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Article ; Online: Two alternative multiplex PCRs for the identification of the seven species of anglerfish (Lophius spp.) using an end-point or a melting curve analysis real-time protocol.

Castigliego, Lorenzo / Armani, Andrea / Tinacci, Lara / Gianfaldoni, Daniela / Guidi, Alessandra

2014 Volume 166, Page(s) 1–9

Abstract: Anglerfish (Lophius spp.) is consumed worldwide and is an important economic resource though its seven species are often fraudulently interchanged due to their different commercial value, especially when sold in the form of fillets or pieces. Molecular ... ...

Abstract	Anglerfish (Lophius spp.) is consumed worldwide and is an important economic resource though its seven species are often fraudulently interchanged due to their different commercial value, especially when sold in the form of fillets or pieces. Molecular analysis is the only possible mean to verify traceability and counteract fraud. We developed two multiplex PCRs, one end-point and one real-time with melting curve post-amplification analysis, which can even be run with the simplest two-channel thermocyclers. The two methods were tested on seventy-five reference samples. Their specificity was checked in twenty more species of those most commonly available on the market and in other species of the Lophiidae family. Both methods, the choice of which depends on the equipment and budget of the lab, provide a rapid and easy-to-read response, improving both the simplicity and cost-effectiveness of existing methods for identifying Lophius species.
MeSH term(s)	Animals ; Fishes ; Multiplex Polymerase Chain Reaction/methods ; Sensitivity and Specificity
Language	English
Publishing date	2014-06-12
Publishing country	England
Document type	Journal Article
ZDB-ID	243123-3
ISSN	1873-7072 ; 0308-8146
ISSN (online)	1873-7072
ISSN	0308-8146
DOI	10.1016/j.foodchem.2014.06.014
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Article ; Online: A Conventional Multiplex PCR Assay for the Detection of Toxic Gemfish Species (Ruvettus pretiosus and Lepidocybium flavobrunneum): A Simple Method To Combat Health Frauds.

Giusti, Alice / Castigliego, Lorenzo / Rubino, Rossella / Gianfaldoni, Daniela / Guidi, Alessandra / Armani, Andrea

Journal of agricultural and food chemistry

2016 Volume 64, Issue 4, Page(s) 960–968

Abstract: The meat of Ruvettus pretiosus and Lepidocybium flavobrunneum (gemfishes) contains high amounts of indigestible wax esters that provoke gastrointestinal disorders. Although some countries have banned the sale of these species, mislabeling cases have been ...

Abstract	The meat of Ruvettus pretiosus and Lepidocybium flavobrunneum (gemfishes) contains high amounts of indigestible wax esters that provoke gastrointestinal disorders. Although some countries have banned the sale of these species, mislabeling cases have been reported in sushi catering. This work developed a simple conventional multiplex PCR, which discriminates the two toxic gemfishes from other potentially replaced species, such as tunas, cod, and sablefish. A common degenerate forward primer and three species-specific reverse primers were designed to amplify cytochrome oxidase subunit I (COI) gene regions of different lengths (479, 403, and 291 bp) of gemfishes, tunas, and sablefish, respectively. A primer pair was designed to amplify a fragment (193 bp) of the cytb gene of cod species. Furthermore, a primer pair targeting the 16S rRNA gene was intended as common positive control (115 bp). The method developed in this study, by producing the expected amplicon for all of the DNA samples tested (reference and commercial), provides a rapid and reliable response in identifying the two toxic species to combat health frauds.
MeSH term(s)	Animals ; DNA Primers/genetics ; Fish Proteins/genetics ; Food Contamination/analysis ; Multiplex Polymerase Chain Reaction/methods ; Perciformes/genetics ; Seafood/analysis
Chemical Substances	DNA Primers ; Fish Proteins
Language	English
Publishing date	2016-02-03
Publishing country	United States
Document type	Evaluation Studies ; Journal Article
ZDB-ID	241619-0
ISSN	1520-5118 ; 0021-8561
ISSN (online)	1520-5118
ISSN	0021-8561
DOI	10.1021/acs.jafc.5b04899
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Article ; Online: Serum responsiveness to recombinant bovine somatotropin in buffalo: a three-month lactation study using an acid-stripping ELISA for screening.

Castigliego, Lorenzo / Tinacci, Lara / Armani, Andrea / Boselli, Carlo / Grifoni, Goffredo / Mazzi, Marco / Guidi, Alessandra

Drug testing and analysis

2016 Volume 9, Issue 4, Page(s) 646–656

Abstract: The misuse of recombinant bovine somatotropin (rbST) to increase milk yield involves buffalo not just cows. Screening methods to identify rbST-treated cattle have already been proposed. However, there have been no studies on prolonged periods with a high ...

Abstract	The misuse of recombinant bovine somatotropin (rbST) to increase milk yield involves buffalo not just cows. Screening methods to identify rbST-treated cattle have already been proposed. However, there have been no studies on prolonged periods with a high number of animals. In this study, we developed a new enzyme-linked immunosorbent assay (ELISA) to measure the serum responsiveness towards rbST, based on an acid-stripping procedure and relatively simple integral calculation dilution curves. We also applied the analysis to 640 serum and 96 milk samples collected from 16 buffalo treated with rbST and 16 controls, over a period of approximately three months. Its suitability as a screening method, in compliance with EU law, was also assessed. A bi-factorial approach was also evaluated, including the measurement of insulin-like growth factor 1 concentration in serum. Results showed that our ELISA could be used on its own for screening purposes, without the need to assess other biomarkers. Copyright © 2016 John Wiley & Sons, Ltd.
MeSH term(s)	Animals ; Buffaloes/blood ; Buffaloes/physiology ; Cattle ; Enzyme-Linked Immunosorbent Assay/methods ; Female ; Growth Hormone/analysis ; Growth Hormone/blood ; Insulin-Like Growth Factor I/analysis ; Lactation ; Limit of Detection ; Milk/chemistry ; Substance Abuse Detection/methods
Chemical Substances	Insulin-Like Growth Factor I (67763-96-6) ; Growth Hormone (9002-72-6)
Language	English
Publishing date	2016-06-14
Publishing country	England
Document type	Journal Article
ZDB-ID	2462336-2
ISSN	1942-7611 ; 1942-7603
ISSN (online)	1942-7611
ISSN	1942-7603
DOI	10.1002/dta.1994
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Article: A LC-MS-MS method to detect recombinant bovine somatotropin misuse in buffalos

Castigliego, Lorenzo / Andrea Armani / Goffredo Grifoni / Marco Mazzi / Carlo Boselli / Alessandra Guidi / Riccardo Donzelli / Alessandro Saba

Analytical and bioanalytical chemistry. 2016 July, v. 408, no. 18

2016

Abstract: Recombinant bovine somatotropin (rbST) is a peptide hormone used to increase milk yield in cows and buffalos. In Europe, its use has been banned. However, rbST is sometimes illegally included in zootechnical practices for profit purposes, undermining the ...

Abstract	Recombinant bovine somatotropin (rbST) is a peptide hormone used to increase milk yield in cows and buffalos. In Europe, its use has been banned. However, rbST is sometimes illegally included in zootechnical practices for profit purposes, undermining the fair trade and the law prescriptions. For this reason, efficient and reliable analytical techniques are required to contrast rbST misuse. A few LC-MS-MS methods have been developed to detect, in cow serum, methyonil-rbST, one of the two main rbST forms available on the market. The other form, which is widespread, is identical to the most abundant variant of bovine somatotropin (bST) and differs from the buffalo somatotropin for one amino acid in the N-terminus. For this reason, it is technically possible to distinguish both rbST forms in serum of buffalos. In this work, we describe a novel LC-MS-MS-based method, capable to quantify, with a high sensitivity and selectivity, the methyonil-rbST and the other bST-identical recombinant form in buffalo serum, previously purified using a solid-phase extraction procedure. The method was internally validated and used to analyse 152 serum samples, collected from eight buffalos administered with rbST for a period of 3Â months, according to conventional protocols. The obtained results confirmed the suitability of the method in the detection of illegal hormonal treatments. Graphical abstract á
Keywords	amino acids ; blood serum ; buffaloes ; cows ; liquid chromatography ; markets ; milk yield ; solid phase extraction ; somatotropin ; tandem mass spectrometry ; Europe
Language	English
Dates of publication	2016-07
Size	p. 4917-4926.
Publishing place	Springer Berlin Heidelberg
Document type	Article
ISSN	1618-2642
DOI	10.1007/s00216-016-9578-9
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Pentaplex PCR as screening assay for jellyfish species identification in food products.

Armani, Andrea / Giusti, Alice / Castigliego, Lorenzo / Rossi, Aurelio / Tinacci, Lara / Gianfaldoni, Daniela / Guidi, Alessandra

Journal of agricultural and food chemistry

2014 Volume 62, Issue 50, Page(s) 12134–12143

Abstract: Salted jellyfish, a traditional food in Asian Countries, is nowadays spreading on the Western markets. In this work, we developed a Pentaplex PCR for the identification of five edible species (Nemopilema nomurai, Rhopilema esculentum, Rhizostoma pulmo, ... ...

Abstract	Salted jellyfish, a traditional food in Asian Countries, is nowadays spreading on the Western markets. In this work, we developed a Pentaplex PCR for the identification of five edible species (Nemopilema nomurai, Rhopilema esculentum, Rhizostoma pulmo, Pelagia noctiluca, and Cotylorhiza tuberculata), which cannot be identified by a mere visual inspection in jellyfish products sold as food. A common degenerated forward primer and five specie-specific reverse primers were designed to amplify COI gene regions of different lengths. Another primer pair targeted the 28SrRNA gene and was intended as common positive reaction control. Considering the high level of degradation in the DNA extracted from acidified and salted products, the maximum length of the amplicons was set at 200 bp. The PCR was developed using 66 reference DNA samples. It gave successful amplifications in 85.4% of 48 ready to eat products (REs) and in 60% of 30 classical salted products (CPs) collected on the market.
MeSH term(s)	Animals ; DNA Primers/genetics ; Polymerase Chain Reaction/methods ; RNA, Ribosomal, 28S/genetics ; Scyphozoa/classification ; Scyphozoa/genetics ; Seafood/analysis
Chemical Substances	DNA Primers ; RNA, Ribosomal, 28S
Language	English
Publishing date	2014-12-17
Publishing country	United States
Document type	Evaluation Studies ; Journal Article
ZDB-ID	241619-0
ISSN	1520-5118 ; 0021-8561
ISSN (online)	1520-5118
ISSN	0021-8561
DOI	10.1021/jf504654b
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Article: What is inside the jar? Forensically informative nucleotide sequencing (FINS) of a short mitochondrial COI gene fragment reveals a high percentage of mislabeling in jellyfish food products

Armani, Andrea / Tinacci, Lara / Giusti, Alice / Castigliego, Lorenzo / Gianfaldoni, Daniela / Guidi, Alessandra

Food research international. 2013 Dec., v. 54, no. 2

2013

Abstract: The jellyfish belonging to the Rhizostomeae order are a typical Asian seafood and are nowadays also marketed in the Western countries. The jellyfish species used for food cannot be identified due to the loss of the morphological characteristics during ... ...

Abstract	The jellyfish belonging to the Rhizostomeae order are a typical Asian seafood and are nowadays also marketed in the Western countries. The jellyfish species used for food cannot be identified due to the loss of the morphological characteristics during processing. In this work, a pre-extraction treatment consisting of desalting under running water for 48h, followed by incubation in 400mM EDTA solution for 1h, was developed for products containing high concentration of salts. Moreover, considering the DNA degradation observed in both classical (CPs) and ready to eat (RE) commercial samples, five sets of primers for the amplification of fragments with different lengths belonging to the mitochondrial COI gene were designed and used to obtain the sequences from 54 reference specimens and 70 market samples. A phylogenetic analysis was then performed using the neighbor-joining method. It was found that a fragment of 142bp was enough informative to allow identification at the species level. The results showed that most of the products were made of Nemopilema nomurai (94% of RE and 45.5% of CPs). The analysis permitted to uncover an alarming level of mislabeling which reaches 79% for CPs and 100% for ready-to-eat products.
Keywords	DNA ; EDTA (chelating agent) ; Scyphozoa ; genes ; markets ; phylogeny ; ready-to-eat foods ; salts ; seafoods ; species identification ; water
Language	English
Dates of publication	2013-12
Size	p. 1383-1393.
Publishing place	Elsevier Ltd
Document type	Article
ZDB-ID	1111695-x
ISSN	0963-9969
ISSN	0963-9969
DOI	10.1016/j.foodres.2013.10.003
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: A LC-MS-MS method to detect recombinant bovine somatotropin misuse in buffalos.

Castigliego, Lorenzo / Armani, Andrea / Grifoni, Goffredo / Mazzi, Marco / Boselli, Carlo / Guidi, Alessandra / Donzelli, Riccardo / Saba, Alessandro

Analytical and bioanalytical chemistry

2016 Volume 408, Issue 18, Page(s) 4917–4926

Abstract	Recombinant bovine somatotropin (rbST) is a peptide hormone used to increase milk yield in cows and buffalos. In Europe, its use has been banned. However, rbST is sometimes illegally included in zootechnical practices for profit purposes, undermining the fair trade and the law prescriptions. For this reason, efficient and reliable analytical techniques are required to contrast rbST misuse. A few LC-MS-MS methods have been developed to detect, in cow serum, methyonil-rbST, one of the two main rbST forms available on the market. The other form, which is widespread, is identical to the most abundant variant of bovine somatotropin (bST) and differs from the buffalo somatotropin for one amino acid in the N-terminus. For this reason, it is technically possible to distinguish both rbST forms in serum of buffalos. In this work, we describe a novel LC-MS-MS-based method, capable to quantify, with a high sensitivity and selectivity, the methyonil-rbST and the other bST-identical recombinant form in buffalo serum, previously purified using a solid-phase extraction procedure. The method was internally validated and used to analyse 152 serum samples, collected from eight buffalos administered with rbST for a period of 3 months, according to conventional protocols. The obtained results confirmed the suitability of the method in the detection of illegal hormonal treatments. Graphical abstract ᅟ.
MeSH term(s)	Animals ; Buffaloes/blood ; Cattle/genetics ; Chromatography, High Pressure Liquid/methods ; Chromatography, High Pressure Liquid/veterinary ; Growth Hormone/blood ; Growth Hormone/genetics ; Reproducibility of Results ; Sensitivity and Specificity ; Substance Abuse Detection/methods ; Substance Abuse Detection/veterinary ; Tandem Mass Spectrometry/methods ; Tandem Mass Spectrometry/veterinary
Chemical Substances	Growth Hormone (9002-72-6) ; sometribove (PBK5EQG5CQ)
Language	English
Publishing date	2016-05-05
Publishing country	Germany
Document type	Journal Article
ZDB-ID	201093-8
ISSN	1618-2650 ; 0016-1152 ; 0372-7920
ISSN (online)	1618-2650
ISSN	0016-1152 ; 0372-7920
DOI	10.1007/s00216-016-9578-9
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Article: Multiplex conventional and real-time PCR for fish species identification of Bianchetto (juvenile form of Sardina pilchardus), Rossetto (Aphia minuta), and Icefish in fresh, marinated and cooked products

Armani, Andrea / Castigliego, Lorenzo / Tinacci, Lara / Gianfaldoni, Daniela / Guidi, Alessandra

Food chemistry. 2012 July 1, v. 133, no. 1

2012

Abstract: A conventional and a realtime multiplex PCR were developed to detect fraudulent substitutions of Bianchetto (juvenile form of Sardina philcardus) and Rossetto (Aphia minuta) with Icefish (Neosalanx spp.), which show similar morphological characteristics. ...

Abstract	A conventional and a realtime multiplex PCR were developed to detect fraudulent substitutions of Bianchetto (juvenile form of Sardina philcardus) and Rossetto (Aphia minuta) with Icefish (Neosalanx spp.), which show similar morphological characteristics. Since it is the major by-catch species, Engraulis encrasicolus was also included in the analytical procedure. A common reverse primer and forward species-specific primer were designed on the mitochondrial cytochrome b gene to amplify sequences of different lengths by conventional PCR. Specific peaks were therefore also provided after melting temperature analysis in real-time PCR, thus enabling each species to be clearly differentiated. The two PCR methods were validated on fresh and processed products after preparing four typical dishes: two marinades (from raw or lightly boiled fish), a pasta sauce, and batter-fried fish cakes. All samples were correctly identified, although there was some DNA degradation after processing.
Keywords	DNA ; Engraulis encrasicolus ; Sardina pilchardus ; cooked foods ; cytochrome b ; fish ; fish cakes ; marinating ; melting point ; nucleotide sequences ; pasta ; polymerase chain reaction ; sauces ; taxonomy
Language	English
Dates of publication	2012-0701
Size	p. 184-192.
Publishing place	Elsevier Ltd
Document type	Article
ZDB-ID	243123-3
ISSN	1873-7072 ; 0308-8146
ISSN (online)	1873-7072
ISSN	0308-8146
DOI	10.1016/j.foodchem.2011.12.076
Database	NAL-Catalogue (AGRICOLA)

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Article: A rapid PCR–RFLP method for the identification of Lophius species

Armani, Andrea / Castigliego, Lorenzo / Tinacci, Lara / Gandini, Gabriele / Gianfaldoni, Daniela / Guidi, Alessandra

European food research & technology. 2012 Aug., v. 235, no. 2

2012

Abstract: The seven Anglerfish species, which belong to the genus Lophius, have a different value on the market, worldwide. If whole fishes can be identified by their morphological characteristics, they become indistinguishable when prepared or processed. In this ... ...

Abstract	The seven Anglerfish species, which belong to the genus Lophius, have a different value on the market, worldwide. If whole fishes can be identified by their morphological characteristics, they become indistinguishable when prepared or processed. In this study, a rapid method based on polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) was developed for the authentication of the seven Lophius species, using a cytochrome b gene fragment of 566 bp. After a genus-specific PCR, a fast digestion with the restriction enzyme BfaI, followed by agarose gel electrophoresis, allowed a clear species identification by producing specific restriction patterns. The total time required was as low as 6 h, DNA extraction included. The method was then used to analyse 48 commercial samples, whose phylogenetic analysis confirmed the PCR–RFLP response at 100 %. Results showed that mislabelling occurs on the market regardless the kind of processing.
Keywords	DNA ; Lophius ; cytochrome b ; digestion ; enzymes ; fish ; gel electrophoresis ; genes ; market value ; markets ; phylogeny ; polymerase chain reaction ; rapid methods ; taxonomy
Language	English
Dates of publication	2012-08
Size	p. 253-263.
Publishing place	Springer-Verlag
Document type	Article
ZDB-ID	1359456-4
ISSN	1431-4630 ; 1438-2377
ISSN	1431-4630 ; 1438-2377
DOI	10.1007/s00217-012-1754-3
Database	NAL-Catalogue (AGRICOLA)

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Article: Mislabeling of an “unlabelable” seafood sold on the European market: The jellyfish

Armani, Andrea / D’Amico, Priscilla / Castigliego, Lorenzo / Sheng, Gan / Gianfaldoni, Daniela / Guidi, Alessandra

Food control. 2012 Aug., v. 26, no. 2

2012

Abstract: The jellyfish is a popular seafood in South-East Asia. It can also be found throughout Europe in many Chinese shops, which despite being well-organized from a commercial perspective, often disregard rules on product traceability. Therefore because ... ...

Abstract	The jellyfish is a popular seafood in South-East Asia. It can also be found throughout Europe in many Chinese shops, which despite being well-organized from a commercial perspective, often disregard rules on product traceability. Therefore because jellyfish has still not been officially defined by the European Legislation on Food Hygiene, we performed a survey on fifty-six jellyfish products sold on the Italian market. This was done by assessing label compliance with European law and with the new Chinese Food Safety Law of 2009. Our survey found many incongruences and shortfalls including the presence of a trade name referring to vegetables or a lack of an unequivocal specification of ingredients. Focus is therefore needed on the possible hazards associated with the consumption of jellyfish products. In addition, regulations need adjusting in order to create more transparent marketing and sales practices.
Keywords	Scyphozoa ; compliance ; food law ; food sanitation ; ingredients ; markets ; sales ; seafoods ; surveys ; traceability ; trade ; vegetables
Language	English
Dates of publication	2012-08
Size	p. 247-251.
Publishing place	Elsevier Ltd
Document type	Article
ZDB-ID	1027805-9
ISSN	0956-7135
ISSN	0956-7135
DOI	10.1016/j.foodcont.2012.01.059
Database	NAL-Catalogue (AGRICOLA)

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