Abstract
Conventional internal amplification controls (IAC) are DNA-based controls which monitor the amplification reaction of real-time PCR in food pathogen detection. Food pathogen detection using real-time PCR, however, includes necessarily sample preparation and DNA isolation/purification. This modular structure leads to an analytical chain. To cover the whole analytical chain, the concept of the IAC has to be extended to internal sample process controls (ISPCs) which include supporting pre-analytical steps. One concept for such ISPCs is the use of recombinant bacterial cells comprising a deleted target and an artificial competitive target instead, which are derived from the actual target strain. In this work, we present an ISPC for the molecular detection of Listeria monocytogenes. A Δ-prfA L. monocytogenes EGDe strain was cloned with a pPL2 phage insertion vector to include a single copy artificial DNA target, resulting in a fluorescence signal not interfering with the respective signal of the L. monocytogenes EGDe wild-type strain during real-time PCR. The recombinant strain was confirmed and characterized with conventional and real-time PCR including sequencing. Microbiological examination revealed a distinct phenotype pattern on selective plate media which enables discrimination of Δ-prfA L. monocytogenes EGDe from wild-type L. monocytogenes EGDe and Listeria innocua. The ISPC was applied in an examination of artificially contaminated ultra high temperature-treated milk to demonstrate its analytical suitability. The resulting corrected recovery values of the ISPC as obtained by the whole molecular quantification procedure correspond to the respective values determined for the actual target strain (P ≤ 0.05).
Similar content being viewed by others
References
Abdulmawjood A, Roth S, Bülte M (2002) Two methods for construction of internal amplification controls for the detection of Escherichia coli O157 by polymerase chain reaction. Mol Cell Probes 16:335–339
Böckmann R, Dickneite C, Middendorf B, Goebel W, Sokolovic Z (1996) Specific binding of the Listeria monocytogenes transcriptional regulator prfA to target sequences requires additional factor(s) and is influenced by iron. Mol Microbiol 22:643–653
Border PM, Howard JJ, Plastow GS, Siggens KW (1990) Detection of Listeria species and Listeria monocytogenes using polymerase chain reaction. Lett Appl Microbiol 11:158–162
Brehm-Stecher B, Young C, Jaykus LA, Tortorello ML (2009) Sample preparation: the forgotten beginning. J Food Prot 72:1774–1789
Bubert A, Hein I, Rauch M, Lehner A, Yoon B, Goebel W, Wagner M (1999) Detection and differentiation of Listeria spp. by a single reaction based on multiplex PCR. Appl Environ Microbiol 65:4688–4692
Croci L, Dubois E, Cook N, De Medici D, Schultz AC, China B, Rutjes SA, Hoorfar J, Van der Poel WHM (2008) Current methods for extraction and concentration of enteric viruses from fresh fruit and vegetables: towards international standards. Food Anal Methods 1:73–84
D'Agostino M, Wagner M, Vazquez-Boland JA, Kuchta T, Karpiskova R, Hoorfar J, Novella S, Scortti M, Ellison J, Murray A, Fernandes I, Kuhn M, Pazlarova J, Heuvelink A, Cook N (2004) A validated PCR-based method to detect Listeria monocytogenes using raw milk as a food model—towards an international standard. J Food Prot 67:1646–1655
Di Pasquale S, Paniconi M, Auricchio B, Orefice L, Schultz AC, De Medici D (2010) Comparison of different concentration methods for the detection of hepatitis A virus and calicivirus from bottled natural mineral waters. J Virol Methods 165:57–63
Dreier J, Störmer M, Kleesiek K (2005) Use of bacteriophage MS2 as an internal control in viral reverse transcription-PCR assays. J Clin Microbiol 43:4551–4557
Espy MJ, Uhl JR, Sloan LM, Buckwalter SP, Jones MF, Vetter EA, Yao JD, Wengenack NL, Rosenblatt JE, Cockerill FR III, Smith TF (2006) Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clin Microbiol Rev 19:165–256
Glaser P, Frangeul L, Buchrieser C, Rusniok C, Amend A, Baquero F, Berche P, Bloecker H, Brandt P, Chakraborty T, Charbit A, Chetouani F, Couvé E, de Daruvar A, Dehoux P, Domann E, Domínguez-Bernal G, Duchaud E, Durant L, Dussurget O, Entian KD, Fsihi H, Garcia-del Portillo F, Garrido P, Gautier L, Goebel W, Gómez-López N, Hain T, Hauf J, Jackson D, Jones LM, Kaerst U, Kreft J, Kuhn M, Kunst F, Kurapkat G, Madueño E, Maitournam A, Vicente JM, Ng E, Nedjari H, Nordsiek G, Novella S, de Pablos B, Pérez-Diaz JC, Purcell R, Remmel B, Rose M, Schlueter T, Simoes N, Tierrez A, Vázquez-Boland JA, Voss H, Wehland J, Cossart P (2001) Comparative genomics of Listeria species. Science 294:849–852
Hoorfar J, Cook N, Malorny B, Wagner M, De Medici D, Abdulmawjood A, Fach P (2004a) Diagnostic PCR: making internal amplification control mandatory. J Appl Microbiol 96:221–222
Hoorfar J, Malorny B, Abdulmawjood A, Cook N, Wagner M, Fach P (2004b) Practical considerations in design of internal amplification controls for diagnostic PCR assays. J Clin Microbiol 42:1863–1868
Hoorfar J, Wolffs P, Rådström P (2004c) Diagnostic PCR: validation and sample preparation are two sides of the same coin. APMIS 112:808–814
Lauer P, Chow MY, Loessner MJ, Portnoy DA, Calendar R (2002) Construction, characterization, and use of two Listeria monocytogenes site-specific phage integration vectors. J Bacteriol 184:4177–4186
Malorny B, Tassios PT, Radström P, Cook N, Wagner M, Hoorfar J (2003) Standardization of diagnostic PCR for the detection of foodborne pathogens. Int J Food Microbiol 83:39–48
Mattison K, Brassard J, Gagné MJ, Ward P, Houde A, Lessard L, Simard C, Shukla A, Pagotto F, Jones TH, Trottier YL (2009) The feline calicivirus as a sample process control for the detection of food and waterborne RNA viruses. Int J Food Microbiol 132:73–77
Mester P, Wagner M, Rossmanith P (2010) Use of liquid-based extraction for recovery of Salmonella typhimurium and Listeria monocytogenes from food matrices. J Food Prot 73:680–687
Mukherjee K, Altincicek B, Hain T, Domann E, Vilcinskas A, Chakraborty T (2010) Galleria mellonella as a model system for studying Listeria pathogenesis. Appl Environ Microbiol 76:310–317
Murphy NM, McLauchlin J, Ohai C, Grant KA (2007) Construction and evaluation of a microbiological positive process internal control for PCR-based examination of food samples for Listeria monocytogenes and Salmonella enterica. Int J Food Microbiol 120:110–119
Nelson KE, Fouts DE, Mongodin EF, Ravel J, DeBoy RT, Kolonay JF, Rasko DA, Angiuoli SV, Gill SR, Paulsen IT, Peterson J, White O, Nelson WC, Nierman W, Beanan MJ, Brinkac LM, Daugherty SC, Dodson RJ, Durkin AS, Madupu R, Haft DH, Selengut J, Van Aken S, Khouri H, Fedorova N, Forberger H, Tran B, Kathariou S, Wonderling LD, Uhlich GA, Bayles DO, Luchansky JB, Fraser CM (2004) Whole genome comparisons of serotype 4b and 1/2a strains of the food-borne pathogen Listeria monocytogenes reveal new insights into the core genome components of this species. Nucleic Acids Res 32:2386–2395
Park SF, Stewart GS (1990) High-efficiency transformation of Listeria monocytogenes by electroporation of penicillin-treated cells. Gene 94:129–132
Rip D, Gouws PA (2009) Development of an internal amplification control using multiplex PCR for the detection of Listeria monocytogenes in food products. Food Anal Methods 2:190–196
Rodríguez-Lázaro D, Pla M, Scortti M, Monzó HJ, Vázquez-Boland JA (2005) A novel real-time PCR for Listeria monocytogenes that monitors analytical performance via an internal amplification control. Appl Environ Microbiol 71:9008–9012
Rodríguez-Lázaro D, Lombard B, Smith H, Rzezutka A, D'Agostino M, Helmuth R, Schroeter A, Malorny B, Miko A, Guerra B, Davison J, Kobilinsky A, Hernández M, Bertheau Y, Cook N (2007) Trends in analytical methodology in food safety and quality: monitoring microorganisms and genetically modified organisms. Trends Food Sci Technol 18:306–319
Ross JW, Fraser MD (1993) Analytical goals developed from the inherent error of medical tests. Clin Chem 39:1481–1493
Rossmanith P, Wagner M (2010) Sample preparation for the detection of foodborne pathogens by molecular biological methods. In: Brul S, McMeekin TA (eds) Tracing pathogens in the food chain. Woodhead, Cambridge, pp 237–262
Rossmanith P, Wagner M (2011a) Aspects of systems theory in the analysis and validation of innovative molecular-biological based food pathogen detection methods. Trends Food Sci Technol 22:61–71
Rossmanith P, Wagner M (2011b) The challenge to quantify Listeria monocytogenes—a model leading to new aspects in molecular biological food pathogen detection. J Appl Microbiol 110:605–617
Rossmanith P, Krassnig M, Wagner M, Hein I (2006) Detection of Listeria monocytogenes in food using a combined enrichment/real-time PCR method targeting the prfA gene. Res Microbiol 157:763–771
Sambrook J, Maniatis T, Fritsch EF (1989) Plasmid vectors. In: Sambrook J, Maniatis T, Fritsch EF (eds) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp 74–85
Stevens KA, Jaykus LA (2004) Bacterial separation and concentration from complex sample matrices: a review. Crit Rev Microbiol 30:7–24
Acknowledgement
We gratefully acknowledge the financial support of the Christian Doppler Society for facilitating this work.
Author information
Authors and Affiliations
Corresponding author
Electronic Supplementary Material
Rights and permissions
About this article
Cite this article
Frühwirth, K., Fuchs, S., Mester, P. et al. Cloning and Characterisation of a Δ-prfA Listeria monocytogenes Strain Containing an Artificial Single Copy Genomic Internal Amplification Control (IAC) for Use as Internal Sample Process Control (ISPC). Food Anal. Methods 5, 8–18 (2012). https://doi.org/10.1007/s12161-011-9212-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12161-011-9212-6