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Article ; Online: Decontamination of

Laing, Chad / Janzen, Timothy / Blinov, Vladimir / Volchek, Konstantin / Goji, Noriko / Thomas, Matthew / Telfer, Melissa / Rohonczy, Elizabeth / Amoako, Kingsley K

Applied biosafety : journal of the American Biological Safety Association

2021 Volume 26, Issue 1, Page(s) 6–13

Abstract: Introduction: ...

Abstract	Introduction:
Language	English
Publishing date	2021-03-19
Publishing country	United States
Document type	Journal Article
ISSN	2470-1246
ISSN (online)	2470-1246
DOI	10.1089/apb.20.0067
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Article ; Online: Rapid SNP Detection and Genotyping of Bacterial Pathogens by Pyrosequencing.

Amoako, Kingsley K / Thomas, Matthew C / Janzen, Timothy W / Goji, Noriko

Methods in molecular biology (Clifton, N.J.)

2017 Volume 1492, Page(s) 203–220

Abstract: Bacterial identification and typing are fixtures of microbiology laboratories and are vital aspects of our response mechanisms in the event of foodborne outbreaks and bioterrorist events. Whole genome sequencing (WGS) is leading the way in terms of ... ...

Abstract	Bacterial identification and typing are fixtures of microbiology laboratories and are vital aspects of our response mechanisms in the event of foodborne outbreaks and bioterrorist events. Whole genome sequencing (WGS) is leading the way in terms of expanding our ability to identify and characterize bacteria through the identification of subtle differences between genomes (e.g. single nucleotide polymorphisms (SNPs) and insertions/deletions). Modern high-throughput technologies such as pyrosequencing can facilitate the typing of bacteria by generating short-read sequence data of informative regions identified by WGS analyses, at a fraction of the cost of WGS. Thus, pyrosequencing systems remain a valuable asset in the laboratory today. Presented in this chapter are two methods developed in the Amoako laboratory that detail the identification and genotyping of bacterial pathogens. The first targets canonical single nucleotide polymorphisms (canSNPs) of evolutionary importance in Bacillus anthracis, the causative agent of Anthrax. The second assay detects Shiga-toxin (stx) genes, which are associated with virulence in Escherichia coli and Shigella spp., and differentiates the subtypes of stx-1 and stx-2 based on SNP loci. These rapid methods provide end users with important information regarding virulence traits as well as the evolutionary and biogeographic origin of isolates.
Language	English
Publishing date	2017
Publishing country	United States
Document type	Journal Article
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-4939-6442-0_15
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Article: First Complete Genome Sequence of Yersinia massiliensis.

Thomas, Matthew C / Arling, Victoria / Goji, Noriko / Janzen, Timothy W / Duceppe, Marc-Olivier / Mathews, Amit / Carrillo, Catherine / Amoako, Kingsley K

Genome announcements

2018 Volume 6, Issue 20

Abstract: Using a combination of Illumina paired-end sequencing, Pacific Biosciences RS II sequencing, and OpGen Argus whole-genome optical mapping, we report here the first complete genome sequence ... ...

Abstract	Using a combination of Illumina paired-end sequencing, Pacific Biosciences RS II sequencing, and OpGen Argus whole-genome optical mapping, we report here the first complete genome sequence of
Language	English
Publishing date	2018-05-17
Publishing country	United States
Document type	Journal Article
ZDB-ID	2704277-7
ISSN	2169-8287
ISSN	2169-8287
DOI	10.1128/genomeA.00416-18
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Article: Rapid detection and serovar identification of common Salmonella enterica serovars in Canada using a new pyrosequencing assay

Furukawa, Maika / Amoako, Kingsley K / Blais, Burton / Goji, Noriko / Janzen, Timothy W / Misawa, Naoaki / Ogunremi, Dele / Thomas, Matthew C

Canadian journal of microbiology. 2018, v. 64, no. 1

2018

Abstract: Serotyping of Salmonella enterica subsp. enterica is a critical step for foodborne salmonellosis investigation. To identify Salmonella enterica subsp. enterica serovars, we have developed a new assay based on a triplex polymerase chain reaction (PCR) ... ...

Abstract	Serotyping of Salmonella enterica subsp. enterica is a critical step for foodborne salmonellosis investigation. To identify Salmonella enterica subsp. enterica serovars, we have developed a new assay based on a triplex polymerase chain reaction (PCR) with pyrosequencing for amplicon confirmation and phylogenetic discrimination of strains. The top 54 most prevalent serovars of S. enterica in Canada were examined with a total of 23 single-nucleotide polymorphisms (SNPs) and (or) single-nucleotide variations (SNVs) located on 3 genes (fliD, sopE2, and spaO). Seven of the most common serovars, Newport, Typhi, Javiana, Infantis, Thompson, Heidelberg, and Enteritidis, were successfully distinguished from the other serovars based on their unique SNP–SNV combinations. The remaining serovars, including Typhimurium, ssp I:4,[5],12:i:–, and Saintpaul, were further divided into 47 subgroups that demonstrate the relatedness to phylogenetic classifications of each serovar. This pyrosequencing assay is not only cost-effective, rapid, and user-friendly, but also provides phylogenetic information by analyzing 23 selected SNPs. With the added layer of confidence in the PCR results and the accuracy and speed of pyrosequencing, this novel method would benefit the food industry and provides a tool for rapid outbreak investigation through quick detection and identification of common S. enterica serovars in Canada.
Keywords	cost effectiveness ; food industry ; genes ; outbreak investigation ; phylogeny ; polymerase chain reaction ; rapid methods ; Salmonella enterica subsp. enterica ; salmonellosis ; sequence analysis ; serotypes ; single nucleotide polymorphism ; Canada
Language	English
Size	p. 75-86.
Publishing place	NRC Research Press
Document type	Article
ZDB-ID	280534-0
ISSN	1480-3275 ; 0008-4166
ISSN (online)	1480-3275
ISSN	0008-4166
DOI	10.1139/cjm-2017-0496
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Article ; Online: Rapid detection and serovar identification of common Salmonella enterica serovars in Canada using a new pyrosequencing assay.

Furukawa, Maika / Goji, Noriko / Janzen, Timothy W / Thomas, Matthew C / Ogunremi, Dele / Blais, Burton / Misawa, Naoaki / Amoako, Kingsley Kwaku

Canadian journal of microbiology

2017

Abstract: Serotyping of Salmonella enterica subspecies enterica is a critical step for foodborne salmonellosis investigation. We have developed a new assay to identify Salmonella enterica subsp. enterica (S. enterica) serovars based on a triplex polymerase chain ... ...

Abstract	Serotyping of Salmonella enterica subspecies enterica is a critical step for foodborne salmonellosis investigation. We have developed a new assay to identify Salmonella enterica subsp. enterica (S. enterica) serovars based on a triplex polymerase chain reaction (PCR) with pyrosequencing for amplicon confirmation and phylogenetic discrimination of strain. The top 54 most prevalent serovars of S. enterica in Canada were examined with a total of 23 single nucleotide polymorphisms (SNPs) and/or variations (SNVs) located on three genes (fliD, sopE2, and spaO). Seven of the most common serovars, including Newport, Typhi, Javiana, Infantis, Thompson, Heidelberg and Enteritidis are successfully distinguished from the other serovars based on their unique SNP/SNV combinations. The remaining serovars, including Typhimurium, ssp I 4,[5],12:i:-, and Saintpaul are further divided into 47 subgroups that demonstrate the relatedness to phylogenetic classifications of each serovar. This pyrosequencing assay is not only cost-effective, rapid, and user-friendly, but also provides phylogenetic information by analysing 23 selected SNPs. With the additional layer of confidence to the PCR results and the accuracy and speed of pyrosequencing, this novel method would benefit the food industry and provides a tool for rapid outbreak investigation through quick detection and identification of common S. enterica serovars in Canada.
Language	English
Publishing date	2017-10-31
Publishing country	Canada
Document type	Journal Article
ZDB-ID	280534-0
ISSN	1480-3275 ; 0008-4166
ISSN (online)	1480-3275
ISSN	0008-4166
DOI	10.1139/cjm-2017-0496
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Article ; Online: Rapid detection method for Bacillus anthracis using a combination of multiplexed real-time PCR and pyrosequencing and its application for food biodefense.

Janzen, Timothy W / Thomas, Matthew C / Goji, Noriko / Shields, Michael J / Hahn, Kristen R / Amoako, Kingsley K

Journal of food protection

2015 Volume 78, Issue 2, Page(s) 355–361

Abstract: Bacillus anthracis, the causative agent of anthrax, has the capacity to form highly resilient spores as part of its life cycle. The potential for the dissemination of these spores using food as a vehicle is a huge public health concern and, hence, ... ...

Abstract	Bacillus anthracis, the causative agent of anthrax, has the capacity to form highly resilient spores as part of its life cycle. The potential for the dissemination of these spores using food as a vehicle is a huge public health concern and, hence, requires the development of a foodborne bioterrorism response approach. In this work, we address a critical gap in food biodefense by presenting a novel, combined, sequential method involving the use of real-time PCR and pyrosequencing for the rapid, specific detection of B. anthracis spores in three food matrices: milk, apple juice, and bottled water. The food samples were experimentally inoculated with 40 CFU ml(-1), and DNA was extracted from the spores and analyzed after immunomagnetic separation. Applying the combination of multiplex real-time PCR and pyrosequencing, we successfully detected the presence of targets on both of the virulence plasmids and the chromosome. The results showed that DNA amplicons generated from a five-target multiplexed real-time PCR detection using biotin-labeled primers can be used for single-plex pyrosequencing detection. The combined use of multiplexed real-time PCR and pyrosequencing is a novel, rapid detection method for B. anthracis from food and provides a tool for accurate, quantitative identification with potential biodefense applications.
MeSH term(s)	Animals ; Bacillus anthracis/genetics ; Bacillus anthracis/isolation & purification ; Beverages/microbiology ; Bioterrorism ; DNA Primers ; DNA, Bacterial/isolation & purification ; Drinking Water/microbiology ; Food Microbiology/methods ; Immunomagnetic Separation ; Milk/microbiology ; Real-Time Polymerase Chain Reaction/methods ; Sequence Analysis, DNA/methods ; Spores, Bacterial/isolation & purification
Chemical Substances	DNA Primers ; DNA, Bacterial ; Drinking Water
Language	English
Publishing date	2015-01-20
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	243284-5
ISSN	1944-9097 ; 0362-028X
ISSN (online)	1944-9097
ISSN	0362-028X
DOI	10.4315/0362-028X.JFP-14-216
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Article ; Online: Single nucleotide repeat analysis of B. anthracis isolates in Canada through comparison of pyrosequencing and Sanger sequencing.

Hahn, Kristen R / Janzen, Timothy W / Thomas, Matthew C / Shields, Michael J / Goji, Noriko / Valle, Edith / Amoako, Kingsley K

Veterinary microbiology

2014 Volume 169, Issue 3-4, Page(s) 228–232

Abstract: Several technology platforms have been developed to resolve the phylogenetic placement of B. anthracis. However, these methods lack the resolution to identify differences between closely related strains within an outbreak due to the highly clonal nature ... ...

Abstract	Several technology platforms have been developed to resolve the phylogenetic placement of B. anthracis. However, these methods lack the resolution to identify differences between closely related strains within an outbreak due to the highly clonal nature of B. anthracis. Single Nucleotide Repeats (SNRs) are a type of rapidly evolving genetic marker that can be used to track epidemiological distribution in the event of an outbreak. Four SNR targets were used to detect and type 35 B. anthracis isolates in our collection; 18 from across Canada obtained between 1972 and 2005 and 17 from the 2006 Anthrax outbreak in north eastern Saskatchewan. A control sequence was developed for pyrosequencing which yielded consistent and accurate reads of SNRs. However, when DNA from the isolates was tested using pyrosequencing the results were inconsistent and did not reflect the number of SNRs obtained by Sanger sequencing. The SNR numbers derived from the Sanger sequencing show two of the four SNR loci could provide information on subtype, whereas the other two were not discriminatory. There is variation in SNRs between strains isolated from different outbreaks, the subset of 2006 outbreak strains showed very little difference in SNR number, and thus suggests low diversity among the strains sampled from the same outbreak.
MeSH term(s)	Animals ; Anthrax/microbiology ; Bacillus anthracis/classification ; Bacillus anthracis/genetics ; Bacillus anthracis/isolation & purification ; Canada ; Genetic Markers/genetics ; Genetic Variation ; Genotype ; Nucleotides/genetics ; Phylogeny ; Sequence Analysis, DNA/standards ; Species Specificity
Chemical Substances	Genetic Markers ; Nucleotides
Language	English
Publishing date	2014-03-14
Publishing country	Netherlands
Document type	Comparative Study ; Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	753154-0
ISSN	1873-2542 ; 0378-1135
ISSN (online)	1873-2542
ISSN	0378-1135
DOI	10.1016/j.vetmic.2013.12.020
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Article ; Online: Rapid detection and identification of Bacillus anthracis in food using pyrosequencing technology.

Amoako, Kingsley K / Janzen, Timothy W / Shields, Michael J / Hahn, Kristen R / Thomas, Matthew C / Goji, Noriko

International journal of food microbiology

2013 Volume 165, Issue 3, Page(s) 319–325

Abstract: The development of advanced methodologies for the detection of Bacillus anthracis has been evolving rapidly since the release of the anthrax spores in the mail in 2001. Recent advances in detection and identification techniques could prove to be an ... ...

Abstract	The development of advanced methodologies for the detection of Bacillus anthracis has been evolving rapidly since the release of the anthrax spores in the mail in 2001. Recent advances in detection and identification techniques could prove to be an essential component in the defense against biological attacks. Sequence based such as pyrosequencing, which has the capability to determine short DNA stretches in real-time using biotinylated PCR amplicons, has potential biodefense applications. Using markers from the virulence plasmids (pXO1 and pXO2) and chromosomal regions, we have demonstrated the power of this technology in the rapid, specific and sensitive detection of B. anthracis spores in food matrices including milk, juice, bottled water, and processed meat. The combined use of immunomagnetic separation and pyrosequencing showed positive detection when liquid foods (bottled water, milk, juice), and processed meat were experimentally inoculated with 6CFU/mL and 6CFU/g, respectively, without an enrichment step. Pyrosequencing is completed in about 60min (following PCR amplification) and yields accurate and reliable results with an added layer of confidence. The entire assay (from sample preparation to sequencing information) can be completed in about 7.5h. A typical run on food samples yielded 67-80bp reads with 94-100% identity to the expected sequence. This sequence based approach is a novel application for the detection of anthrax spores in food with potential application in foodborne bioterrorism response and biodefense involving the use of anthrax spores.
MeSH term(s)	Animals ; Bacillus anthracis/genetics ; Bacillus anthracis/isolation & purification ; DNA, Bacterial/genetics ; Dairy Products/microbiology ; Food Microbiology/methods ; Immunomagnetic Separation ; Milk/microbiology ; Polymerase Chain Reaction ; Sequence Analysis, DNA ; Spores, Bacterial ; Water Microbiology
Chemical Substances	DNA, Bacterial
Language	English
Publishing date	2013-08-01
Publishing country	Netherlands
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	87122-9
ISSN	1879-3460 ; 0168-1605
ISSN (online)	1879-3460
ISSN	0168-1605
DOI	10.1016/j.ijfoodmicro.2013.05.028
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Article ; Online: Rapid detection and antimicrobial resistance gene profiling of Yersinia pestis using pyrosequencing technology.

Amoako, Kingsley K / Thomas, Matthew C / Kong, Fanliang / Janzen, Timothy W / Hahn, Kristen R / Shields, Michael J / Goji, Noriko

Journal of microbiological methods

2012 Volume 90, Issue 3, Page(s) 228–234

Abstract: When a bioterrorism attack is attempted or perpetrated there is considerable risk for public health and large scale socioeconomic consequences. It is imperative that we possess established assays for the rapid identification of biothreat agents with high ...

Abstract	When a bioterrorism attack is attempted or perpetrated there is considerable risk for public health and large scale socioeconomic consequences. It is imperative that we possess established assays for the rapid identification of biothreat agents with high sensitivity and specificity to ensure emergency response measures can be deployed appropriately. Highly trustworthy information within a relevant timeframe is required to make a rapid and informed decision. Obtaining DNA sequence data from a suspected agent provides an added layer of confidence compared to a presumptive positive PCR amplicon. Sequencing based technologies, such as pyrosequencing, have sufficient discrimination potential to be used for microbial identification and can also be used to identify antimicrobial resistance (AMR) genes. We have shown in this study the power of pyrosequencing in the unambiguous detection and identification of nine Yersinia pestis strains based on virulence genes. Furthermore, we developed assays to characterize their AMR gene profiles. Sequence results ranging from 40 to 84bp were generated in about 60 min following initial PCR amplification and provide a rapid method for determining the AMR profile as compared to the conventional plate method which takes several days. The high sequence identities (95-100%) and specificity observed indicate the high level of accuracy of pyrosequencing technology. In addition, the read lengths of up to 84 bp observed in this study are unprecedented for pyrosequencing using the Pyromark Q24. We propose this method as a novel, rapid, sequence based detection and identification tool for Y. pestis with a potential application in biodefence.
MeSH term(s)	Base Sequence ; DNA, Bacterial/genetics ; DNA, Bacterial/isolation & purification ; Drug Resistance, Bacterial/genetics ; Molecular Sequence Data ; Molecular Typing ; R Factors/genetics ; Sensitivity and Specificity ; Sequence Analysis, DNA ; Yersinia pestis/genetics
Chemical Substances	DNA, Bacterial
Language	English
Publishing date	2012-09
Publishing country	Netherlands
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	604916-3
ISSN	1872-8359 ; 0167-7012
ISSN (online)	1872-8359
ISSN	0167-7012
DOI	10.1016/j.mimet.2012.05.012
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Article ; Online: Rapid Detection and Identification of Yersinia pestis from Food Using Immunomagnetic Separation and Pyrosequencing.

Amoako, Kingsley K / Shields, Michael J / Goji, Noriko / Paquet, Chantal / Thomas, Matthew C / Janzen, Timothy W / Bin Kingombe, Cesar I / Kell, Arnold J / Hahn, Kristen R

Journal of pathogens

2012 Volume 2012, Page(s) 781652

Abstract: Interest has recently been renewed in the possible use of Y. pestis, the causative agent of plague, as a biological weapon by terrorists. The vulnerability of food to intentional contamination coupled with reports of humans having acquired plague through ...

Abstract	Interest has recently been renewed in the possible use of Y. pestis, the causative agent of plague, as a biological weapon by terrorists. The vulnerability of food to intentional contamination coupled with reports of humans having acquired plague through eating infected animals that were not adequately cooked or handling of meat from infected animals makes the possible use of Y. pestis in a foodborne bioterrorism attack a reality. Rapid, efficient food sample preparation and detection systems that will help overcome the problem associated with the complexity of the different matrices and also remove any ambiguity in results will enable rapid informed decisions to be made regarding contamination of food with biothreat agents. We have developed a rapid detection assay that combines the use of immunomagnetic separation and pyrosequencing in generating results for the unambiguous identification of Y. pestis from milk (0.9 CFU/mL), bagged salad (1.6 CFU/g), and processed meat (10 CFU/g). The low detection limits demonstrated in this assay provide a novel tool for the rapid detection and confirmation of Y. pestis in food without the need for enrichment. The combined use of the iCropTheBug system and pyrosequencing for efficient capture and detection of Y. pestis is novel and has potential applications in food biodefence.
Language	English
Publishing date	2012-10-03
Publishing country	Egypt
Document type	Journal Article
ZDB-ID	2662334-1
ISSN	2090-3065 ; 2090-3065
ISSN (online)	2090-3065
ISSN	2090-3065
DOI	10.1155/2012/781652
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