Cargando…

Droplet digital polymerase chain reaction (ddPCR) assays integrated with an internal control for quantification of bovine, porcine, chicken and turkey species in food and feed

Food adulteration and feed contamination are significant issues in the food/feed industry, especially for meat products. Reliable techniques are needed to monitor these issues. Droplet Digital PCR (ddPCR) assays were developed and evaluated for detection and quantification of bovine, porcine, chicke...

Descripción completa

Detalles Bibliográficos
Autores principales: Shehata, Hanan R., Li, Jiping, Chen, Shu, Redda, Helen, Cheng, Shumei, Tabujara, Nicole, Li, Honghong, Warriner, Keith, Hanner, Robert
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5552122/
https://www.ncbi.nlm.nih.gov/pubmed/28796824
http://dx.doi.org/10.1371/journal.pone.0182872
_version_ 1783256413796040704
author Shehata, Hanan R.
Li, Jiping
Chen, Shu
Redda, Helen
Cheng, Shumei
Tabujara, Nicole
Li, Honghong
Warriner, Keith
Hanner, Robert
author_facet Shehata, Hanan R.
Li, Jiping
Chen, Shu
Redda, Helen
Cheng, Shumei
Tabujara, Nicole
Li, Honghong
Warriner, Keith
Hanner, Robert
author_sort Shehata, Hanan R.
collection PubMed
description Food adulteration and feed contamination are significant issues in the food/feed industry, especially for meat products. Reliable techniques are needed to monitor these issues. Droplet Digital PCR (ddPCR) assays were developed and evaluated for detection and quantification of bovine, porcine, chicken and turkey DNA in food and feed samples. The ddPCR methods were designed based on mitochondrial DNA sequences and integrated with an artificial recombinant plasmid DNA to control variabilities in PCR procedures. The specificity of the ddPCR assays was confirmed by testing both target species and additional 18 non-target species. Linear regression established a detection range between 79 and 33200 copies of the target molecule from 0.26 to 176 pg of fresh animal tissue DNA with a coefficient of determination (R(2)) of 0.997–0.999. The quantification ranges of the methods for testing fortified heat-processed food and feed samples were 0.05–3.0% (wt/wt) for the bovine and turkey targets, and 0.01–1.0% (wt/wt) for pork and chicken targets. Our methods demonstrated acceptable repeatability and reproducibility for the analytical process for food and feed samples. Internal validation of the PCR process was monitored using a control chart for 74 consecutive ddPCR runs for quantifying bovine DNA. A matrix effect was observed while establishing calibration curves with the matrix type under testing, and the inclusion of an internal control in DNA extraction provides a useful means to overcome this effect. DNA degradation caused by heating, sonication or Taq I restriction enzyme digestion was found to reduce ddPCR readings by as much as 4.5 fold. The results illustrated the applicability of the methods to quantify meat species in food and feed samples without the need for a standard curve, and to potentially support enforcement activities for food authentication and feed control. Standard reference materials matching typical manufacturing processes are needed for future validation of ddPCR assays for absolute quantification of meat species.
format Online
Article
Text
id pubmed-5552122
institution National Center for Biotechnology Information
language English
publishDate 2017
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-55521222017-08-25 Droplet digital polymerase chain reaction (ddPCR) assays integrated with an internal control for quantification of bovine, porcine, chicken and turkey species in food and feed Shehata, Hanan R. Li, Jiping Chen, Shu Redda, Helen Cheng, Shumei Tabujara, Nicole Li, Honghong Warriner, Keith Hanner, Robert PLoS One Research Article Food adulteration and feed contamination are significant issues in the food/feed industry, especially for meat products. Reliable techniques are needed to monitor these issues. Droplet Digital PCR (ddPCR) assays were developed and evaluated for detection and quantification of bovine, porcine, chicken and turkey DNA in food and feed samples. The ddPCR methods were designed based on mitochondrial DNA sequences and integrated with an artificial recombinant plasmid DNA to control variabilities in PCR procedures. The specificity of the ddPCR assays was confirmed by testing both target species and additional 18 non-target species. Linear regression established a detection range between 79 and 33200 copies of the target molecule from 0.26 to 176 pg of fresh animal tissue DNA with a coefficient of determination (R(2)) of 0.997–0.999. The quantification ranges of the methods for testing fortified heat-processed food and feed samples were 0.05–3.0% (wt/wt) for the bovine and turkey targets, and 0.01–1.0% (wt/wt) for pork and chicken targets. Our methods demonstrated acceptable repeatability and reproducibility for the analytical process for food and feed samples. Internal validation of the PCR process was monitored using a control chart for 74 consecutive ddPCR runs for quantifying bovine DNA. A matrix effect was observed while establishing calibration curves with the matrix type under testing, and the inclusion of an internal control in DNA extraction provides a useful means to overcome this effect. DNA degradation caused by heating, sonication or Taq I restriction enzyme digestion was found to reduce ddPCR readings by as much as 4.5 fold. The results illustrated the applicability of the methods to quantify meat species in food and feed samples without the need for a standard curve, and to potentially support enforcement activities for food authentication and feed control. Standard reference materials matching typical manufacturing processes are needed for future validation of ddPCR assays for absolute quantification of meat species. Public Library of Science 2017-08-10 /pmc/articles/PMC5552122/ /pubmed/28796824 http://dx.doi.org/10.1371/journal.pone.0182872 Text en © 2017 Shehata et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Shehata, Hanan R.
Li, Jiping
Chen, Shu
Redda, Helen
Cheng, Shumei
Tabujara, Nicole
Li, Honghong
Warriner, Keith
Hanner, Robert
Droplet digital polymerase chain reaction (ddPCR) assays integrated with an internal control for quantification of bovine, porcine, chicken and turkey species in food and feed
title Droplet digital polymerase chain reaction (ddPCR) assays integrated with an internal control for quantification of bovine, porcine, chicken and turkey species in food and feed
title_full Droplet digital polymerase chain reaction (ddPCR) assays integrated with an internal control for quantification of bovine, porcine, chicken and turkey species in food and feed
title_fullStr Droplet digital polymerase chain reaction (ddPCR) assays integrated with an internal control for quantification of bovine, porcine, chicken and turkey species in food and feed
title_full_unstemmed Droplet digital polymerase chain reaction (ddPCR) assays integrated with an internal control for quantification of bovine, porcine, chicken and turkey species in food and feed
title_short Droplet digital polymerase chain reaction (ddPCR) assays integrated with an internal control for quantification of bovine, porcine, chicken and turkey species in food and feed
title_sort droplet digital polymerase chain reaction (ddpcr) assays integrated with an internal control for quantification of bovine, porcine, chicken and turkey species in food and feed
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5552122/
https://www.ncbi.nlm.nih.gov/pubmed/28796824
http://dx.doi.org/10.1371/journal.pone.0182872
work_keys_str_mv AT shehatahananr dropletdigitalpolymerasechainreactionddpcrassaysintegratedwithaninternalcontrolforquantificationofbovineporcinechickenandturkeyspeciesinfoodandfeed
AT lijiping dropletdigitalpolymerasechainreactionddpcrassaysintegratedwithaninternalcontrolforquantificationofbovineporcinechickenandturkeyspeciesinfoodandfeed
AT chenshu dropletdigitalpolymerasechainreactionddpcrassaysintegratedwithaninternalcontrolforquantificationofbovineporcinechickenandturkeyspeciesinfoodandfeed
AT reddahelen dropletdigitalpolymerasechainreactionddpcrassaysintegratedwithaninternalcontrolforquantificationofbovineporcinechickenandturkeyspeciesinfoodandfeed
AT chengshumei dropletdigitalpolymerasechainreactionddpcrassaysintegratedwithaninternalcontrolforquantificationofbovineporcinechickenandturkeyspeciesinfoodandfeed
AT tabujaranicole dropletdigitalpolymerasechainreactionddpcrassaysintegratedwithaninternalcontrolforquantificationofbovineporcinechickenandturkeyspeciesinfoodandfeed
AT lihonghong dropletdigitalpolymerasechainreactionddpcrassaysintegratedwithaninternalcontrolforquantificationofbovineporcinechickenandturkeyspeciesinfoodandfeed
AT warrinerkeith dropletdigitalpolymerasechainreactionddpcrassaysintegratedwithaninternalcontrolforquantificationofbovineporcinechickenandturkeyspeciesinfoodandfeed
AT hannerrobert dropletdigitalpolymerasechainreactionddpcrassaysintegratedwithaninternalcontrolforquantificationofbovineporcinechickenandturkeyspeciesinfoodandfeed