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Development of a novel multiplexed qPCR and Pyrosequencing method for the detection of human pathogenic yersiniae.
Thomas, M C; Janzen, T W; Huscyzynsky, G; Mathews, A; Amoako, K K.
Afiliación
  • Thomas MC; Canadian Food Inspection Agency, Lethbridge Laboratory, Township Rd 9-1, Lethbridge, Alberta T1J 3Z4, Canada.
  • Janzen TW; Canadian Food Inspection Agency, Lethbridge Laboratory, Township Rd 9-1, Lethbridge, Alberta T1J 3Z4, Canada.
  • Huscyzynsky G; Canadian Food Inspection Agency, Greater Toronto Area Laboratory, 2301 Midland Ave., Scarborough, Ontario M1P 4R7, Canada.
  • Mathews A; Canadian Food Inspection Agency, Greater Toronto Area Laboratory, 2301 Midland Ave., Scarborough, Ontario M1P 4R7, Canada.
  • Amoako KK; Canadian Food Inspection Agency, Lethbridge Laboratory, Township Rd 9-1, Lethbridge, Alberta T1J 3Z4, Canada. Electronic address: kingsley.amoako@inspection.gc.ca.
Int J Food Microbiol ; 257: 247-253, 2017 Sep 18.
Article en En | MEDLINE | ID: mdl-28704728
The purpose of this study was to develop a novel and robust molecular assay for the detection of human pathogenic yersiniae (i.e. Yersinia enterocolitica, Y. pseudotuberculosis and Y. pestis) in complex food samples. The assay combines multiplexed real-time PCR (qPCR) and Pyrosequencing for detecting and differentiating human pathogenic yersiniae with high confidence through sequence based confirmation. The assay demonstrated 100% specificity and inclusivity when tested against a panel of 14 Y. enterocolitica, 22 Y. pestis, 24 Y. pseudotuberculosis and a diverse selection of 17 other non-Yersinia bacteria. Pyrosequencing reads ranged from 28 to 40bp in length and had 94-100% sequence identity to the correct species in the GenBank nr database. Microbial enrichments of 48 ready-to-eat foods collected in the Greater Toronto Area from March 2014 to May 2014, including 46 fresh sprout and 2 salad products, were then tested using the assay. All samples were negative for Y. pestis and Y. pseudotuberculosis. Both salads (n=2) and 35% of sprout products (n=46) including 7.1% of alfalfa sprouts (n=14), 81% of bean sprouts (n=16), 12% of mixed sprouts (n=8) tested positive for Y. enterocolitica which was not detected in broccoli sprouts (n=5), onion sprouts (n=1), and pea sprouts (n=2). Cycle thresholds (Ct) of positive samples for Y. enterocolitica were between 23.0 and 37.9 suggesting post enrichment concentrations of approximately 1×102 to 1×106Y. enterocolitica per 1mL of enriched broth. An internal amplification control which was coamplified with targets revealed PCR inhibition in five samples which was resolved following a one in ten dilution. Pyrosequencing of qPCR amplicons suggests monoclonality and revealed a single nucleotide polymorphism that is present in Y. enterocolitica biotype 1A suggesting low pathogenicity of the detected strains. This study is the first to combine Pyrosequencing and qPCR for the detection of human pathogenic yersiniae and is applicable to a broad range of complex samples including ready-to-eat food samples.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Yersinia enterocolitica / Yersinia pestis / Yersinia pseudotuberculosis / Contaminación de Alimentos / Reacción en Cadena de la Polimerasa Multiplex / Microbiología de Alimentos Tipo de estudio: Diagnostic_studies Límite: Humans Idioma: En Revista: Int J Food Microbiol Asunto de la revista: CIENCIAS DA NUTRICAO / MICROBIOLOGIA Año: 2017 Tipo del documento: Article País de afiliación: Canadá Pais de publicación: Países Bajos

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Yersinia enterocolitica / Yersinia pestis / Yersinia pseudotuberculosis / Contaminación de Alimentos / Reacción en Cadena de la Polimerasa Multiplex / Microbiología de Alimentos Tipo de estudio: Diagnostic_studies Límite: Humans Idioma: En Revista: Int J Food Microbiol Asunto de la revista: CIENCIAS DA NUTRICAO / MICROBIOLOGIA Año: 2017 Tipo del documento: Article País de afiliación: Canadá Pais de publicación: Países Bajos