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

doi:10.1016/j.mimet.2012.05.012

Cited by 22 publications

(18 citation statements)

References 38 publications

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“…The pyrosequencing of Y. pestis cells captured from the three different food matrices, conducted for targets Ypc4, Ypcaf1M1, and Yppst1, yielded reads identical to those observed in our previous report [21] (Figure 2). As previously observed, they yielded BLAST results exclusive to Y. pestis and thus confirmed the identification of Y. pestis .…”

Section: Resultssupporting

confidence: 72%

“…Following a brief centrifugation, 3.5 μ L of the supernatant was used as template for PCR amplification and then followed by pyrosequencing analysis. The PCR primers and reaction conditions are indicated in our previous work [21]. …”

Section: Methodsmentioning

confidence: 99%

“…Genomic DNA from Y. pestis samples isolated from food were analysed using our previously described pyrosequencing assay [21]. Briefly, biotinylated PCR products were bound to streptavidin-coated sepharose beads (GE Healthcare, Piscataway, NJ) and the beads were then resuspended in annealing buffer containing 0.3 μ M of the sequencing primer.…”

Section: Methodsmentioning

confidence: 99%

“…We have recently developed an immunomagnetic separation (IMS) assay for the efficient concentration of Bacillus anthracis spores from different food matrices [20] and a novel sequence-based assay using pyrosequencing for the specific detection and antimicrobial resistance gene profiling of Y. pestis [21]. Here, we present the application of IMS and pyrosequencing based assays for the rapid, specific, and sensitive detection and identification of Y. pestis from food matrices such as milk, bagged salad, and processed meat.…”

Section: Introductionmentioning

confidence: 99%

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Rapid Detection and Identification ofYersinia pestisfrom Food Using Immunomagnetic Separation and Pyrosequencing

Amoako

Shields

Goji

et al. 2012

Journal of Pathogens

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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.

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Section: Resultssupporting

confidence: 72%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Rapid Detection and Identification ofYersinia pestisfrom Food Using Immunomagnetic Separation and Pyrosequencing

Amoako

Shields

Goji

et al. 2012

Journal of Pathogens

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“…The simplicity and error tolerance of detection methods should also be considered for nonprofessional operations. Polymerase chain reaction (PCR) [4] and other techniques [9]–[13] have been developed, but these methods cannot be used as POCT assays because of expensive equipment, complex sample pretreatment, and complicated and time-consuming operations. Lateral flow (LF) immunoassay is a widely used and comprehensively applied POCT technology [14], [15] to rapidly detect various pathogens, such as Y. pestis [16], B. anthracis [17], and Brucella spp.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of Up-Converting Phosphor Technology-Based Lateral Flow Strips for Rapid Detection of Bacillus anthracis Spore, Brucella spp., and Yersinia pestis

et al. 2014

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Bacillus anthracis, Brucella spp., and Yersinia pestis are zoonotic pathogens and biowarfare- or bioterrorism-associated agents that must be detected rapidly on-site from various samples (e.g., viscera and powders). An up-converting phosphor technology-based lateral flow (UPT–LF) strip was developed as a point-of-care testing (POCT) to satisfy the requirements of first-level emergency response. We developed UPT–LF POCT to quantitatively detect the three pathogens within 15 min. Sample and operation-error tolerances of the assay were comprehensively evaluated. The sensitivity of UPT–LF assay to bacterial detection reached 104 cfu·mL−1 (100 cfu/test), with a linear quantitative range of 4 to 6 orders of magnitude. Results revealed that the UPT–LF assay exhibited a high specificity with the absence of false-positive results even at 109 cfu·mL−1 of non-specific bacterial contamination. The assay could tolerate samples with a wide pH range (2 to 12), high ion strengths (≥4 mol·L−1 of NaCl), high viscosities (≤25 mg·mL−1 of PEG20000 or ≥20% of glycerol), and high concentrations of bio-macromolecule (≤200 mg·mL−1 of bovine serum albumin or ≥80 mg·mL−1 of casein). The influence of various types of powders and viscera (fresh and decomposed) on the performance of UPT–LF assay was determined. The operational error of liquid measurement exhibited few effects on sensitivity and specificity. The developed UPT–LF POCT assay is applicable under field conditions with excellent tolerance to sample complexity and operational error.

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Application of Pyrosequencing® in Food Biodefense

Amoako

2015

Methods in Molecular Biology

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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.

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Rapid detection and antimicrobial resistance gene profiling of Yersinia pestis using pyrosequencing technology

Cited by 22 publications

References 38 publications

Rapid Detection and Identification ofYersinia pestisfrom Food Using Immunomagnetic Separation and Pyrosequencing

Rapid Detection and Identification ofYersinia pestisfrom Food Using Immunomagnetic Separation and Pyrosequencing

Evaluation of Up-Converting Phosphor Technology-Based Lateral Flow Strips for Rapid Detection of Bacillus anthracis Spore, Brucella spp., and Yersinia pestis

Application of Pyrosequencing® in Food Biodefense

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