Pyrosequencing pp 363-375 | Cite as

Application of Pyrosequencing® in Food Biodefense

  • Kingsley Kwaku Amoako
Part of the Methods in Molecular Biology book series (MIMB, volume 1315)


The perpetration of a bioterrorism attack poses a significant risk for public health with potential socioeconomic consequences. It is imperative that we possess reliable assays for the rapid and accurate identification of biothreat agents to make rapid risk-informed decisions on emergency response. The development of advanced methodologies for the detection of biothreat agents has been evolving rapidly since the release of the anthrax spores in the mail in 2001, and recent advances in detection and identification techniques could prove to be an essential component in the defense against biological attacks. Sequence-based approaches such as Pyrosequencing®, which has the capability to determine short DNA stretches in real time using biotinylated PCR amplicons, have potential biodefense applications. Using markers from the virulence plasmids and chromosomal regions, my laboratory has demonstrated the power of this technology in the rapid, specific, and sensitive detection of B. anthracis spores and Yersinia pestis in food. These are the first applications for the detection of the two organisms in food. Furthermore, my lab has developed a rapid assay to characterize the antimicrobial resistance (AMR) gene profiles for Y. pestis using Pyrosequencing. Pyrosequencing is completed in about 60 min (following PCR amplification) and yields accurate and reliable results with an added layer of confidence, thus enabling rapid risk-informed decisions to be made. A typical run yields 40–84 bp reads with 94–100 % identity to the expected sequence. It also provides a rapid method for determining the AMR profile as compared to the conventional plate method which takes several days. The method described is proposed as a novel detection system for potential application in food biodefense.

Key words

Bacillus anthracis Yersinia pestis Antimicrobial resistance Pyrosequencing® 



Thank you to all the personnel in the Amoako lab who provided technical assistance in the work on the application of Pyrosequencing for the detection of Bacillus anthracis and Yersinia pestis in food. The assistance of Matthew Thomas in compiling this work is acknowledged. This work was funded by the Defence Research Development Canada Centre for Security Science, Chemical, Biological, Radiological, Nuclear and Explosive Research Technology Initiative (CRTI) funding from CRTI 08-0203RD.


  1. 1.
    Torok TJ, Tauxe RV, Wise RP et al (1997) A large community outbreak of salmonellosis caused by intentional contamination of restaurant salad bars. JAMA 278:389–395PubMedCrossRefGoogle Scholar
  2. 2.
    Kennedy S (2008) Epidemiology. Why can’t we test our way to absolute food safety? Science 322:1641–1643PubMedCrossRefGoogle Scholar
  3. 3.
    Amoako KK, Thomas MC, Kong F et al (2012) Rapid detection and antimicrobial resistance gene profiling of Yersinia pestis using pyrosequencing technology. J Microbiol Methods 90:228–234PubMedCrossRefGoogle Scholar
  4. 4.
    Ronaghi M, Karamohamed S, Pettersson B et al (1996) Real-time DNA sequencing using detection of pyrophosphate release. Anal Biochem 242:84–89PubMedCrossRefGoogle Scholar
  5. 5.
    Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 74:5463–5467PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Ronaghi M (2001) Pyrosequencing sheds light on DNA sequencing. Genome Res 11:3–11PubMedCrossRefGoogle Scholar
  7. 7.
    Ronaghi M, Elahi E (2002) Pyrosequencing for microbial typing. J Chromatogr B Analyt Technol Biomed Life Sci 782:67–72PubMedCrossRefGoogle Scholar
  8. 8.
    Hahn KR, Janzen TW, Thomas MC et al (2014) Single nucleotide repeat analysis of B. anthracis isolates in Canada through comparison of pyrosequencing and Sanger sequencing. Vet Microbiol 169:228–232PubMedCrossRefGoogle Scholar
  9. 9.
    Alderborn A, Kristofferson A, Hammerling U (2000) Determination of single-nucleotide polymorphisms by real-time pyrophosphate DNA sequencing. Genome Res 10:1249–1258PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Unnerstad H, Ericsson H, Alderborn A et al (2001) Pyrosequencing as a method for grouping of Listeria monocytogenes strains on the basis of single-nucleotide polymorphisms in the inlB gene. Appl Environ Microbiol 67:5339–5342PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Jonasson J, Olofsson M, Monstein HJ (2002) Classification, identification and subtyping of bacteria based on pyrosequencing and signature matching of 16S rDNA fragments. APMIS 110:263–272PubMedCrossRefGoogle Scholar
  12. 12.
    Bravo LT, Tuohy MJ, Ang C et al (2009) Pyrosequencing for rapid detection of Mycobacterium tuberculosis resistance to rifampin, isoniazid, and fluoroquinolones. J Clin Microbiol 47:3985–3990PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Arnold C, Westland L, Mowat G et al (2005) Single-nucleotide polymorphism-based differentiation and drug resistance detection in Mycobacterium tuberculosis from isolates or directly from sputum. Clin Microbiol Infect 11:122–130PubMedCrossRefGoogle Scholar
  14. 14.
    Gharizadeh B, Akhras M, Unemo M et al (2005) Detection of gyrA mutations associated with ciprofloxacin resistance in Neisseria gonorrhoeae by rapid and reliable pre-programmed short DNA sequencing. Int J Antimicrob Agents 26:486–490PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Naas T, Poirel L, Nordmann P (2006) Pyrosequencing for rapid identification of carbapenem-hydrolysing OXA-type ß-lactamases in Acinetobacter baumannii. Clin Microbiol Infect 12:1236–1240PubMedCrossRefGoogle Scholar
  16. 16.
    Borman AM, Petch R, Linton CJ et al (2008) Candida nivariensis, an emerging pathogenic fungus with multidrug resistance to antifungal agents. J Clin Microbiol 46:933–938PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Haanperä M, Huovinen P, Jalava J (2005) Detection and quantification of macrolide resistance mutations at positions 2058 and 2059 of the 23S rRNA gene by pyrosequencing. Antimicrob Agents Chemother 49:457–460PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Kruckeberg KE, Thibodeau SN (2004) Pyrosequencing technology as a method for the diagnosis of multiple endocrine neoplasia type 2. Clin Chem 50:522–529PubMedCrossRefGoogle Scholar
  19. 19.
    Söderbäck E, Zackrisson AL, Lindblom B et al (2005) Determination of CYP2D6 gene copy number by pyrosequencing. Clin Chem 51:522–531PubMedCrossRefGoogle Scholar
  20. 20.
    Kobayashi N, Bauer TW, Tuohy MJ et al (2006) The comparison of pyrosequencing molecular Gram stain, culture, and conventional Gram stain for diagnosing orthopaedic infections. J Orthop Res 24:1641–1649PubMedCrossRefGoogle Scholar
  21. 21.
    García-Sierra N, Lacoma A, Prat C et al (2011) Pyrosequencing for rapid molecular detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis strains and clinical specimens. J Clin Microbiol 49:3683–3686PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Helgason E, Okstad OA, Caugant DA et al (2000) Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis–one species on the basis of genetic evidence. Appl Enviorn Microbiol 66:2627–2630CrossRefGoogle Scholar
  23. 23.
    Ash C, Farrow JA, Dorsch M et al (1991) Comparative analysis of Bacillus anthracis, Bacillus cereus, and related species on the basis of reverse transcriptase sequencing of 16S rRNA. Int J Syst Bacteriol 41:343–346PubMedCrossRefGoogle Scholar
  24. 24.
    Priest FG, Goodfellow M, Todd C (1988) A numerical classification of the genus Bacillus. J Gen Microbiol 134:1847–1882PubMedGoogle Scholar
  25. 25.
    Harrell LJ, Andersen GL, Wilson KH (1995) Genetic variability of Bacillus anthracis and related species. J Clin Microbiol 33:1847–1850PubMedCentralPubMedGoogle Scholar
  26. 26.
    Jernigan DB, Raghunathan PL, Bell BP et al (2002) Investigation of bioterrorism-related anthrax, United States, 2001: epidemiologic findings. Emerg Infect Dis 8:1019–1028PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Pearson T, Busch JD, Ravel J et al (2004) Phylogenetic discovery bias in Bacillus anthracis using single-nucleotide polymorphisms from whole-genome sequencing. Proc Natl Acad Sci U S A 101:13536–13541PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Van Ert MN, Hofstadler SA, Jiang Y et al (2004) Mass spectrometry provides accurate characterization of two genetic marker types in Bacillus anthracis. Biotechniques 37:642–648PubMedGoogle Scholar
  29. 29.
    Hurtle W, Bode E, Kulesh DA et al (2004) Detection of the Bacillus anthracis gyrA gene by using a minor groove binder probe. J Clin Microbiol 42:179–185PubMedCentralPubMedCrossRefGoogle Scholar
  30. 30.
    Hill KK, Ticknor LO, Okinaka RT et al (2004) Fluorescent amplified fragment length polymorphism analysis of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis isolates. Appl Enviorn Microbiol 70:1068–1080CrossRefGoogle Scholar
  31. 31.
    Ellerbrok H, Nattermann H, Ozel M et al (2002) Rapid and sensitive identification of pathogenic and apathogenic Bacillus anthracis by real-time PCR. FEMS Microbiol Lett 214:51–59PubMedCrossRefGoogle Scholar
  32. 32.
    Qi Y, Patra G, Liang X et al (2001) Utilization of the rpoB gene as a specific chromosomal marker for real-time PCR detection of Bacillus anthracis. Appl Enviorn Microbiol 67:3720–3727CrossRefGoogle Scholar
  33. 33.
    Joung KB, Cote JC (2002) Evaluation of ribosomal RNA gene restriction patterns for the classification of Bacillus species and related genera. J Appl Microbiol 92:97–108PubMedCrossRefGoogle Scholar
  34. 34.
    Yamazaki K, Okubo T, Inoue N et al (1997) Randomly amplified polymorphic DNA (RAPD) for rapid identification of spoilage bacterium Alicyclobacillus acidoterrestris. Biosci Biotechnol Biochem 61:1016–1019CrossRefGoogle Scholar
  35. 35.
    Goto K, Omura T, Hara Y et al (2000) Application of the partial 16S rDNA sequence as an index for rapid identification of species in the genus Bacillus. J Gen Appl Microbiol 46:1–8PubMedCrossRefGoogle Scholar
  36. 36.
    Turenne CY, Tschetter L, Wolfe J et al (2001) Necessity of quality-controlled 16S rRNA gene sequence databases: identifying nontuberculous Mycobacterium species. J Clin Microbiol 39:3637–3648PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    Amoako KK, Shields MJ, Goji N et al (2012) Rapid detection and identification of Yersinia pestis from food using immunomagnetic separation and pyrosequencing. J Pathog 2012:781652PubMedCentralPubMedCrossRefGoogle Scholar
  38. 38.
    Amoako KK, Janzen TW, Shields MJ et al (2013) Rapid detection and identification of Bacillus anthracis in food using pyrosequencing technology. Int J Food Microbiol 165:319–325PubMedCrossRefGoogle Scholar
  39. 39.
    Loveless BM, Yermakova A, Christensen DR et al (2010) Identification of ciprofloxacin resistance by simpleprobe, high resolution melt and pyrosequencing nucleic acid analysis in biothreat agents: Bacillus anthracis, Yersinia pestis and Francisella tularensis. Mol Cell Probes 24:154–160PubMedCrossRefGoogle Scholar
  40. 40.
    Wahab T, Hjalmarsson S, Wollin R et al (2005) Pyrosequencing Bacillus anthracis. Emerg Infect Dis 11:1527–1531PubMedCentralPubMedCrossRefGoogle Scholar
  41. 41.
    Ahmod NZ, Gupta RS, Shah HN (2011) Identification of a Bacillus anthracis specific indel in the yeaC gene and development of a rapid pyrosequencing assay for distinguishing B. anthracis from the B. cereus group. J Microbiol Methods 87:278–285PubMedCrossRefGoogle Scholar
  42. 42.
    Janzen Timothy, Michael Shields, Noriko Goji, Matthew Thomas, Kristen Hahn and Amoako K (2015) Rapid detection method for B. anthracis using a combination of multiplexed real-time PCR and pyrosequencing and its application for food biodefense. J Food Prot 78:355–361Google Scholar
  43. 43.
    Shields MJ, Hahn KR, Janzen TW et al (2012) Immunomagnetic capture of Bacillus anthracis spores from food. J Food Prot 75:1243–1248PubMedCrossRefGoogle Scholar
  44. 44.
    Amoako KK, Goji N, Macmillan T et al (2010) Development of multitarget real-time PCR for the rapid, specific, and sensitive detection of Yersinia pestis in milk and ground beef. J Food Prot 73:18–25PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.National Centers for Animal Disease, Lethbridge LaboratoryCanadian Food Inspection AgencyLethbridgeCanada

Personalised recommendations