Abstract
Background: The Pacific white shrimp is one of the world’s most economically significant aquatic species, being one of the top three species cultured globally. However, the increasing incidence of diseases such as acute hepatopancreatic necrosis disease and hepatopancreatic microsporidia has led to a serious decline in shrimp production and severe economic losses. With the increasing demand for pathogen detection in shrimp farms, rapid DNA extraction technology has become more sophisticated. In this study, a rapid and crude method of extracting genomic DNA from shrimp muscle and hepatopancreas using Chelex-100 was established.
Results: DNA was successfully extracted from muscle and hepatopancreatic tissues using both the Chelex-100 method and commercial kits. The internal reference genes of shrimp were successfully amplified via PCR and real-time PCR using the obtained DNA samples. Moreover, a field assay was successfully conducted using real-time PCR and real-time enzymatic recombinase amplification (real-time ERA), indicating that the quality of the DNA extracted using Chelex-100 is sufficient for use in conjunction with nucleic acid amplification to detect pathogens in shrimps.
Conclusions: Chelex-100 is an efficient method for extracting DNA from shrimp muscle or hepatopancreas tissues, with a short extraction time, high extraction efficiency, and simple operation, making it appropriate for use in the detection of pathogens in shrimp.
References
Dhar AK, Piamsomboon P, Aranguren Caro LF, et al. First report of acute hepatopancreatic necrosis disease (AHPND) occurring in the USA. Diseases of Aquatic Organisms 2019;132(3):241-247. https://doi.org/10.3354/dao03330 PMid: 31019129
Kongrueng J, Yingkajorn M, Bunpa S, et al. Characterization of Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease in southern Thailand. Journal of Fish Diseases 2015;38(11):957-966. https://doi.org/10.1111/jfd.12308 PMid: 25287127
Han JE, Tang KFJ, Tran LH, et al. Photorhabdus insect-related (Pir) toxin-like genes in a plasmid of Vibrio parahaemolyticus, the causative agent of acute hepatopancreatic necrosis disease (AHPND) of shrimp. Diseases of Aquatic Organisms 2015;113(1):33-40. https://doi.org/10.3354/dao02830 PMid: 25667334
Kumar R, Ng TH, Wang HC. Acute hepatopancreatic necrosis disease in penaeid shrimp. Reviews in Aquaculture 2020;12(3):1867-1880. https://doi.org/10.1111/raq.12414
Kumar V, De Bels L, Couck L, et al. PirABVP toxin binds to epithelial cells of the digestive tract and produce pathognomonic AHPND lesions in germ-free brine shrimp. Toxins 2019;11(12):717. https://doi.org/10.3390/toxins11120717 PMid: 31835437
Biju N, Sathiyaraj G, Raj M, et al. High prevalence of Enterocytozoon hepatopenaei in shrimps Penaeus monodon and Litopenaeus vannamei sampled from slow growth ponds in India. Diseases of Aquatic Organisms 2016;120(3):225-230. https://doi.org/10.3354/dao03036 PMid: 27503918
Thitamadee S, Prachumwat A, Srisala J, et al. Review of current disease threats for cultivated penaeid shrimp in Asia. Aquaculture 2016;452(69-87. https://doi.org/10.1016/j.aquaculture.2015.10.028
Jaroenlak P, Boakye DW, Vanichviriyakit R, et al. Identification, characterization and heparin binding capacity of a spore-wall, virulence protein from the shrimp microsporidian, Enterocytozoon hepatopenaei (EHP). Parasites & Vectors 2018;11:177. https://doi.org/10.1186/s13071-018-2758-z PMid: 29530076
Tang KFJ, Han JE, Aranguren LF, et al. Dense populations of the microsporidian Enterocytozoon hepatopenaei (EHP) in feces of Penaeus vannamei exhibiting white feces syndrome and pathways of their transmission to healthy shrimp. Journal of Invertebrate Pathology 2016;140:1-7. https://doi.org/10.1016/j.jip.2016.08.004 PMid: 27530403
Quintana M, de-León L, Cubero J, et al. Assessment of psyllid handling and DNA extraction methods in the detection of 'Candidatus Liberibacter solanacearum' by qPCR. Microorganisms 2022;10(6):1104. https://doi.org/10.3390/microorganisms10061104 PMid: 35744622
Kang M, Yang JS, Kim Y, et al. Comparison of DNA extraction methods for drug susceptibility testing by allele-specific primer extension on a microsphere-based platform: Chelex-100 (in-house and commercialized) and MagPurix TB DNA extraction kit. J Microbiol Methods 2018;152:105-108. https://doi.org/10.1016/j.mimet.2018.07.019 PMid: 30075237
Schrader C, Schielke A, Ellerbroek L, et al. PCR inhibitors - occurrence, properties and removal. J Appl Microbiol 2012;113(5):1014-1026. https://doi.org/10.1111/j.1365-2672.2012.05384.x PMid: 22747964
Tan SC, Yiap BC. DNA, RNA, and protein extraction: the past and the present. J Biomed Biotechnol 2009;2009:574398. https://doi.org/10.1155/2009/574398 PMid: 20011662
Willard, J.M., Lee, D.A., Holland, M.M. (1998). Recovery of DNA for PCR Amplification from Blood and Forensic Samples Using a Chelating Resin. In: Lincoln, P.J., Thomson, J. (eds) Forensic DNA Profiling Protocols. Methods in Molecular Biology, vol 98. Humana Press. https://doi.org/10.1385/0-89603-443-7:9
Srirungruang S, Mahajindawong B, Nimitpanya P, et al. Comparative study of DNA extraction methods for the PCR detection of intestinal parasites in human stool samples. Diagnostics 2022;12(11):2588. https://doi.org/10.3390/diagnostics12112588 PMid: 36359432
Maksimov P, Schares G, Press S, et al. Comparison of different commercial DNA extraction kits and PCR protocols for the detection of Echinococcus multilocularis eggs in faecal samples from foxes. Vet Parasitol 2017;237:83-93. https://doi.org/10.1016/j.vetpar.2017.02.015 PMid: 28268038
Kennedy NA, Walker AW, Berry SH, et al. The impact of different DNA extraction kits and laboratories upon the assessment of human gut microbiota composition by 16S rRNA gene sequencing. Plos One 2014;9(2):e88982. https://doi.org/10.1371/journal.pone.0088982 PMid: 24586470
Angthong P, Uengwetwanit T, Pootakham W, et al. Optimization of high molecular weight DNA extraction methods in shrimp for a long-read sequencing platform. PeerJ 2020;8:e10340. https://doi.org/10.7717/peerj.10340 PMid: 33240651
Iovieno A, Miller D, Lonnen J, et al. Extraction of Acanthamoeba DNA by use of Chelex resin. J Clin Microbiol 2011;49(1):476-7. https://doi.org/10.1128/JCM.01795-10 PMid: 21084505
Rodríguez-Riveiro R, Velasco A, Sotelo CG. The influence of DNA extraction methods on species identification results of seafood products. Foods 2022;11(12):1739. https://doi.org/10.3390/foods11121739 PMid: 35741937
Ip SCY, Lin SW, Lai KM. An evaluation of the performance of five extraction methods: Chelex® 100, QIAamp® DNA Blood Mini Kit, QIAamp® DNA Investigator Kit, QIAsymphony® DNA Investigator® Kit and DNA IQTM. Sci Justice 2015;55(3):200-208. https://doi.org/10.1016/j.scijus.2015.01.005 PMid: 25934373
Konakandla B, Park Y, Margolies D. Whole genome amplification of Chelex-extracted DNA from a single mite: a method for studying genetics of the predatory mite Phytoseiulus persimilis. Exp Appl Acarol 2006;40(3-4):241-247. https://doi.org/10.1007/s10493-006-9042-1 PMid: 17237968
Martín-Platero AM, Peralta-Sánchez JM, Soler JJ, et al. Chelex-based DNA isolation procedure for the identification of microbial communities of eggshell surfaces. Anal Biochem 2010;397(2):253-255. https://doi.org/10.1016/j.ab.2009.10.041 PMid: 19887062
Panda BB, Meher AS, Hazra RK. Comparison between different methods of DNA isolation from dried blood spots for determination of malaria to determine specificity and cost effectiveness. J Parasit Dis 2019;43(3):337-342. https://doi.org/10.1007/s12639-019-01136-0 PMid: 31406397
Phillips K, McCallum N, Welch L. A comparison of methods for forensic DNA extraction: Chelex-100® and the QIAGEN DNA Investigator Kit (manual and automated). Forensic Sci Int Genet 2012;6(2):282-285. https://doi.org/10.1016/j.fsigen.2011.04.018 PMid: 21703957
Turan C, Nanni IM, Brunelli A, et al. New rapid DNA extraction method with Chelex from Venturia inaequalis spores. J Microbiol Methods 2015;115:139-143. https://doi.org/10.1016/j.mimet.2015.06.005 PMid: 26079986
Mao J, Lv J, Miao Y, et al. Development of a rapid and efficient method for non-lethal DNA sampling and genotyping in scallops. PLoS One 2013;8(7):e68096. https://doi.org/10.1371/journal.pone.0068096 PMid: 23874509
Zhou QQ, Wang Y, Hu JJ, et al. Development of a real-time enzymatic recombinase amplification assay (RT-ERA) and an ERA combined with lateral flow dipsticks (LFD) assay (ERA-LFD) for rapid detection of acute hepatopancreatic necrosis disease (AHPND) in shrimp Penaeus vannamei. Aquaculture 2023;566:739205. https://doi.org/10.1016/j.aquaculture.2022.739205
Bachère E. Shrimp immunity and disease control. Aquaculture 2000;191(1-3):3-11. https://doi.org/10.1016/S0044-8486(00)00413-0
Yang JL, Wang MS, Cheng AC, et al. A simple and rapid method for extracting bacterial DNA from intestinal microflora for ERIC-PCR detection. World J Gastroenterol 2008;14(18):2872-2876. https://doi.org/10.3748/wjg.14.2872 PMid: 18473413
Zou Y, Mason MG, Wang Y, et al. Nucleic acid purification from plants, animals and microbes in under 30 seconds. PLOS Biology 2017;15(11):e2003916. https://doi.org/10.1371/journal.pbio.2003916 PMid: 29161268
Omazic ML, Hamer M, Bustos CP, et al. Use of Chelex-100 for the molecular diagnosis of five animal pathogens. FAVE Sección Ciencias Veterinarias 2021;20(1):26-33. https://doi.org/10.14409/favecv.v20i1.9724
Reyes-Escogido L, Balam-Chi M, Rodríguez-Buenfil I, et al. Purification of bacterial genomic DNA in less than 20 min using Chelex-100 microwave: examples from strains of lactic acid bacteria isolated from soil samples. Antonie Van Leeuwenhoek 2010;98(4):465-474. https://doi.org/10.1007/s10482-010-9462-0 PMid: 20556655
Pingcuo S, Gao J, Jiang ZR, et al. Duplication polymorphisms in exon 4 of ?-casein gene in yak breeds/populations. Genet Mol Res 2015;14(3):10242-10248. https://doi.org/10.4238/2015.August.28.8 PMid: 26345961
Singh UA, Kumari M, Iyengar S. Method for improving the quality of genomic DNA obtained from minute quantities of tissue and blood samples using Chelex 100 resin. Biol Proced Online 2018;20:12. https://doi.org/10.1186/s12575-018-0077-6 PMid: 2988133
Xu L, Sun L, Guan GY, et al. The effects of pH and salts on nucleic acid partitioning during phenol extraction. Nucleosides Nucleotides & Nucleic Acids 2019;38(4):305-320. https://doi.org/10.1080/15257770.2018.1533138 PMid: 30922157

This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.
Copyright (c) 2025 Electronic Journal of Biotechnology