环介导等温扩增（LAMP）技术检测蜜蜂球囊菌

doi:10.3864/j.issn.0578-1752.2016.04.015

Abstract

Abstract: 【Objective】 The objective of this study is to establish a rapid and sensitive method for detecting the Ascosphaera apis in epidemiological investigations by the loop-mediated isothermal amplification (LAMP), and to provide technical supports for monitoring and prevention of chalkbrood in honeybee.【Method】Four specific primers were designed based on the particular sequence of ITS of the A. apis using the software of PrimerExplorer V4.0 online. The primers included A. apis-F3 (5′-ACATTGC GCCCTCTGGTA-3′), A. apis-B3 (5′-TGGTTAGACCGGACAGTCG-3′), A. apis-FIP (5′-TAAGACGGGACGATCGCCCAACC TGTCCGAGCGTCATTG-3′) and A. apis-BIP (5′-GAAAGGCAGTGACGGCGTCGGGCCACTAGAGCGAAAGAC-3′). LAMP were performed under different concentrations such as Mg²⁺ (0, 2, 4, 6, 8, 10, 12, and 14 mmol·L^-1), dNPTs (0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, and 1.8 mmol?L^-1), FIB/BIF (0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, and 1.8 mmol·L^-1), betaine (0, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mol?L^-1), reaction temperatures (58, 60, 63, and 65℃) and reaction times (30, 40, 50, and 60 min), respectively. The LAMP result was determined by both the turbidimeter and agarose gel electrophoresis in order to optimize the reaction conditions. The specificity of LAMP was tested by using genomic DNA of Nosema ceranae, Nosema apis, Deformed wing virus (DWV), Sacbrood virus (SBV), Black queen cell virus (BQCV) and Israel acute paralysis virus (IAPV). In addition, the LAMP amplified products were digested with PvuⅠrestriction enzyme. The sensitivity of LAMP was compared with normal PCR by employing ten-fold serially diluted DNA templates of A. apis (including 0.2231, 0.2231×10^-1, 0.2231×10^-2, 0.2231×10^-3, 0.2231×10^-4, 0.2231×10^-5, 0.2231×10^-6, 0. 2231×10^-7, and 0.2231×10^-8 μg·μL^-1). The practical reliability was achieved in clinical trials.【Result】A rapid and specific method for detction of A. apis was established. The optimum conditions for LAMP reaction were as follows: 4 mmol·L^-1 Mg²⁺, 1.2 mmol·L^-1dNTPs, 1.6 mmol·L^-1FIP/BIP, 0.4 mol·L^-1betaine, and the method was performed at 63℃ for 60 min. The templates of A. apis, N. ceranae, N. apis, DWV, SBV, BQCV and IAPV were tested, the result showed that only the A. apis DNA template could produce typical ladder-like bands. The sensitivity test showed that the PCR could detect the DNA templates as low as 0.2231×10^-5 μg·μL^-1. While the LAMP could detect ten times lower DNA templates of 0.2231×10^-6μg·μL^-1. The LAMP is effective, simple and time saving for detecting the A. apis infect honeybee. 【Conclusion】The method of LAMP was proved to be a precise, faster, lower cost in diagnosis of chalkbrood in honeybee. It also could be applied in both research institutions and field.

Key words: loop-mediated isothermal amplification (LAMP), Ascosphaera apis, rapidly detective, real-time turbidity

XI Wei-jun, LI Jiang-hong, CHEN Da-fu, LIANG Qin. Diagnosis of the Ascosphaera apis by the Loop-Mediated Isothermal Amplification[J].Scientia Agricultura Sinica, 2016, 49(4): 765-774.

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References

[1]    Gupta R K, Reybroeck W. Honeybee pathogens and their management// Beekeeping for Poverty Alleviation and Livelihood Security, Springer, 2014: 297-320.
[2]    Vojvodic S, Jensen A B, James R R, Boomsma J J, Eilenberg J. Temperature dependent virulence of obligate and facultative fungal pathogens of honeybee brood. Veterinary Microbiology, 2011, 149: 200-205.

[3]    梁勤, 陈大福. 蜜蜂保护学. 2版. 北京: 中国农业出版社, 2009: 83-84.

Liang Q, Chen D F. Bee Protection. 2nd ed. Beijing: China Agriculture Press, 2009: 83-84. (in Chinese)

[4]    晏励民, 刘元珍, 刘芳, 苏松坤. 蜜蜂抗白垩病机制的研究进展. 应用昆虫学报, 2013, 50(6): 1700-1705.

Yan L M, Liu Y Z, Liu F, Su S K. Progress in research on resistance to chalkbrood in honeybees. Chinese Journal of Applied Entomology, 2013, 50(6): 1700-1705. (in Chinese)

[5]    Aizen M A, Garibaldi L A, Cunningham S A, Klein A M. How much does agriculture depend on pollinators? Lessons from long-term trends in crop production. Annals of Botany, 2009, 103(9): 1579-1588.

[6]    Aronstein K A, Murray K D. Chalkbrood disease in honey bees. Journal of Invertebrate Pathology, 2010, 103: S20-S29.

[7]    Chorbiński P, Rypu?a K. Studies on the morphology of strains Ascosphaera apis isolated from chalkbrood disease of the honey bees. Electronic Journal of Polish Agricultural Universities, 2003, 6(2): Series veterinary medicine.

[8]    Bissett J. Contribution toward a monograph of the genus Ascosphaera. Canadian Journal of Botany, 1988, 66: 2541-2560.

[9]    Gilliam M, Lorenz B J, Wenner A M, Thorp R W. Occurrence and distribution of Ascosphaera apis in North America: chalkbrood in feral honey bee colonies that had been in isolation on Santa Cruz Island, California for over 110 years. Apidologie, 1997, 28: 329-338.

[10]   James R R, Skinner J S. PCR diagnostic methods for Ascosphaera infections in bees. Journal of Invertebrate Pathology, 2005, 90(2): 98-103.

[11]   Garrido-Bailon E, Higes M, Martinez-Salvador A, Antunez K, Botias C, Meana, A, Prieto L, Martin-Hernandez R. The prevalence of the honeybee brood pathogens Ascosphaera apis, Paenibacillus larvae and Melissococcus plutonius in Spanish apiaries determined with a new multiplex PCR assay. Microbe Biotechnology, 2013, 6(6): 731-739.

[12]   宋战昀, 王振国, 冯新, 王伟利, 罗雁非, 刘金华, 姜永莉. 蜜蜂球囊菌荧光PCR快速检测方法的建立及应用. 动物医学进展, 2010, 31(12): 49-53.

Song Z Y, Wang Z G, Feng X, Wang W L, Luo Y F, Liu J H, Jiang Y L. Establishment and application of real-time PCR for detection of Ascosphaera apis. Progress in Veterinary Medicine, 2010, 31(12): 49-53. (in Chinese)

[13]   Lee H M, Nam S H, Yoon B S. PCR detection method of Ascosphera apis, Aspergillus flavus for rapid identification of fungal disease in honeybee. Journal of Apiculture, 2004, 19(2): 1-10.

[14]   Murray K D, Aronstein K A, Jones W A. A molecular diagnostic method for selected Ascosphaera species using PCR amplification of internal transcribed spacer regions of rDNA. Journal of Apicultural Research, 2005, 44(2): 61-64.

[15]   Aronstein K A, Murray K D, de León J H, Qin X, Weinstock G M. High mobility group (HMG-box) genes in the honey bee fungal pathogen Ascosphaera apis. Mycologia, 2007, 99(4): 553-561.

[16]   Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T. Loop-mediated isothermal amplifictaion of DNA. Nucleic Acids Research, 2000, 28(12): e63.

[17]   庄明亮, 李江红, 陈大福, 梁勤. 应用环介导等温扩增技术 (LAMP)检测蜜蜂黑蜂王台病毒的研究. 应用昆虫学报, 2014, 51(6): 1612-1619.

Zhuang M L, Li J H, Chen D F, Liang Q. Diagnosis of the black queen cell virus (BQCV) by loop-mediated isothermal amplification. Chinese Journal of Applied Entomology,2014, 51(6): 1612-1619. (in Chinese)

[18]   No J N, Lee B R, Yoo M S, Yoon B S. Development of RT-LAMP (reverse transcription loop-mediated isothermal amplification) for detection of capsid protein in IAPV (Israel acute paralysis virus). Journal of Apiculture, 2011, 26(2): 119-127.

[19]   Nguyen P V, Lee B, Yoo M S, Yoon B S. Development and clinical validation of a DNA gyrase subunit B gene-based loop-mediated isothermal amplification method for detection of Melissococcus plutonius. Journal of Apiculture, 2012, 27(1): 51-58.

[20]   Nguyen P V, Han S H, Yoon B S. Development of a metalloproteinase gene based loop mediated isothermal amplification assay for detection of Paenibacillus larvae. Journal of Apiculture, 2011, 26(3): 213-220.

[21]   Lee S J, Yong S J, Lim H Y, Yoon B S. A simple sensitive gene-based diagnosis of Aspergillus flavus by loop-mediated isothermal amplification in honeybee. Journal of Apiculture, 2015, 30(1): 53-59.

[22] Ptaszynska A A, Borsuk G, Wozniakowski G, Gnat S, Malek W. Loop-mediated isothermal amplification (LAMP) assays for rapid detection and differentiation of Nosema apis and N. ceranae in honeybees. FEMA Microbiology Letters, 2014, 357(1): 40-48.

[23]   Lee B R, Yoo M S, Phu N V, No J N, Yoon B U. Development of method for the detection of Ascophaera apis by loop-mediated isothermal amplification. Journal of Apiculture, 2011, 26(2): 103-111.

[24]   Lee G J, Lim H, Yoo B S. Development of specific detection method for fungal pathogens in honeybee by loop-mediated isothermal amplification. Journal of Apiculture, 2013, 28(4): 245-250.

[25]   Anderson D L, Gibbs A J, Gibson N L. Identification and phylogeny of spore-cyst fungi (Ascosphaera spp.) using ribosomal DNA sequences. Mycological Research, 1998, 102(5): 541-547.

[26]   Chorbinski P. Identification of Ascosphaera apis strains using ribosomal DNA sequences. Medycyna Weterynaryjna, 2004, 60(2): 190-192.

[27]   Jensen A B, Aronstein K, Manuel Flores J, Vojvodic S, Alejandra Palacio M, Spivak M. Standard methods for fungal brood disease research. Journal of Apicultural Research, 2013, 52(1): http://dx.doi. org/10.3896/IBRA. 1.52.1.13.

[28]   熊翠玲. 蜜蜂白垩病分子诊断技术的研究[D]. 福州: 福建农林大学, 2010.

Xiong C L. Development of molecular diagnostic technique for chalkbrood disease[D]. Fuzhou: Fujian Agriculture and Forestry University, 2010. (in Chinese)

[29]   李江红, 郑志阳. 蜜蜂球囊菌在蜜蜂体内的生长涉及多种胞外酶对蜜蜂幼虫组织的降解作用//“蜂之巢”2010年全国蜂产品市场信息交流会暨中国（武汉）蜂业博览会, 武汉, 2010.

Li J H, Zheng Z Y. The growth of Ascosphaera apis in vivo involving in the extracellular enzymes degradated tissues of infecting the honeybee larvae//“Honeycomb” The National Information Semminar of Bee Products Market and China’s Apiculture Fair in 2010. Wuhan, 2010. (in Chinese)

[30]   Crailsheim K, Brodschneider R, Aupinel P, Behrens D, Genersch E, Vollman J, Riessberger-Galle U. Standard methods for artificial rearing of Apis mellifera larvae. Journal of Apicultural Research, 2013, 52(1): http://dx.doi.org/10.3896/IBRA. 1.52.1.05.

[31]   Gardes M, Bruns T D. ITS primers with enhanced specificity for basidiomycetes-application to the identification of mycorrhizae and rusts. Molecular Ecology, 1993, 2: 113-118.

[32]   刘春来, 文景芝, 杨明秀, 李永刚. rDNA-ITS在植物病原真菌分子检测中的应用. 东北农业大学学报, 2007, 38(1): 101-106.

Liu C L,Wen J Z, Yang M X, Li Y G. Application of rDNA- ITS in molecular test of phytopathogenic fungi. Journal of Northeast Agricultural University, 2007, 38(1): 101-106. (in Chinese)

[33]   Hershkovitz M A, Lewis L A. Deep-level diagnostic value of the rDNA-ITS region. Molecular Biology and Evolution, 1996, 13(9): 1276-1295.

[34]   孙广宇, 彭友良, 李振歧, 张天宇. 核苷酸序列分析在真菌系统学研究中的应用. 西北农林科技大学学报: 自然科学版, 2003, 31(6): 187-192.

Sun G Y, Peng Y L, Li Z Q, Zhang T Y. Application of sequencing technique on fungal systematics. Joural of Northwest Sci-Tech University of Agriculture and Forestry: Natural Science Edition, 2003, 31(6): 187-192. (in Chinese)

[35]   Tomita N, Mori Y, Kanda H, Notomi T. Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nature Protocols, 2008, 5(3): 877-882.

[36]   匡燕云. 环介导等温扩增核酸技术检测嗜水气单胞菌方法的建立[D]. 南昌: 南昌大学, 2007.

Kuang Y Y. Establishment of loop-mediated isothermal amplification method in detection of Aeromonas hydrophila[D]. Nanchang: Nanchang University, 2007. (in Chinese)

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