14146935 PCR Protocol for Fish and Seafood Authentication MSc Thesis

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Transcript of 14146935 PCR Protocol for Fish and Seafood Authentication MSc Thesis

ERASMUS MUNDUS MASTER COURSE SEFOTECH.NUT

The Optimization and Validation of a Polymerase Chain Reaction Protocol for Fish and Seafood Authenticity based on the Cytochrome b Gene

M.Sc. thesis submitted by: Dwiyitno

Dwiyitno

Digitally signed by Dwiyitno DN: cn=Dwiyitno, c=ID, ou=SefotechNUT, email=dwiyitno@yahoo.com Date: 2009.04.11 18:47:54 +07'00'

Catholic University of Applied Science (KaHo) Sint Lieven, Belgium Dublin Institute of Technology, Ireland Universidade Catlica Portuguesa, Portugal Anhalt University of Applied Sciences, Germany

2008

KaHo-Sint Lieven School for Engineering Gebr. Desmetstraat 1 9000 Gent - Belgium

The Optimization and Validation of a Polymerase Chain Reaction Protocol for Fish and Seafood Authenticity based on the Cytochrome b Gene

M.Sc. thesis submitted by: Dwiyitno

Project Coordinator: Prof. Dr. Chris Van Keer Supervisors: Prof. Dr. Chris Van Keer Dr. Koen Parmentier Co-supervisor: Stefan Hoffman, M.Sc.

February 2008

The optimization & validation of a PCR protocol for fish & seafood authenticity based on the cyt b gene

ABSTRACTDwiyitno. The optimization and validation of a polymerase chain reaction protocol for fish and seafood authenticity based on the cytochrome b gene. (Under direction of Prof. Dr. Chris Van Keer and Dr. Koen Parmentier; supervised by Stefan Hoffman, M.Sc) Cytochrome b mtDNA has been widely applied for identification of fish and seafood, either in fresh or processed products. The successful application of product authentication based on genomic profiling considerably depends on the primer design which is used to amplify the targeted DNA fragment. Several primers have been developed specifically to identify particular groups of fish, crustaceans and molluscs. However, universal primers for identification of most fish, crustaceans, and molluscs based on the cyt b region have not been established yet. This study focused on the development of universal primers for fish and seafood authenticity based on the cyt b gene. Universal primers are essential, particularly for identification of unrecognizable samples such as fish fillet, surimi and mixed products. In addition, since DNA quality plays an important contribution on PCR amplification, investigation on the different DNA isolation methods was carried out. Firstly, CytBL1 and CytBH primers which have successfully been used to amplify ~357bp of cyt b gene on various fish were optimized to amplify selected fish, crustaceans and molluscs. Secondly, since this primer couple was not optimum for crustacean, mollusc, and some fishes, degenerate primers were designed by introducing wobbles. Notably, other degenerate primers were evaluated to amplify ~410bp of expected fragment. Evaluation of 3 classical DNA extraction methods and a commercial kit was studied to isolate total DNA of selected samples. The results showed that the CytBL1 and CytBH failed to amplify crustacean and mollusc. Likewise, some species of fish failed to be amplified by this primer couple. The degenerate primers (CytBL1C and CytBHW) are promising to be employed universally for species identification of crustaceans and molluscs. Other universal primers (UCYTB151BF/UCYTB271R and UCYTB152BF/UCYTB271R) effectively amplified all fish, crustaceans, and molluscs tested in this study and thereby can be considered as the first universal primers applicable for fish and seafood. Sequence analysis proved that all validated primers effectively generate 356-358bp and 398-411bp of cyt b region. The similarity index of PCR products against the libraries varied between 92% and 100%. RTPCR was applicable to differentiate between selected samples based on their melting points (Tm). In comparison to the classical methods, the commercial kit offers simplicity procedure and yielded the better quality of DNA isolate for PCR purposes.

Key words: authenticity, mitochondrial cytochrome b, PCR primers, fish and seafood, DNA sequencing

Dwiyitno MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT)

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The optimization & validation of a PCR protocol for fish & seafood authenticity based on the cyt b gene

ACKNOWLEDGEMENTSThis thesis was a part of my master course in Food Science, Technology, and Nutrition (SEFOTECHnut). It was funded by the European Commission under the Erasmus Mundus framework. The studies were undertaken in 2006-2008 at Catholic University of Applied Science (KaHo) Sint Lieven-Belgium as the host university and partially at Dublin Institute of Technology-Ireland, Universidade Cathlica PortuguesaPortugal, and Anhalt University of Applied Sciences-Germany. Many people took part in my M.Sc studies and without them this thesis would not exist. I am deeply grateful to both of my advisors, Prof. Dr. Chris Van Keer (KaHo Sint Lieven) and Dr. Koen Parmentier (ILVO), who have encouraged and mentored me during this works. Prof. Chris is also the coordinator of SEFOTECHnut, thank you for giving me opportunity to be part of this master course. This thesis project was carried out at Institute for Agriculture and Fisheries Research (ILVO), Belgium. I would like to express my gratitude to Stefan Hoffman M.Sc and his research group (Daphne and Sabrine) for the invaluable support and supervising me during this research and writing the report. They have introduced me to many basic theories and practical application in biomolecular work. I gratefully acknowledge Dr. Kris Cooreman, the Director of ILVO-Fisheries Department, for the opportunity to work at his laboratory. Thank all colleagues in Ankerstraat 1 for creating so enjoyable atmosphere to work in. I also thank my reviewers, Prof. Dr. Dirk Iserentant and Prof. Jan Song (Gent University), for evaluating the manuscript and their comments during my public defense. I wish to thank my present employer at Research Center for Marine and Fisheries Product Processing and Biotechnology-Jakarta (Prof. Dr. Hari Eko Irianto, Prof. Dr. Sumpeno Putro, Prof. Dr. Endang Sri Heruwati, and Dr. Singgih Wibowo, M.S.), my former employer (Dr. Ahmad Dimyati, M.S. and Dr. W. Farid Maruf, M.Sc) and all of my workmates, for their support. My warmest thanks belong to my parents, my parents in law, Om Samso-Bulik Murni, Wa Ann and her family in De Pinte, for all the support and understanding. Finally, I deeply thank my beloved wife, Lusi, and my juniors (Rizan and Fitri) for all their patience and comfort during these years.

Gent-Oostende, February 2008 Dwiyitno

Dwiyitno MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT)

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The optimization & validation of a PCR protocol for fish & seafood authenticity based on the cyt b gene

TABLE OF CONTENTS

ABSTRACT ..................................................................................................................... i ACKNOWLEDGEMENTS ............................................................................................. ii TABLE OF CONTENTS ................................................................................................. iii LIST OF FIGURES .......................................................................................................... v LIST OF TABLES ........................................................................................................... vii I. INTRODUCTION .......................................................................................................... 1 II. REVIEW OF LITERATURE ...................................................................................... 3 2.1. The importance of product authenticity ................................................................ 3 2.2. Analytical methods for product identification ...................................................... 4 2.2.1. Traditional approaches ............................................................................... 5 2.2.2. Protein based methods (Proteomics) .......................................................... 5 2.2.3. DNA based methods (Genomics) ............................................................... 6 2.2.4. Other methods ............................................................................................ 8 2.3. Genomic identification based on mitochondrial DNA ......................................... 8 2.4. Fish and seafood authenticity based on the cytochrome b gene ........................... 11 2.4.1. Specimen and treatment of sample ............................................................. 11 2.4.2. Isolation of DNA ........................................................................................ 12 2.4.2.1. Tissue digestion ............................................................................. 13 2.4.2.2. Separating proteins and contaminants ........................................... 14 2.4.2.3. Precipitation and recovery of DNA ............................................... 15 2.4.2.4. Commercial kits ............................................................................. 16 2.4.3. Determination of DNA yield and purity ..................................................... 17 2.4.4. Polymerase chain reaction (PCR) ............................................................... 19 2.4.4.1. Primer design ................................................................................. 20 2.4.4.2. Components of PCR reaction ........................................................ 22 2.4.5. PCR product analysis ................................................................................. 23 2.4.5.1. DNA sequencing .......................................................................... 24 2.4.5.2. Fingerprinting techniques .............................................................. 25 2.4.5.3. Other techniques .............................................