Loop-mediated isothermal amplification for diagnosing marine pathogens in tissues of Crassostrea spp. and white shrimp, Litopenaeus vannamei, farmed in Mexico

Main Article Content

Ismael Mendoza-Avilés
Carla A Muñoz-Rojas
Mario Rojas
Norma Estrada

Abstract

Loop-mediated isothermal amplification (LAMP) is an accurate, sensitive, rapid, and easy-to-perform method for gene amplification under isothermal conditions, and it has served as a powerful diagnostic tool. In this study, we used LAMP to develop a diagnostic protocol for detecting Vibrio parahaemolyticus and white spot syndrome virus in whiteleg shrimp, and Perkinsus spp. in Crassostrea spp. in Mexico. These pathogens are associated with different diseases and are considered a threat in the aquaculture industry. Infected and uninfected oysters and shrimp were obtained from farms in the northwest coast of Mexico to standardize the LAMP assay. We determined the candidate target genes in the first-round analysis of many sets of primers, and then we chose a set of primers that successfully amplified with Mexican samples. We optimized the LAMP reactions for each pathogen with the chosen primer sets using temperature gradients from 61 to 65 ºC, DNA concentrations from 2.5 pg to 250.0 ng, and reaction times from 10 to 60 min. This study established a diagnostic procedure for detecting pathogens in oysters and shrimp from Mexico. Early diagnosis and treatment of pathogens can immensely reduce disease transmission in aquaculture farms.

Downloads

Download data is not yet available.

Article Details

How to Cite
Mendoza-Avilés, I., Muñoz-Rojas, C. A., Rojas, M., & Estrada, N. (2021). Loop-mediated isothermal amplification for diagnosing marine pathogens in tissues of Crassostrea spp. and white shrimp, Litopenaeus vannamei, farmed in Mexico. Ciencias Marinas, 47(4), 227–239. https://doi.org/10.7773/cm.v47i4.3095
Section
Research Article

Metrics

References

Arunrut N, Prombun P, Saksmerprome V, Flegel TW, Kiatpathomchai W. 2011. Rapid and sensitive detection of infectious hypodermal and hematopoietic necrosis virus by loop-mediated isothermal amplification combined with a lateral flow dipstick. J Virol Methods. 171(1):21–25.

https://doi.org/10.1016/j.jviromet.2010.09.022

Becherer L, Borst N, Bakheit M, Frischmann S, Zengerle R, von Stetten F. 2020. Loop-mediated isothermal amplification (LAMP) – review and classification of methods for sequence-specific detection. Anal Methods. 12(6):717–746.

https://doi.org/10.1039/c9ay02246e

Besuschio SA, Llano-Murcia M, Benatar AF, Monnerat S, Cruz I, Picado A, Curto MA, Kubota Y, Wehrendt DP, Pavia P, et al. 2017. Analytical sensitivity and specificity of a loop-mediated isothermal amplification (LAMP) kit prototype for detection of Trypanosoma cruzi DNA in human blood samples. PLoS Negl Trop Dis. 11(7):e0005779.

https://doi.org/10.1371/journal.pntd.0005779

Boehme CC, Nabeta P, Henostroza G, Raqib R, Rahim Z, Gerhardt M, Sanga E, Hoelscher M, Notomi T, Hase T, et al. 2007. Operational feasibility of using loop-mediated isothermal amplification for diagnosis of pulmonary tuberculosis in microscopy centers of developing countries. J Clin Microbiol. 45(6):1936–1940.

https://doi.org/10.1128/JCM.02352-06

Cáceres-Martínez J, Vásquez-Yeomans R, Padilla-Lardizábal G, del Río Portilla MA. 2008. Perkinsus marinus in pleasure oyster Crassostrea corteziensis from Nayarit, Pacific coast of México. J Invert Pathol. 99(1):66–73.

https://doi.org/10.1016/j.jip.2008.03.005

Chen S, Ge B. 2010. Development of a toxR-based loop-mediated isothermal amplification assay for detecting Vibrio parahaemolyticus. BMC Microbiol. 10(1):41.

https://doi.org/10.1186/1471-2180-10-41

Dhama K, Karthik K, Chakraborty S, Tiwari R, Kapoor S, Kumar A, Thomas P. 2014. Loop-mediated isothermal amplification of DNA (LAMP): a new diagnostic tool lights the world of diagnosis of animal and human pathogens: a review. PJBS. 17(2):151–166.

https://doi.org/10.3923/pjbs.2014.151.166

Enríquez-Espinoza TL, Grijalva-Chon JM, Castro-Longoria R, Ramos-Paredes J. 2010. Perkinsus marinus in Crassostrea gigas in the Gulf of California. Dis Aquat Organ. 89(3):269–273.

https://doi.org/10.3354/dao02199

[FAO] Food and Agriculture Organization of United Nations. 2019. FAO Yearbook of fishery and aquaculture statistics. Rome (Italy): FAO. 84 p. ISBN 978-92-5-131669-6.

Feng C, Wang C, Lin X, Zhang Y, Lv J, Deng J, Yuan X, Mei L, Wu S. 2013. Development of a loop-mediated isothermal amplification method for detection of Perkinsus spp. in mollusks. Dis Aquat Organ. 104(2):141–148.

https://doi.org/10.3354/dao02591

Francois P, Tangomo M, Hibbs J, Bonetti EJ, Boehme CC, Notomi T, Perkins MD, Schrenzel J. 2011. Robustness of a loop-mediated isothermal amplification reaction for diagnostic applications. FEMS Immunol Med Mic. 62(1):41–48.

https://doi.org/10.1111/j.1574-695x.2011.00785.x

Goto M, Honda E, Ogura A, Nomoto A, Hanaki KI. 2009. Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue. BioTechniques. 46(3):167–172.

https://doi.org/10.2144/000113072

Hasson KW, Fan Y, Reisinger T, Venuti J, Varner PW. 2006. White-spot syndrome virus (WSSV) introduction into the Gulf of Mexico and Texas freshwater systems through imported, frozen bait-shrimp. Dis Aquat Organ. 71(2):91–100.

https://doi.org/10.3354/dao071091

Iwamoto T, Sonobe T, Hayashi K. 2003. Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex, M. avium, and M. intracellulare in sputum samples. J Clin Microbiol. 41(6):2616–2622.

https://doi.org/10.1128/JCM.41.6.2616-2622.2003

Jaroenram W, Kiatpathomchai W, Flegel TW. 2009. Rapid and sensitive detection of white spot syndrome virus by loop-mediated isothermal amplification combined with a lateral flow dipstick. Mol Cell Probe. 23(2):65–70.

https://doi.org/10.1016/j.mcp.2008.12.003

Kaneko H, Kawana T, Fukushima E, Suzutani T. 2007. Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances. J Biochem Biophys Methods. 70(3):499–501.

https://doi.org/10.1016/j.jbbm.2006.08.008

Khan M, Li B, Jiang Y, Weng Q, Chen Q. 2017. Evaluation of different PCR-based assays and LAMP method for rapid detection of Phytophthora infestans by targeting the ypt1 gene. Front Microbiol. 8:1920.

https://doi.org/10.3389/fmicb.2017.01920

Khunthong S, Jaroenram W, Arunrut N, Suebsing R, Mungsantisuk I, Kiatpathomchai W. 2013. Rapid and sensitive detection of shrimp yellow head virus by loop-mediated isothermal amplification combined with a lateral flow dipstick. J Virol Methods. 188(1–2):51–56.

https://doi.org/10.1016/j.jviromet.2012.11.041

Kiatpathomchai W, Jaroenram W, Arunrut N, Jitrapakdee S, Flegel TW. 2008. Shrimp Taura syndrome virus detection by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick. J Virol Methods. 153(2):214–217.

https://doi.org/10.1016/j.jviromet.2008.06.025

Kongrueng J, Tansila N, Mitraparp-arthorn P, Nishibuchi M, Vora, GJ, Vuddhakul V. 2015. LAMP assay to detect Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease in shrimp. Aquac Int. 23(5):1179–1188.

https://doi.org/10.1007/s10499-014-9874-3

Kono T, Savan R, Sakai M, Itami T. 2004. Detection of white spot syndrome virus in shrimp by loop-mediated isothermal amplification. J Virol Methods. 115(1):59–65.

https://doi.org/10.1016/j.jviromet.2003.09.015

Li J, Macdonald J. 2015. Advances in isothermal amplification: novel strategies inspired by biological processes. Biosens Bioelectron. 64:196–211.

https://doi.org/10.1016/j.bios.2014.08.069

Maralit BA, Caipang CMA, Santos MD, Calpe AT, Maningas MBB. 2012. Development of primers for loop-mediated isothermal amplification of Philippine white spot syndrome virus isolates. AACL Bioflux. 5(4):274–281.

Mekata T, Kono T, Savan R, Sakai M, Kasornchandra J, Yoshida T, Itami T. 2006. Detection of yellow head virus in shrimp by loop-mediated isothermal amplification (LAMP). J Virol Methods. 135(2):151–156.

https://doi.org/10.1016/j.jviromet.2006.02.012

Mekata T, Sudhakaran R, Kono T, Supamattaya K, Linh NTH, Sakai M, Itami T. 2009. Real-time quantitative loop-mediated isothermal amplification as a simple method for detecting white spot syndrome virus. Lett Appl Microbiol. 48(1):25–32.

https://doi.org/10.1111/j.1472-765x.2008.02479.x

Mori Y, Kanda H, Notomi T. 2013. Loop-mediated isothermal amplification (LAMP): recent progress in research and development. J Infect Chemother. 19(3):404–411.

https://doi.org/10.1007/s10156-013-0590-0

Mori Y, Kitao M, Tomita N, Notomi T. 2004. Real-time turbidimetry of LAMP reaction for quantifying template DNA. J Biochem Biophys Methods. 59(2):145–157.

https://doi.org/10.1016/j.jbbm.2003.12.005

Mori Y, Nagamine K, Tomita N, Notomi T. 2001. Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem Biophys Res Commun. 289(1):150–154.

https://doi.org/10.1006/bbrc.2001.5921

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.

https://doi.org/10.1007/s10156-009-0669-9

Nagamine K, Hase T, Notomi T. 2002. Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probe. 16(3):223–229.

https://doi.org/10.1006/mcpr.2002.0415

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.

https://doi.org/10.1093/clinchem/47.9.1742

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.

https://doi.org/10.1007/s12275-015-4656-9

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.

https://doi.org/10.1093/nar/28.12.e63

Nzelu CO, Kato H, Peters NC. 2019. Loop-mediated isothermal amplification (LAMP): An advanced molecular point-of-care technique for the detection of Leishmania infection. PLoS Negl Trop Dis. 13(11):e0007698.

https://doi.org/10.1371/journal.pntd.0007698

[OIE] World Organisation for Animal Health. 2019. Manual of Diagnostic Tests for Aquatic Animals = Manual de las Pruebas de Diagnóstico para los Animales Acuáticos. [place unknown]: OIE; accessed Nov 2019.

https://www.oie.int/es/normas/manual-acuatico/acceso-en-linea/

Palomino-Camargo C, González-Muñoz Y. 2014. Técnicas moleculares para la detección e identificación de patógenos en alimentos: Ventajas y limitaciones = Molecular techniques for detection and identification of pathogens in food: advantages and limitations. Rev Peru Med Exp. Salud Publica. 31(3):535–546.

[SAGARPA] Secretaria de Agricultura, Ganadería, Desarrollo rural, Pesca y Alimentación. 2017. Anuario Estadístico de Acuicultura y Pesca 2017. México: CONAPESCA; accessed Jan 2019. https://www.conapesca.gob.mx/work/sites/cona/dgppe/2017/ ANUARIO_ESTADISTICO_2017.pdf

Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular Cloning: A Laboratory Manual Vol. 1, 2 and 3. 2nd. ed. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press. 1669 p.

Soto-Rodriguez SA, Gomez-Gil B, Lozano R, Roque A. 2010. Density of vibrios in hemolymph and hepatopancreas of diseased Pacific white shrimp, Litopenaeus vannamei, from northwestern Mexico. J World Aquacult Soc. 41(S1):76–83.

https://doi.org/10.1111/j.1749-7345.2009.00335.x

Tanner NA, Zhang Y, Evans TC. 2015. Visual detection of isothermal nucleic acid amplification using pH-sensitive dyes. BioTechniques. 58(2):59–68.

https://doi.org/10.2144/000114253

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.

https://doi.org/10.1038/nprot.2008.57

Tomlinson J. 2013. In-field diagnostics using loop-mediated isothermal amplification. In: Dickinson M, Hodgetts J (eds.), Phytoplasma. Methods in Molecular Biology (Methods and Protocols), vol. 938. Totowa (NJ): Humana Press.

https://doi.org/10.1007/978-1-62703-089-2_25

Wong YP, Othman S, Lau YL, Radu S, Chee HY. 2018. Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms. J Appl Microbiol. 124(3):626–643.

https://doi.org/10.1111/jam.13647

Xu H, He L, Lv S, Gong Q, Li S. 2012. Establishment of universal loop-mediated isothermal amplification method (LAMP) for rapid detection of pathogenic Vibrio spp. in aquatic organisms. African J Microbiol Res. 6(14):3447– 3454.

https://doi.org/10.5897/AJMR11.1417

Yamazaki W, Ishibashi M, Kawahara R, Inoue K. 2008. Development of a loop-mediated isothermal amplification assay for sensitive and rapid detection of Vibrio parahaemolyticus. BMC Microbiol. 8(1):163.

https://doi.org/10.1186/1471-2180-8-163