lawrence, C.B. and Pryor, B.M. 2005. Alt a 1 allergen homologs from Alternaria and related texa: analysis of phylogenetic content and secondary structure. Fungal Genet. Biol. 42: 119-129.
Horn, B.W. and Dorner, J.W. 1999. Regional difference in production of aflatoxin B1 and cyclopiazonic acid by soil isolates of Aspergillus flavus along a transect within the United States. Appl. Environ. Microbiol. 65(4): 1444-1449.
Hussein, H.S. and Brasel, J.M. 2001. Toxicity, metabolism, and impact of mycotoxins on humans and animals. Toxicology. 167: 101-134.
Ikadai, H., Tanaka, H., Shibahara, N., Matsuu, A., Uechi, M., Itoh, N., Oshiro, S., Kudo, N., Igarachi, I. and Oyamada, T. 2004. Molecular evidence of infections with Babesia gibsoni parasites in Japan and evaluation of the diagnostic potential of a loop-mediated isothermal amplification method. J. Clin. Microbiol. 42: 2465-2469.
Iwamoto, R., Yamazaki, S., Asakura, M., Takashima, S., Hasuwa, H., Miyado, K., Adachi, S., Kitakaze, M., Hashimoto, K., Raab, G., Nanba, D., Higashiyama, S., Hori, M., Klagsbrun, M, and Mekada, E. 2003. Heparin-binding EGF-like growth factor and ErbB signaling is essential for heart function. Proc. Natl. Acad. Sci. USA. 100: 3221–3226
Jiufeng, S., Najafzadeh, M.J., Vicente, V., Xi, L. and de Hoog, G.S. 2010. Rapid detection of pathogenic fungi using loop-mediated isothermal amplification, exemplified by Fonsecaea Agents of chromoblastomycosis. J. Microbiol. Methods. 80: 19-24.
Klich, M.A. and Pitt, J.I. 1988. Differentiation of Aspergillus flavus and Aspergillus parasiticus and other closely related species. 91(1): 99-108.
Kuboki, N., Inouc, N., Sakurai, T., Di Cello, F., Grab, D.J., Suzuki, H., Sugimoto, C., Igarashi, I. 2003. Loop-mediated isothermal amplification for detection of African trypanosomes. J. Clin. Microbiol. 41: 5517-5524.
Levin, R.E. 2012. PCR detection of aflatoxin producing fungi and its limitations. Int. J. Food Microbiol. 156: 1-6.
Lindemann, M.D., Blodgett, D.J., Kornegay, E.T. and Schurig, G.G. 1993. Potential ameliorators of aflatoxicosis in weaning growing swine. .J. Anim. Sci. 71: 171-178.
Masclaux, F., Gueho, E., Hoog, G.S.D. and Christien, R. 1995. Phylogenetic relationships of human pathogenic Clasdosporium (Xylohypha) species inferred from partial LS rRNA sequences. J. Med. Vet. Mycol. 33: 327-338.
Medina, A., Rodriguez, A., and Magan, N. 2014. Effect of climate change on Aspergillus flavus and aflatoxin B1 production. Front. Microbiol. 5: 348.
Mori, Y., Nagamine, K., Tomita, N. and Notomi, T. 2001. Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem. Biophys. Res. Commun. 289: 150-154.
Nagamine, K., Hase, T. and Notomi, T. 2002. Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol. Cell Probe. 16: 223–229.
Niessen, L., Luo, J., Denschlag, C. and Vogel, R.F. 2013. The application of loop-mediated isothermal amplification (LAMP) in food testing for bacterial pathogens and fungal contaminants. Food Microbiol. 36(2): 191–206.
Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K, Amino, N. and Hase, T. 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 15:28 (12): E63.
Peterson S.W., Vega, F.E., Posada, F. and Nagai, C. 2005. Penicillium coffeae, a new endophytic species isolated from a coffee plant and its phylogenetic relationship to P. fellutanum, P. thiersii and P. brocaeon parsimony analysis of multilocus DNA sequences. Mycologia. 97: 659–666.
Priyanka, S.R., Ramana, M.V., Balakrishna, K., Murali, H.S. and Batra, H.V. 2012. A novel non radioactive PCR- DNA Probes for detection of Aflatoxins producing Aspergillus species from major crops grown in India. Adv. Microbiol. 2: 577-586.
Rahimi, P., Sharifnabi, B. and Bahar, M. 2008. Detection of aflatoxin in Aspergillus species isolated from pistachio in Iran. Phytopathology. 156: 15–20.
Rodrigues, P., Soares, C., Kozakiewicz, Z., Paterson, R.R.M., Lima, N. and Venancio, A. 2007. Identification and characterization of Aspergillus flavus and aflatoxins. Commun. Curr. Res. Educ. Top. Trends Appl. Microbiol. A Mendez–Vilas (ed). 527-534.
Shapira, R., Paster, N., Eyal, O., Menasherov, M., Mett, A. and Salomon, R. 1996. Detection of aflatoxigenic molds in grains by PCR. Appl. Environ Microbiol. 62: 3270-3273.
Squire, R.A. 1981. Ranking animal carcinogens-a proposed regulatory approach. Science. 214: 877-880.
Varga, J., Frisvad, J.C. and Samson, R.A. 2011. The new aflatoxin producing species, and overview of Aspergillus section Flavi. Stud. Mycol. 69(1): 57-80.
Wacoo, A.P., Wendiro, D., Vuzi, P.C. and Hawumba, J.F. 2014. Methods for detection of aflatoxins in agricultural food crops. J. Appl. Chem. 2014(Article ID 706291): 1-15.
Yin, H., Chu, A., Li, W., Wang, B., Shelton, F. and Otero, F. 2009. Lipid G-protein-coupled receptor ligand identification using β-arrestin PathHunterTM assay. J. Biol. Chem. 284: 12328–12338.
Yu, J., Whitelaw, C.A., Nierman, W.C., Bhatnagar, D. and Cleveland, T.E. 2004. Aspergillus flavus expressed sequence tags for identification of genes with putative roles in aflatoxin contamination of crops. FEMS Microbiol. Lett. 237: 333-340.
White, T.J., Bruns, T., Lee, S. and Taylor, J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protocols: A Guide to Methods and Applications. p. 315-322.
Zhang, Y, Fu., P., Li, J., Jiang, F., Li, J. and Wu, W. 2011. Development of loop-mediated isothermal amplification method for visualization detection of the highly virulent strains of porcine reproductive and respiratory syndrome virus (PRRSV) in China. Afr. J. Biotechnol. 10(6): 13278-13283.
Zhou, S., Han, S., Shi, J., Wu, J., Yuan, X., Cong, X., Xu, S., Wu, X., Li, J. and Wang, J. 2011. Loop-mediated isothermal amplification for detection of porcine circovirus type 2. Virol. J. 8: 497-501.
Zhuang, L., Gong, J., Li, Q., Zhu, C., Yu, Y., Dou, X., Liu, X., Xu, B. and Wang, C. 2014. Detection of Salmonella spp. by a loop-mediated isothermal amplification (LAMP) method targeting bcfD gene. Lett. Appl. Microbiol. 59(6): 658-664.
