Use of Chelex-100 for the molecular diagnosis of five animal pathogens
DOI:
https://doi.org/10.14409/favecv.v20i1.9724Keywords:
Veterinary, Diagnosis, DNA, Chelex-100, PCRAbstract
Molecular tools have improved conventional veterinary diagnosis. Acid nucleic extraction is a key step for downstream applications. This work aimed to compare the DNA extraction method Chelex-100 resin (M1) with Whatman® cards (M2), phenol-chloroform (M3), or commercial kits (M4), and to determine the most sensitive and inexpensive one for its diagnosis of animal pathogens that, despite their economic or zoonotic relevance, receive little attention. DNA was isolated from urine, organs, semen, blood and intestinal mucous, from the bacteria Leptospira interrogans serovar Pomona Pomona (by M1 and M2), Brucella melitensis (by M1, M3 and M4), and Salmonella ser. Abortusequi (by M1 and M4), and the parasites Leishmania spp. (by M1, M3 and M4), and Eimeria spp. (by M1 and M3), respectively. The sensitivity of each method was assayed by Polymerase Chain Reaction (PCR). The M1 showed similar sensitivity for Salmonella ser. Abortusequi, Leishmania spp., and Eimeria spp., being better for L. interrogans serovar Pomona Pomona and slightly lower for B. melitensis. For the first time, a simple and economic method was successfully employed for extracting DNA from these animal pathogens, especially important in low-resource settings, contributing to the diagnosis of leptospirosis, brucellosis, leishmaniasis, and coccidiosis; as well as to the molecular epidemiology of salmonellosis in stallion from semen samples.
References
Ascencio ME, Sarmiento NF, Schnittger L, Florin-Christensen M, and Rodriguez AE. 2019. Molecular Diagnosis of Leishmania Spp. in Dogs of a Subtropical Locality of Argentina. Transboun. Emerg. Dis. 67 Suppl 2: 106-110.
Bal AE, Gravekamp C, Hartskeerl R, De Meza-Brewster J, Korver H, Terpstra W. 1994. Detection of Leptospires in Urine by PCR for Early Diagnosis of Leptospirosis. J. Clin. Microbiol. 32: 1894-1898.
Blake DP, Smith AL, Shirley MW. 2003. Amplified Fragment Length Polymorphism Analyses of Eimeria Spp.: An Improved Process for Genetic Studies of Recombinant Parasites. Parasitol. Res. 90: 473-475.
Bricker BJ, Halling SM. 1994. Differentiation of Brucella Abortus Bv. 1, 2, and 4, Brucella Melitensis, Brucella Ovis, and Brucella Suis Bv. 1 by PCR. J. Clin. Microbiol. 32: 2660-2666.
Bustos CP, Moroni M, Caffer MI, Ivanissevich A, Herrera M, Moreira AR, Guida N, Chacana P. 2020. Genotypic Diversity of Salmonella Ser. Abortusequi Isolates from Argentina. Equine Vet. J. 52: 98-103.
Chapman HD, Shirley MW. 2003. The Houghton Strain of Eimeria tenella: A Review of the Type Strain Selected for Genome Sequencing. Avian Pathol. 32: 115-127.
Chen ML, Shieh YS, Shim KS, Gerber MA. 1991. Comparative Studies on the Detection of Hepatitis B Virus DNA in Frozen and Paraffin Sections by the Polymerase Chain Reaction. Mod. Pathol. 4: 555-558.
Dalloul RA, Lillehoj HS. 2006. Poultry coccidiosis: recent advancements in control measures and vaccine development. Expert Rev. Vaccines 5: 143-163.
Dantas-Torres, F. 2007. The Role of Dogs as Reservoirs of Leishmania Parasites, with Emphasis on Leishmania (Leishmania) Infantum and Leishmania (Viannia) Braziliensis. Vet. Parasitol. 10;149: 139-146.
Gill P, Kimpton CP, Sullivan K. 1992. A rapid polymerase chain reaction method for identifying fixed specimens. Electrophoresis 13: 173-175.
Godfroid J, Scholz HC, Barbier T, Nicolas C, Wattiau P, Fretin D, Whatmore AM, Cloeckaert A, Blasco JM, Moriyon I, Saegerman C, Muma JB, Al Dahouk S, Neubauer H, Letesson JJ. 2011. Brucellosis at the animal/ecosystem/human interface at the beginning of the 21st century. Prev. Vet. Med. 102: 118-131.
Godfroid J, Klaus N, Saegerman. 2010. Diagnosis of Brucellosis in Livestock and Wildlife. Croat. Med. J. 51: 296-305.
Gwida MM, Al-Ashmawy MA. 2014. Culture versus PCR for Salmonella Species Identification in Some Dairy Products and Dairy Handlers with Special Concern to Its Zoonotic Importance. Vet. Med. Int. 2014: 1-5.
Hamer M, Saraullo V, Brihuega B, Watanave O, Martinez M, and Grune Loffler S. 2019. Comparación de Métodos de Extracción de ADN Simples y Económicos Para El Diagnóstico Molecular de Leptospirosis Animal. FAVE Sección Ciencias Veterinarias 18: 68-73.
Kumar S, Garg R, Moftah A, Clark EL, Macdonald SE, Chaudhry AS, Sparagano O, Banerjee PS, Kundu K, Tomley FM, Blake DP. 2014. An optimised protocol for molecular identification of Eimeria from chickens. Vet. Parasitol. 199: 24-31.
Lachaud L, Marchergui-Hammami S, Chabbert E, Dereure J, Dedet JP, Bastien P. 2002. Comparison of six PCR methods using peripheral blood for detection of canine visceral leishmaniasis. J. Clin. Microbiol. 40: 210-215.
Levett PN. 2001. Leptospirosis. Clin. Microbiol. Rev. 14: 296-326.
Malorny B, Hoorfar J, Bunge C, Helmuth R. 2003. Multicenter validation of the analytical accuracy of Salmonella PCR: towards an international standard. Appl. Environ. Microbiol. 69: 290-296.
Miles AA, Misra SS, Irwin JO. 1938. The Estimation of the Bactericidal Power of the Blood. J. Hyg. 38: 732-749.
Natarajan G, Kabir M, Perin J, Hossain B, Debes A, Haque R, George CM. 2018. Whatman protein saver cards for storage and detection of parasitic enteropathogens. Am. J. Trop. Med. Hyg. 99: 1613-1618.
Noh CS, Kim SS, Park SY, Moon HS, Hong Y, Ryu JS. 2019. Comparison of Two PCR Assays for Trichomonas vaginalis. Korean J. Parasitol. 57: 27-31.
Pastor-Fernández I, Kim S, Billington K, Bumstead J, Marugán-Hernández V, Küster T, Ferguson DJP, Vervelde L, Blake DP, Tomley FM. Development of cross-protective Eimeria-vectored vaccines based on apical membrane antigens. Int. J. Parasitol. 48: 505-518.
Pathmanathan SG, Cardona-Castro N, Sánchez-Jiménez MM, Correa-Ochoa MM, Puthucheary SD, Thong KL. 2003. Simple and rapid detection of Salmonella strains by direct PCR amplification of the hilA gene. J. Med. Microbiol. 52: 773-776.
Rodgers MR, Popper SJ, Wirth DF. 1990. Amplification of Kinetoplast DNA as a Tool in the Detection and Diagnosis of Leishmania. Exp. Parasitol. 71: 267-275.
Rossetti CA., Arenas-Gamboa AM., Maurizio E. 2017. Caprine brucellosis: A historically neglected disease with significant impact on public health. PLoS Negl. Trop. Dis. 11:1-8.
Schwarz RS, Jenkins MC, Klopp S, Miska KB. 2009. Genomic analysis of Eimeria spp. populations in relation to performance levels of broiler chicken farms in Arkansas and North Carolina. J. Parasitol. 95: 871-80.
Singh UA, Kumari M, Iyengar S. 2018. Method for Improving the Quality of Genomic DNA Obtained from Minute Quantities of Tissue and Blood Samples Using Chelex 100 Resin. Biological Procedures Online 20: 1-8.
Turan C, Nanni IM, Brunelli A, Collina M. 2015. New Rapid DNA Extraction Method with Chelex from Venturia Inaequalis Spores. J. Microbiol. Methods. 115:139-43.
Vejarano MP, Matrone M, Keid L, Rocha MV, Ikuta CY, Rodriguez C, Salgado V, Ferreira F, Dias RA, Telles O, Ferreira Neto JS. 2013. Evaluation of four DNA extraction protocols for Brucella Abortus detection by PCR in tissues from experimentally infected cows with the 2308 strain. Vector Borne Zoon. Dis. 13: 237-242.
Worl Organisation for Animal Health (OIE). 2018. Leptospirosis. In Terrestrial Manual. 8th Ed. 1833 pp.
World Health Organization. 2010. Control of the Leishmaniases. In World Health Organization Technical Report Series. 186 pp.
Downloads
Published
Issue
Section
License
FAVE Sección Ciencias Veterinarias ratifies the open access model, in which contents (in full) are available free to anyone in the internet. The costs of production and publication are not transfered to the authors. This policy intends to break social and economical barriers that generate inequities in the access to information, and for the publication of research results.
All articles can be accessed at http://bibliotecavirtual.unl.edu.ar/publicaciones/index.php/FAVEveterinaria/issue/current/, under license Creative CommonsAtribución-NoComercial-Compartir Igual 4.0 Internacional.
