Abstract
Single-nucleotide polymorphisms (SNPs) are powerful molecular markers for the identification and differentiation of closely related organisms. A variety of methods can be used to determine the allele that is present at a specific locus in the genome, including real-time PCR by using an allele-specific primer. In order to increase the selectivity for the target allele, deliberate mismatch bases at the 3′ end of the allele-specific primer may be introduced. This strategy has already been used for the identification and differentiation of microorganisms and plants. We have recently developed real-time PCR assays involving mismatch primers for the identification and differentiation of closely related deer species (red deer, fallow deer, sika deer) or the discrimination of wild boar and domestic pig in game meat products. These methods are applicable to detect meat species adulteration in food products.
In this chapter, we offer a protocol for the design of PCR primer/probe systems suitable for meat species authentication in food. We address the retrieval and alignment of sequences, primer design by using a commercial software and the introduction of deliberate mismatch bases. In addition, we describe how the suitability of primer/probe systems can be tested in silico and in practice. We use the design of PCR primer/probe systems for wild boar and domestic pig as example.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Vignal A, Milan D, SanCristobal M, Eggen A (2002) A review on SNP and other types of molecular markers and their use in animal genetics. Genet Sel Evol 34(3):275–305
Beissinger TM, Hirsch CN, Sekhon RS, Foerster JM, Johnson JM, Muttoni G, Vaillancourt B, Buell CR, Kaeppler SM, de Leon N (2013) Marker density and read depth for genotyping populations using genotyping-by-sequencing. Genetics 193(4):1073–1081
Gibson NJ (2006) The use of real-time PCR methods in DNA sequence variation analysis. Clin Chim Acta 363(1-2):32–47
Cha RS, Zarbl H, Keohavong P, Thilly WG (1992) Mismatch amplification mutation assay (MAMA): application to the c-H-ras gene. Genome Res 2(1):14–20
Sabui S, Dutta S, Debnath A, Ghosh A, Hamabata T, Rajendran K, Ramamurthy T, Nataro JP, Sur D, Levine MM, Chatterjee NS (2012) Real-time PCR-based mismatch amplification mutation assay for specific detection of CS6-expressing allelic variants of enterotoxigenic Escherichia coli and its application in assessing diarrheal cases and asymptomatic controls. J Clin Microbiol 50(4):1308–1312
Easterday WR, Van Ert MN, Zanecki S, Keim P (2005) Specific detection of Bacillus anthracis using a TaqMan® mismatch amplification mutation assay. BioTechniques 38(5):731–735
Kreizinger Z, Sulyok KM, Grózner D, Beko K, Dán A, Szabó Z, Gyuranecz M (2017) Development of mismatch amplification mutation assays for the differentiation of MS1 vaccine strain from wild-type Mycoplasma synoviae and MS-H vaccine strains. PLoS One 12(4):e0175969
Morita M, Ohnishi M, Arakawa E, Bhuiyan NA, Nusrin S, Alam M, Siddique AK, Qadri F, Izumiya H, Nair GB, Watanabe H (2008) Development and validation of a mismatch amplification mutation PCR assay to monitor the dissemination of an emerging variant of Vibrio cholerae O1 biotype El Tor. Microbiol Immunol 52(6):314–317
Syverson RL, Bradeen JM (2011) A novel class of simple PCR markers with SNP-level sensitivity for mapping and haplotype characterization in Solanum species. Am J Pot Res 88(3):269–282
Han EH, Lee SJ, Kim MB, Shin YW, Kim YH, Lee SW (2017) Molecular marker analysis of Cynanchum wilfordii and C. auriculatum using the simple ARMS-PCR method with mismatched primers. Plant Biotechnol Rep 11(2):127–133
Kaltenbrunner M, Hochegger R, Cichna-Markl M (2018) Red deer (Cervus elaphus)-specific real-time PCR assay for the detection of food adulteration. Food Control 89:157–166
Kaltenbrunner M, Hochegger R, Cichna-Markl M (2018) Development and validation of a fallow deer (Dama dama)-specific TaqMan real-time PCR assay for the detection of food adulteration. Food Chem 243:82–90
Kaltenbrunner M, Hochegger R, Cichna-Markl M (2018) Sika deer (Cervus nippon)-specific real-time PCR method to detect fraudulent labelling of meat and meat products. Sci Rep 8(1):7236
Kaltenbrunner M, Mayer W, Kerkhoff K, Epp R, Rüggeberg H, Hochegger R, Cichna-Markl M (2019) Differentiation between wild boar and domestic pig in food by targeting two gene loci by real-time PCR. Sci Rep 9(1):9221
Ballin NZ (2010) Authentication of meat and meat products. Meat Sci 86(3):577–587
Laube I (2010) Meat. In: Popping B, Diaz-Amigo C, Hoenicke K (eds) Molecular biological and immunological techniques and applications for food chemists. John Wiley & Sons, Inc., Hoboken, New Jersey, pp 135–156
Broll H (2010) Quantitative Real-Time PCR. In: Popping B, Diaz-Amigo C, Hoenicke K (eds) Molecular biological and immunological techniques and applications for food chemists. John Wiley & Sons, Inc, Hoboken, New Jersey, pp 59–83
Kaltenbrunner M, Hochegger R, Cichna-Markl M (2018) Tetraplex real-time PCR assay for the simultaneous identification and quantification of roe deer, red deer, fallow deer and sika deer for deer meat authentication. Food Chem 269:486–494
Beugin M-P, Baubet E, Dufaure De Citres C, Kaerle C, Muselet L, Klein F, Queney G (2017) A set of 20 multiplexed single-nucleotide polymorphism (SNP) markers specifically selected for the identification of the wild boar (Sus scrofa scrofa) and the domestic pig (Sus scrofa domesticus). Conserv Genet Resour 9(4):671–675
Codex Alimentarius Austriacus, Österreichisches Lebensmittelbuch, Codexkapitel/B14/Fleisch und Fleischerzeugnisse. 2005
Druml B, Kaltenbrunner M, Hochegger R, Cichna-Markl M (2016) A novel reference real-time PCR assay for the relative quantification of (game) meat species in raw and heat-processed food. Food Control 70:392–400
Dobrovolny S, Blaschitz M, Weinmaier T, Pechatschek J, Cichna-Markl M, Indra A, Hufnagl P, Hochegger R (2019) Development of a DNA metabarcoding method for the identification of fifteen mammalian and six poultry species in food. Food Chem 272:354–361
Livak KJ (1999) Allelic discrimination using fluorogenic probes and the 5′ nuclease assay. Genet Anal 14(5-6):143–149
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Kaltenbrunner, M., Hochegger, R., Cichna-Markl, M. (2022). Design of Mismatch Primers to Identify and Differentiate Closely Related (Sub)Species: Application to the Authentication of Meat Products. In: Basu, C. (eds) PCR Primer Design. Methods in Molecular Biology, vol 2392. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1799-1_5
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1799-1_5
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1798-4
Online ISBN: 978-1-0716-1799-1
eBook Packages: Springer Protocols