Multiplex Detection of <em>Salmonella</em> spp., <em>E. coli</em> O157 and <em>L. monocytogenes</em> by qPCR Melt Curve Analysis in Spiked Infant Formula
Food poisoning continue to be a threat in the food industry showing a need to improve the detection of the pathogen responsible for the hospitalization cases and death. DNA-based techniques represent a real advantage and allow the detection of several targets at the same time, reducing cost and time...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2020-09-01
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| Series: | Microorganisms |
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| Online Access: | https://www.mdpi.com/2076-2607/8/9/1359 |
| _version_ | 1827706838929375232 |
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| author | Sarah Azinheiro Joana Carvalho Marta Prado Alejandro Garrido-Maestu |
| author_facet | Sarah Azinheiro Joana Carvalho Marta Prado Alejandro Garrido-Maestu |
| author_sort | Sarah Azinheiro |
| collection | DOAJ |
| description | Food poisoning continue to be a threat in the food industry showing a need to improve the detection of the pathogen responsible for the hospitalization cases and death. DNA-based techniques represent a real advantage and allow the detection of several targets at the same time, reducing cost and time of analysis. The development of new methodology using SYBR Green qPCR for the detection of <i>L. monocytogenes</i>, <i>Salmonella</i> spp. and <i>E. coli</i> O157 simultaneously was developed and a non-competitive internal amplification control (NC-IAC) was implemented to detect reaction inhibition. The formulation and supplementation of the enrichment medium was also optimized to allow the growth of all pathogens. The limit of detection (LoD) 95% obtained was <1 CFU/25 g for <i>E. coli</i> O157, and 2 CFU/25 g for <i>Salmonella</i> spp. and <i>L. monocytogenes</i> and regarding the multiplex detection a LoD 95% of 1.7 CFU/25 g was observed. The specificity, relative sensitivity and accuracy of full methodology were 100% and the use of the NC-IAC allowed the reliability of the results without interfering with the sensitivity of the methodology. The described study proved to obtain results comparable to those of probe-based qPCR, and more economically than classical high resolution melting qPCR, being both important aspects for its implementation in the food industry. |
| first_indexed | 2024-03-10T16:34:21Z |
| format | Article |
| id | doaj.art-e3a257d59be1415e8dfd7b51bdc70b7c |
| institution | Directory Open Access Journal |
| issn | 2076-2607 |
| language | English |
| last_indexed | 2024-03-10T16:34:21Z |
| publishDate | 2020-09-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Microorganisms |
| spelling | doaj.art-e3a257d59be1415e8dfd7b51bdc70b7c2023-11-20T12:38:12ZengMDPI AGMicroorganisms2076-26072020-09-0189135910.3390/microorganisms8091359Multiplex Detection of <em>Salmonella</em> spp., <em>E. coli</em> O157 and <em>L. monocytogenes</em> by qPCR Melt Curve Analysis in Spiked Infant FormulaSarah Azinheiro0Joana Carvalho1Marta Prado2Alejandro Garrido-Maestu3Food Quality and Safety Research Group, International Iberian Nanotechnology Laboratory, Av. Mestre José Veiga s/n, 4715-330 Braga, PortugalFood Quality and Safety Research Group, International Iberian Nanotechnology Laboratory, Av. Mestre José Veiga s/n, 4715-330 Braga, PortugalFood Quality and Safety Research Group, International Iberian Nanotechnology Laboratory, Av. Mestre José Veiga s/n, 4715-330 Braga, PortugalFood Quality and Safety Research Group, International Iberian Nanotechnology Laboratory, Av. Mestre José Veiga s/n, 4715-330 Braga, PortugalFood poisoning continue to be a threat in the food industry showing a need to improve the detection of the pathogen responsible for the hospitalization cases and death. DNA-based techniques represent a real advantage and allow the detection of several targets at the same time, reducing cost and time of analysis. The development of new methodology using SYBR Green qPCR for the detection of <i>L. monocytogenes</i>, <i>Salmonella</i> spp. and <i>E. coli</i> O157 simultaneously was developed and a non-competitive internal amplification control (NC-IAC) was implemented to detect reaction inhibition. The formulation and supplementation of the enrichment medium was also optimized to allow the growth of all pathogens. The limit of detection (LoD) 95% obtained was <1 CFU/25 g for <i>E. coli</i> O157, and 2 CFU/25 g for <i>Salmonella</i> spp. and <i>L. monocytogenes</i> and regarding the multiplex detection a LoD 95% of 1.7 CFU/25 g was observed. The specificity, relative sensitivity and accuracy of full methodology were 100% and the use of the NC-IAC allowed the reliability of the results without interfering with the sensitivity of the methodology. The described study proved to obtain results comparable to those of probe-based qPCR, and more economically than classical high resolution melting qPCR, being both important aspects for its implementation in the food industry.https://www.mdpi.com/2076-2607/8/9/1359multiplex qPCRmelting analysisfood analysis<i>Listeria monocytogenes</i><i>Salmonella</i> spp.<i>E coli</i> O157 |
| spellingShingle | Sarah Azinheiro Joana Carvalho Marta Prado Alejandro Garrido-Maestu Multiplex Detection of <em>Salmonella</em> spp., <em>E. coli</em> O157 and <em>L. monocytogenes</em> by qPCR Melt Curve Analysis in Spiked Infant Formula Microorganisms multiplex qPCR melting analysis food analysis <i>Listeria monocytogenes</i> <i>Salmonella</i> spp. <i>E coli</i> O157 |
| title | Multiplex Detection of <em>Salmonella</em> spp., <em>E. coli</em> O157 and <em>L. monocytogenes</em> by qPCR Melt Curve Analysis in Spiked Infant Formula |
| title_full | Multiplex Detection of <em>Salmonella</em> spp., <em>E. coli</em> O157 and <em>L. monocytogenes</em> by qPCR Melt Curve Analysis in Spiked Infant Formula |
| title_fullStr | Multiplex Detection of <em>Salmonella</em> spp., <em>E. coli</em> O157 and <em>L. monocytogenes</em> by qPCR Melt Curve Analysis in Spiked Infant Formula |
| title_full_unstemmed | Multiplex Detection of <em>Salmonella</em> spp., <em>E. coli</em> O157 and <em>L. monocytogenes</em> by qPCR Melt Curve Analysis in Spiked Infant Formula |
| title_short | Multiplex Detection of <em>Salmonella</em> spp., <em>E. coli</em> O157 and <em>L. monocytogenes</em> by qPCR Melt Curve Analysis in Spiked Infant Formula |
| title_sort | multiplex detection of em salmonella em spp em e coli em o157 and em l monocytogenes em by qpcr melt curve analysis in spiked infant formula |
| topic | multiplex qPCR melting analysis food analysis <i>Listeria monocytogenes</i> <i>Salmonella</i> spp. <i>E coli</i> O157 |
| url | https://www.mdpi.com/2076-2607/8/9/1359 |
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