A new SYBR green real-time PCR assay for semi-quantitative detection of Echinococcus multilocularis and Echinococcus canadensis DNA on bilberries (Vaccinium myrtillus)
Berries and vegetables are potential transmission vehicles for eggs of pathogenic parasites, such as Echinococcus spp. We developed a SYBR Green based semi-quantitative real-time PCR (qPCR) method for detection of Echinococcus multilocularis and Echinococcus canadensis DNA from berry samples. A set...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2019-12-01
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| Series: | Food and Waterborne Parasitology |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2405676619300290 |
| _version_ | 1818908407855316992 |
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| author | Sanna Malkamäki Anu Näreaho Antti Lavikainen Antti Oksanen Antti Sukura |
| author_facet | Sanna Malkamäki Anu Näreaho Antti Lavikainen Antti Oksanen Antti Sukura |
| author_sort | Sanna Malkamäki |
| collection | DOAJ |
| description | Berries and vegetables are potential transmission vehicles for eggs of pathogenic parasites, such as Echinococcus spp. We developed a SYBR Green based semi-quantitative real-time PCR (qPCR) method for detection of Echinococcus multilocularis and Echinococcus canadensis DNA from berry samples. A set of primers based on the mitochondrial NADH dehydrogenase subunit 1 (nad1) gene was designed and evaluated. To assess the efficacy of the assay, we spiked bilberries (Vaccinium myrtillus) with a known amount of E. multilocularis eggs. The detection limit for the assay using the NAD1_88 primer set was 4.37 × 10−5 ng/μl of E. multilocularis DNA. Under artificial contamination of berries, 50 E. multilocularis eggs were reliably detected in 250 g of bilberries. Analytical sensitivity of the assay was determined to be 100% with three eggs. As an application of the assay, 21 bilberry samples from Finnish market places and 21 bilberry samples from Estonia were examined. Previously described sieving and DNA extraction methods were used, and the samples were analyzed for E. multilocularis and E. canadensis DNA using semi-quantitative real-time PCR and a melting curve analysis of the amplified products. Echinococcus DNA was not detected in any of the commercial berry samples. This easy and fast method can be used for an efficient detection of E. multilocularis and E. canadensis in bilberries or other berries, and it is applicable also for fruits and vegetables. Keywords: Cestoda, Echinococcus, qPCR, SYBR green, Bilberry |
| first_indexed | 2024-12-19T22:10:32Z |
| format | Article |
| id | doaj.art-3a0081a2fda74a038bf34ac8070317ec |
| institution | Directory Open Access Journal |
| issn | 2405-6766 |
| language | English |
| last_indexed | 2024-12-19T22:10:32Z |
| publishDate | 2019-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Food and Waterborne Parasitology |
| spelling | doaj.art-3a0081a2fda74a038bf34ac8070317ec2022-12-21T20:03:54ZengElsevierFood and Waterborne Parasitology2405-67662019-12-0117A new SYBR green real-time PCR assay for semi-quantitative detection of Echinococcus multilocularis and Echinococcus canadensis DNA on bilberries (Vaccinium myrtillus)Sanna Malkamäki0Anu Näreaho1Antti Lavikainen2Antti Oksanen3Antti Sukura4Department of Veterinary Biosciences (FINPAR), Faculty of Veterinary Medicine, University of Helsinki, Finland; Corresponding author.Department of Veterinary Biosciences (FINPAR), Faculty of Veterinary Medicine, University of Helsinki, FinlandDepartment of Veterinary Biosciences (FINPAR), Faculty of Veterinary Medicine, University of Helsinki, FinlandFinnish Food Authority (FINPAR), Oulu, FinlandDepartment of Veterinary Biosciences (FINPAR), Faculty of Veterinary Medicine, University of Helsinki, FinlandBerries and vegetables are potential transmission vehicles for eggs of pathogenic parasites, such as Echinococcus spp. We developed a SYBR Green based semi-quantitative real-time PCR (qPCR) method for detection of Echinococcus multilocularis and Echinococcus canadensis DNA from berry samples. A set of primers based on the mitochondrial NADH dehydrogenase subunit 1 (nad1) gene was designed and evaluated. To assess the efficacy of the assay, we spiked bilberries (Vaccinium myrtillus) with a known amount of E. multilocularis eggs. The detection limit for the assay using the NAD1_88 primer set was 4.37 × 10−5 ng/μl of E. multilocularis DNA. Under artificial contamination of berries, 50 E. multilocularis eggs were reliably detected in 250 g of bilberries. Analytical sensitivity of the assay was determined to be 100% with three eggs. As an application of the assay, 21 bilberry samples from Finnish market places and 21 bilberry samples from Estonia were examined. Previously described sieving and DNA extraction methods were used, and the samples were analyzed for E. multilocularis and E. canadensis DNA using semi-quantitative real-time PCR and a melting curve analysis of the amplified products. Echinococcus DNA was not detected in any of the commercial berry samples. This easy and fast method can be used for an efficient detection of E. multilocularis and E. canadensis in bilberries or other berries, and it is applicable also for fruits and vegetables. Keywords: Cestoda, Echinococcus, qPCR, SYBR green, Bilberryhttp://www.sciencedirect.com/science/article/pii/S2405676619300290 |
| spellingShingle | Sanna Malkamäki Anu Näreaho Antti Lavikainen Antti Oksanen Antti Sukura A new SYBR green real-time PCR assay for semi-quantitative detection of Echinococcus multilocularis and Echinococcus canadensis DNA on bilberries (Vaccinium myrtillus) Food and Waterborne Parasitology |
| title | A new SYBR green real-time PCR assay for semi-quantitative detection of Echinococcus multilocularis and Echinococcus canadensis DNA on bilberries (Vaccinium myrtillus) |
| title_full | A new SYBR green real-time PCR assay for semi-quantitative detection of Echinococcus multilocularis and Echinococcus canadensis DNA on bilberries (Vaccinium myrtillus) |
| title_fullStr | A new SYBR green real-time PCR assay for semi-quantitative detection of Echinococcus multilocularis and Echinococcus canadensis DNA on bilberries (Vaccinium myrtillus) |
| title_full_unstemmed | A new SYBR green real-time PCR assay for semi-quantitative detection of Echinococcus multilocularis and Echinococcus canadensis DNA on bilberries (Vaccinium myrtillus) |
| title_short | A new SYBR green real-time PCR assay for semi-quantitative detection of Echinococcus multilocularis and Echinococcus canadensis DNA on bilberries (Vaccinium myrtillus) |
| title_sort | new sybr green real time pcr assay for semi quantitative detection of echinococcus multilocularis and echinococcus canadensis dna on bilberries vaccinium myrtillus |
| url | http://www.sciencedirect.com/science/article/pii/S2405676619300290 |
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