Development and assessment of multiplex high resolution melting assay as a tool for rapid single-tube identification of five Brucella species

BMC Research Notes

Adrian M Whatmore⁴  Jeffrey T Foster¹  Stephen M Beckstrom- Sternberg²  Robin Lee⁴  Jessica Sells³  Krishna K Gopaul⁴ 

[1] Current address: Department of Molecular, Cellular, & Biomedical Sciences, University of New Hampshire, College Road, Durham, NH 03824, USA;Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, AZ 86011-4073, USA;Current address: MRC Prion Unit, Queen Square House, Queen Square, London WC1N 3BG, UK;Department of Bacteriology, Animal and Plant Health Agency, Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB, UK

关键词: Real time PCR;    Typing;    Species identification;    Brucella;    High Resolution Melting (HRM);   

Others  :  1092159
DOI  :  10.1186/1756-0500-7-903

 received in 2014-04-25, accepted in 2014-11-28,  发布年份 2014

【 摘 要 】

Background

The zoonosis brucellosis causes economically significant reproductive problems in livestock and potentially debilitating disease of humans. Although the causative agent, organisms from the genus Brucella, can be differentiated into a number of species based on phenotypic characteristics, there are also significant differences in genotype that are concordant with individual species. This paper describes the development of a five target multiplex assay to identify five terrestrial Brucella species using real-time polymerase chain reaction (PCR) and subsequent high resolution melt curve analysis. This technology offers a robust and cost effective alternative to previously described hydrolysis-probe Single Nucleotide Polymorphism (SNP)-based species defining assays.

Results

Through the use of Brucella whole genome sequencing five species defining SNPs were identified. Individual HRM assays were developed to these target these changes and, following optimisation of primer concentrations, it was possible to multiplex all five assays in a single tube. In a validation exercise using a panel of 135 Brucella strains of terrestrial and marine origin, it was possible to distinguish the five target species from the other species within this panel.

Conclusion

The HRM multiplex offers a number of diagnostic advantages over previously described SNP-based typing approaches. Further, and uniquely for HRM, the successful multiplexing of five assays in a single tube allowing differentiation of five Brucella species in the diagnostic laboratory in a cost-effective and timely manner is described. However there are possible limitations to using this platform on DNA extractions direct from clinical material.

【 授权许可】

   
2014 Gopaul et al.; licensee BioMed Central.

【 图 表 】

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【 参考文献 】

• [1]Godfroid J, Nielsen K, Saegerman C: Diagnosis of brucellosis in livestock and wildlife. Croat Med J 2010, 51:296-305.
• [2]Seleem MN, Boyle SM, Sriranganathan N: Brucellosis: a re-emerging zoonosis. Vet Microbiol 2012, 140:392-398.
• [3]Whatmore AM: Current understanding of the genetic diversity of Brucella, an expanding genus of zoonotic pathogens. Infect Genet Evol 2009, 9:1168-1184.
• [4]Schlabritz-Loutsevich NE, Whatmore AM, Quance CR, Koylass MS, Cummins LB, Dick EJ Jr, Snider CL, Cappelli D, Ebersole JL, Nathanielsz PW, Hubbard GB: A novel Brucella isolate in association with two cases of stillbirth in non-human primates-first report. J Med Primatol 2009, 38:70-73.
• [5]Hofer E, Revilla-Fernández S, Al Dahouk S, Riehm JM, Nöckler K, Zygmunt MS, Cloeckaert A, Tomaso H, Scholz HC: A potential novel Brucella species isolated from mandibular lymph nodes of red foxes in Austria. Vet Microbiol 2012, 155:93-99.
• [6]Eisenberg T, Hamann HP, Kaim U, Schlez K, Seeger H, Schauerte N, Melzer F, Tomaso H, Scholz HC, Koylass MS, Whatmore AM, Zschöck M: Isolation of potentially novel Brucella spp. from frogs. Appl Environ Microbiol 2012, 78:3753-3755.
• [7]Alvarez J, Sáez JL, García N, Serrat C, Pérez-Sancho M, González S, Ortega MJ, Gou J, Carbajo L, Garrido F, Goyache J, Domínguez L: Management of an outbreak of brucellosis due to B. melitensis in dairy cattle in Spain. Res Vet Sci 2011, 90:208-211.
• [8]Muendo EN, Mbatha PM, Macharia J, Abdoel TH, Janszen PV, Pastoor R, Smits HL: Infection of cattle in Kenya with Brucella abortus biovar 3 and Brucella melitensis biovar 1 genotypes. Trop Anim Health Prod 2012, 44:17-20.
• [9]Probert WS, Schrader KN, Khuong NY, Bystrom SL, Graves MH: Real-time multiplex PCR assay for detection of Brucella spp., B. abortus, and B. melitensis. J Clin Microbiol 2004, 42:1290-1293.
• [10]Bounaadja L, Albert D, Chénais B, Hénault S, Zygmunt MS, Poliak S, Garin-Bastuji B: Real-time PCR for identification of Brucella spp.: a comparative study of IS711, bcsp31 and per target genes. Vet Microbiol 2009, 137:156-164.
• [11]Matero P, Hemmilä H, Tomaso H, Piiparinen H, Rantakokko-Jalava K, Nuotio L, Nikkari S: Rapid field detection assays for Bacillus anthracis, Brucella spp., Francisella tularensis and Yersinia pestis. Clin Microbiol Infect 2011, 17:34-43.
• [12]Bricker BJ, Halling SM: Differentiation of Brucella abortus bv. 1, 2, and 4, Brucella melitensis, Brucella ovis, and Brucella suis bv. 1 by PCR. J Clin Microbiol 1994, 32:2660-2666.
• [13]García-Yoldi D, Marín CM, de Miguel MJ, Muñoz PM, Vizmanos JL, López-Goñi I: Multiplex PCR assay for the identification and differentiation of all Brucella species and the vaccine strains Brucella abortus S19 and RB51 and Brucella melitensis Rev1. Clin Chem 2006, 52:779-781.
• [14]López-Goñi I, García-Yoldi D, Marín CM, de Miguel MJ, Barquero-Calvo E, Guzmán-Verri C, Albert D, Garin-Bastuji B: New Bruce-ladder multiplex PCR assay for the biovar typing of Brucella suis and the discrimination of Brucella suis and Brucella canis. Vet Microbiol 2011, 154:152-155.
• [15]Redkar R, Rose S, Bricker B, DelVecchio V: Real-time detection of Brucella abortus, Brucella melitensis and Brucella suis. Mol Cell Probes 2001, 15:43-52.
• [16]Foster JT, Okinaka RT, Svensson R, Shaw K, De BK, Robison RA, Probert WS, Kenefic LJ, Brown WD, Keim P: Real-time PCR assays of single-nucleotide polymorphisms defining the major Brucella clades. J Clin Microbiol 2008, 46:296-301.
• [17]Gopaul KK, Koylass MS, Smith CJ, Whatmore AM: Rapid identification of Brucella isolates to the species level by real time PCR based single nucleotide polymorphism (SNP) analysis. BMC Microbiol 2008, 8:86. BioMed Central Full Text
• [18]Whatmore AM, Perrett LL, MacMillan AP: Characterisation of the genetic diversity of Brucella by multilocus sequencing. BMC Microbiol 2007, 7:34. BioMed Central Full Text
• [19]Ririe KM, Rasmussen RP, Wittwer CT: Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem 1997, 245:154-160.
• [20]Akey JM, Sosnoski D, Parra E, Dios S, Hiester K, Su B, Bonilla C, Jin L, Shriver MD: Melting curve analysis of SNPs (McSNP): a gel-free and inexpensive approach for SNP genotyping. Biotechniques 2001, 30:358-367.
• [21]Gundry CN, Vandersteen JG, Reed GH, Pryor RJ, Chen J, Wittwer CT: Amplicon melting analysis with labeled primers: a closed-tube method for differentiating homozygotes and heterozygotes. Clin Chem 2003, 49:396-406.
• [22]Wittwer CT, Reed GH, Gundry CN, Vandersteen JG, Pryor RJ: High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem 2003, 49:853-860.
• [23]Tong SY, Giffard PM: Microbiological applications of high-resolution melting analysis. J Clin Microbiol 2012, 50:3418-3421.
• [24]Perng CL, Chen HY, Chiueh TS, Wang WY, Huang CT, Sun JR: Identification of non-tuberculous mycobacteria by real-time PCR coupled with a high-resolution melting system. J Med Microbiol 2012, 61:944-951.
• [25]Touati A, Peuchant O, Jensen JS, Bébéar C, Pereyre S: Direct detection of macrolide resistance in Mycoplasma genitalium in France from clinical specimens using real-time PCR and melting curve analysis. J Clin Microbiol
• [26]Lee TH, Wu TS, Tseng CP, Qiu JT: High-resolution melting molecular signatures for rapid identification of human papillomavirus genotypes. PLoS One 2012, 7:e42051.
• [27]Costa JM, Cabaret O, Moukoury S, Bretagne S: Genotyping of the protozoan pathogen Toxoplasma gondii using high-resolution melting analysis of the repeated B1 gene. J Microbiol Methods 2011, 86:357-363.
• [28]Heideman DA, Lurkin I, Doeleman M, Smit EF, Verheul HM, Meijer GA, Snijders PJ, Thunnissen E, Zwarthoff EC: KRAS and BRAF mutation analysis in routine molecular diagnostics: comparison of three testing methods on formalin-fixed, paraffin-embedded tumor-derived DNA. J Mol Diagn 2012, 14:247-255.
• [29]Vaclavikova R, Ehrlichova M, Hlavata I, Pecha V, Kozevnikovova R, Trnkova M, Adamek J, Edvardsen H, Kristensen VN, Gut I, Soucek P: Detection of frequent ABCB1 polymorphisms by high-resolution melting curve analysis and their effect on breast carcinoma prognosis. Clin Chem Lab Med 2012, 50:1999-2007.
• [30]Seipp MT, Herrmann M, Wittwer CT: Automated DNA extraction, quantification, dilution, and PCR preparation for genotyping by high-resolution melting. J Biomol Tech 2010, 21:163-166.
• [31]Swen JJ, Baak-Pablo RF, Guchelaar HJ, van der Straaten T: Alternative methods to a TaqMan assay to detect a tri-allelic single nucleotide polymorphism rs757210 in the HNF1β gene. Clin Chem Lab Med 2011, 50:279-284.
• [32]Winchell JM, Wolff BJ, Tiller R, Bowen MD, Hoffmaster AR: Rapid identification and discrimination of Brucella isolates by use of real-time PCR and high-resolution melt analysis. J Clin Microbiol 2010, 48:697-702.
• [33]Reed GH, Kent JO, Wittwer CT: High-resolution DNA melting analysis for simple and efficient molecular diagnostics. Pharmacogenomics J 2007, 8:597-608.
• [34]Kagkli DM, Folloni S, Barbau-Piednoir E, Van den Eede G, Van den Bulcke M: Towards a pathogenic Escherichia coli detection platform using multiplex SYBR®Green Real-time PCR methods and high resolution melting analysis. PLoS One 2012, 7:e39287.
• [35]Seipp MT, Pattison D, Durtschi JD, Jama M, Voelkerding KV, Wittwer CT: Quadruplex genotyping of F5, F2 and MTHFR variants in a single closed tube by High-Resolution amplicon Melting. Clin Chem 2008, 54:108-115.
• [36]Whatmore AM, Shankster SJ, Perrett LL, Murphy TJ, Brew SD, Thirlwall RE, Cutler SJ, MacMillan AP: Identification and characterization of variable-number tandem-repeat markers for typing of Brucella spp. J Clin Microbiol 2006, 44:1982-1993.
• [37]Wattam AR, Foster JT, Mane SP, Beckstrom-Sternberg SM, Beckstrom-Sternberg JM, Dickerman AW, Keim P, Pearson T, Shukla M, Ward DV, Williams KP, Sobral BW, Tsolis RM, Whatmore AM, O’Callaghan D: Comparative phylogenomics and evolution of the Brucellae reveal a path to virulence. J Bacteriol 2014, 196:920-930.
• [38]Audic S, Lescot M, Claverie JM, Cloeckaert A, Zygmunt MS: The genome sequence of Brucella pinnipedialis B2/94 sheds light on the evolutionary history of the genus Brucella. BMC Evol Biol 2011, 11:200. BioMed Central Full Text
• [39]Gopaul KK, Sells J, Bricker BJ, Crasta OR, Whatmore AM: Rapid and reliable single nucleotide polymorphism-based differentiation of Brucella live vaccine strains from field strains. J Clin Microbiol 2010, 48:1461-1464.
• [40]Gee JE, De BK, Levett PN, Whitney AM, Novak RT, Popovic T: Use of 16S rRNA gene sequencing for rapid confirmatory identification of Brucella isolates. J Clin Microbiol 2004, 42:3649-3654.