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: D w i y i t n o Catholic University of Applied Science (KaHo) Sint Lieven, Belgium Dublin Institute of Technology, Ireland Universidade Católica Portuguesa, Portugal Anhalt University of Applied Sciences, Germany 2008 ERASMUS MUNDUS MASTER COURSE SEFOTECH.NUT
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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:
D w i y i t n o
Catholic University of Applied Science (KaHo) Sint Lieven, Belgium Dublin Institute of Technology, Ireland
Universidade Católica Portuguesa, Portugal Anhalt University of Applied Sciences, Germany
2008
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:
D w i y i t n o Project Coordinator: Prof. Dr. Chris Van Keer Supervisors: Prof. Dr. Chris Van Keer Dr. Koen Parmentier Co-supervisor: Stefan Hoffman, M.Sc.
February 2008
KaHo-Sint Lieven School for Engineering Gebr. Desmetstraat 1 9000 Gent - Belgium
The optimization & validation of a PCR protocol for fish & seafood authenticity based on the cyt b gene
Dwiyitno – MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT) i
ABSTRACT
Dwiyitno. 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%. RT-PCR 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
The optimization & validation of a PCR protocol for fish & seafood authenticity based on the cyt b gene
Dwiyitno – MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT) ii
ACKNOWLEDGEMENTS
This 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 Cathólica Portuguesa-
Portugal, 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 Ma’ruf, 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
The optimization & validation of a PCR protocol for fish & seafood authenticity based on the cyt b gene
Dwiyitno – MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT) iii
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 ............................................................................ 27
III. OBJECTIVES OF PRESENT STUDY ...................................................................... 28 3.1. Problems .............................................................................................................. 28 3.2. Objectives ............................................................................................................ 28 3.3. Research framework ............................................................................................ 29
IV. MATERIALS AND METHODS ............................................................................... 30 4.1. Construction of reference database of cyt b genes .............................................. 30 4.2. Sample collection and preservation ..................................................................... 30 4.3. Comparison of DNA extraction methods ............................................................ 31 4.3.1. DNA extraction method 1 (Promega) ......................................................... 32 4.3.2. DNA extraction method 2 (Hsieh et al., 2005)............................................ 32 4.3.3. DNA extraction method 3 (Wasko et al., 2003) .......................................... 32 4.3.4. DNA extraction method 4 (Asahida et al., 1996) ........................................ 33
The optimization & validation of a PCR protocol for fish & seafood authenticity based on the cyt b gene
Dwiyitno – MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT) iv
4.3.5. Determination of DNA yield ..................................................................... 33 4.3.5.1. Standard curve .............................................................................. 34 4.3.5.2. Sample Analysis ........................................................................... 35 4.3.6. Evaluation of DNA purity ......................................................................... 35 4.3.7. PCR assay .................................................................................................. 36 4.3.8. Visualization of PCR product .................................................................... 37 4.4. Optimization and validation of a universal PCR protocol ................................... 37 4.4.1. Primer design ............................................................................................. 37 4.4.2. PCR assay .................................................................................................. 38 4.4.3. Direct sequencing of PCR product ............................................................ 38 4.4.4. Real Time PCR application ....................................................................... 39 4.5. Data analysis ........................................................................................................ 40
V. RESULTS .................................................................................................................... 41 5.1. Comparison of DNA extraction methods ............................................................ 41 5.2. Evaluation and optimization of CytBL1 and CytBH primers ............................. 42 5.3. Validation of degenerate primers based on CytBL1 and CytBH ......................... 44 5.4. Optimization of universal primers based on UCYTB151F and UCYTB270R .... 45
5.4.1. Validation of UCYTB151BF/UCYTB271R primers ................................ 46 5.4.2. Validation of UCYTB152BF/UCYTB271R primers ................................ 48 5.5. PCR product analysis .......................................................................................... 50 5.5.1. Direct sequencing ...................................................................................... 50 5.5.2. Melting temperature profiles ..................................................................... 51
VI. DISCUSSION ............................................................................................................ 53 6.1. Comparison of DNA extraction methods ............................................................ 53 6.2. Optimization and validation of universal primers ............................................... 54 6.3. PCR product analysis .......................................................................................... 56
VII. CONCLUSIONS ....................................................................................................... 58
The optimization & validation of a PCR protocol for fish & seafood authenticity based on the cyt b gene
Dwiyitno – MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT) v
LIST OF FIGURES
II. REVIEW OF LITERATURE
Figure 2-1. The application of traceability in fish and seafood product ................................. 4
Figure 2-2. Link between different identification techniques and their tools ........................ 7
Figure 2-3. Illustration of mitochondria in a cell and its genomic map ................................. 10
Figure 2-4. DNA spectra presented by spectrophotometer and spectrofluorometer ................ 18
Figure 2-5. The principle of PCR amplification ..................................................................... 19
III. OBJECTIVES OF PRESENT STUDY ............................................................................ 28
Figure 3-1. The framework of the study ................................................................................. 29
IV. MATERIALS AND METHODS
Figure 4-1. Genomic database obtained from NCBI and FishTrace ...................................... 30
Figure 4-2. An example of standard curve for spectrofluorometric measurement .................. 34
Figure 4-3. The application of NanoDrop and its typical data output ..................................... 35
Figure 4-4. Apparatus for PCR application ............................................................................. 36
Figure 4-5. The scheme of targeted fragments generated by the evaluated primers ............... 38
Figure 4-6. Software for sequence chromatogram analysis: ChromasPro and BioEdit .......... 39
Figure 4-7. Typical outputs of RT-PCR: annealing curve and melting curve ......................... 40
Figure 4-8. An example of BLAST analysis obtained via GenBank and FishTrace................ 40
V. RESULTS
Figure 5-1. Electrophoresis profile of DNA isolates .............................................................. 42
Figure 5-2. Electrophoresis profile of PCR products following different extraction methods ................................................................................................................................ 42
Figure 5-3. Electrophoresis profile of PCR products with CytBL1/CytBH on fish ................. 43
Figure 5-4. Electrophoresis profile of PCR products with CytBL1/CytBH on crustaceans and molluscs ................................................................................. 43
Figure 5-5. Electrophoresis profile of PCR products at different concentrations of MgCl2 ... 43
Figure 5-6. Electrophoresis profile of PCR products produced by degenerate primers based on CuyBL1/CytBH on selected samples ................................................... 44
Figure 5-7. Electrophoresis profile of PCR products with CytBL1C/CytBHW on fish .......... 44
Figure 5-8. Electrophoresis profile of PCR products with CytBL1C/CytBHW on crustaceans ................................................................................................................. 45
Figure 5-9. Electrophoresis profile of PCR products with CytBL1C/CytBHW on molluscs 45
The optimization & validation of a PCR protocol for fish & seafood authenticity based on the cyt b gene
Dwiyitno – MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT) vi
Figure 5-10. Electrophoresis profile of PCR products produced by UCYTB151BF on selected samples ............................................................................................ 45
Figure 5-11. Electrophoresis profile of PCR products produced by UCYTB152BF on selected samples ............................................................................................ 46
Figure 5-12. Electrophoresis profile of PCR products with UCYTB151BF/UCYTB271R on fish-A ............................................................................................................. 46
Figure 5-13. Electrophoresis profile of PCR products with UCYTB151BF/UCYTB271R on fish-B ....................................................................................................................... 47 Figure 5-14. Electrophoresis profile of PCR products with UCYTB151BF/UCYTB271R on crustaceans ............................................................................................................. 47 Figure 5-15. Electrophoresis profile of PCR products with UCYTB151BF/UCYTB271R on molluscs .................................................................................................................. 47
Figure 5-16. Electrophoresis profile of PCR products with UCYTB152BF/UCYTB271R on fish-A ...................................................................................................................... 48
Figure 5-17. Electrophoresis profile of PCR products with UCYTB152BF/UCYTB271R on fish-B ....................................................................................................................... 48
Figure 5-18. Electrophoresis profile of PCR products with UCYTB152BF/UCYTB271R on crustaceans ............................................................................................................. 49
Figure 5-19. Electrophoresis profile of PCR products with UCYTB152BF/UCYTB271R on molluscs .................................................................................................................. 49
Figure 5-20. Variations of melting peak on selected samples ................................................. 52
VI. DISCUSSION
Figure 6-1. Multiple alignment of evaluated primers with cyt b gene of selected references.. 55 APPENDIXES
Figure I. List of samples used in this study ......................................................................... 70
Figure III. Electrophoresis profile of gradient tests ............................................................... 72
Figure IV. Sequence chromatogram profiles of PCR products ............................................. 74
The optimization & validation of a PCR protocol for fish & seafood authenticity based on the cyt b gene
Dwiyitno – MSc Thesis in Food Science, Technology & Nutrition (SefotechNUT) vii
LIST OF TABLES
II. REVIEW OF LITERATURE
Table 2-1. Applicable categorical levels of each molecular marker or gene region ............... 10
Table 2-2. Various primer couples used to amplify cyt b region of fish and seafood ............. 21
IV. MATERIALS AND METHODS
Table 4-1. Protocol used to prepare the standard curve ......................................................... 34
Table 4-2. Composition of PCR reaction ............................................................................... 36
Table 4-3. Set of primers used in this study ........................................................................... 37
Table 4-4. PCR condition of the different primer sets ........................................................... 38
Table 4-5. Reaction components of RT-PCR amplification ................................................... 39
Table 4-6. The thermal profile of RT-PCR amplification ...................................................... 40
V. RESULTS
Table 5-1. DNA concentration and purity yielded by different extraction methods ............... 41
Table 5-2. Optimal annealing temperature (ºC) of validated primer couples .......................... 49
Table 5-3. Sequence analysis of selected samples .................................................................. 51
Table 5-4. Variations of melting point and GC content of selected samples .......................... 52 APPENDIXES
Table II. List of chemicals, solution and equipment ............................................................ 71
The optimization & validation of a PCR protocol for fish & seafood authenticity based on the cyt b gene
mitochondrial genome is arranged very efficiently as it lacks introns and has small
intergenic spacers where the reading frames sometimes overlap. The control region is the
primary non-coding region, and is responsible for the regulation of heavy (H) and light (L)
strand transcription and of H-strand replication (Kleinsmith & Kish, 1995).
Figure 2-3. Illustration of mitochondria in a cell (left) and its genomic map (right)
Table 2-1. Applicable categorical levels of each molecular marker or gene region Kingdom Phylum Class Order Family Genus Species
A. Nuclear DNA 1. SSU (16-18S) +++++++++++++++++++++++++++------ 2. LSU (23-28S) ++++++++++++++++++------- 3. 5.8S +++++++++++++++++++++------ 4. IGS +++++5. ITS +++++++++++6. Rhodopsin*) B. Mitochondrial DNA 1. Ribosomal RNA - 12SrRNA +++++++++++++++++++------ - 16SrRNA +++++++++++++-----2. Protein 3. Coding genes - ND1 -------++++++++++++++++++ - ND2 -------++++++++++++++++++ - COI**) -------++++++++++++++++++ - COII -------++++++++++++++++++ - Cyt b*) -------++++++++++++++++++4. Control region +++++
The bold lines indicate as mostly applicable categorical levels for each molecular marker or gene region, while the dot lines indicate less frequently applicable *) : FishTrace; **) : Barcodinglife
Mitochondria Nucleus
Control region (D-Loop)
16SrRNA
12SrRNA Cyt b
ND6
ND5
ND4
ND4L
ND1
COII ATPase6
COIII ND3
22 tRNA genes
COI
12 protein-coding regions
ATPase8
ND2
The optimization & validation of a PCR protocol for fish & seafood authenticity based on the cyt b gene
Sequence chromatograms were viewed and evaluated by using ChromasPro and
BioEdit software (Figure 4-6). Both forward and reverse sequences were edited by
trimming out the ambiguous bases. The selected sequences were then assembled to analyze
the overlapping bases.
Figure 4-6. Software for sequence analysis: ChromasPro (left) and BioEdit (right)
4.4.4. Real Time PCR application
Prior to the RT-PCR amplification, all reagents (2x QuantiTect SYBR Green RT-
PCR Master Mix and primers) were thawn. The master mix was prepared to a final volume
of 20 μl according to the manufacturer’s instructions (Table 4-5). Into each microplate
well, 15 ul of the mix solution were filled. 5 μl of DNA template (final concentration of
10ng/20μl) was added to each well, except the blank. The microplate was centrifuged for 3
min at 3000 rpm (Sigma 3-18K; Sartorius). The RT-PCR amplification was carried out in a
LightCycler® 480 (Roche) at the conditions as described in Table 4-6. The melting curve
was determined from the thermal profile observed for 30 seconds after elongation (60-90ºC
with ramp rate 2.2-4.4ºC/s). The melting points (Tm) generated from the melting curve
(Figure 4-7) were used to discriminate between closely-related species.
Table 4-5. Reaction components of RT-PCR amplification
Component Volume/reaction Final concentration SYBR Green RT-PCR Master Mix 2x 10μl 1x Forward and reverse primer 10μM 1μl 0.5μM DNA template 5μl 10ng PCR grade H2O 3μl To make final volume Final volume 20 μl 1 x Reaction Mix
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APPENDIX II. List of chemicals, solutions, and equipment
Table II-A. List of chemicals and reagents
Chemical Company Ordering number Ethanol p.a. Merck 8.18760.2500 EDTA-Na2 dihydrate BDH 443885J Tris Base Promega A5135 Nuclei Lysis Solution Promega A7943 Proteinase K recombinant PCR Roche 3115879 RNAase Roche 10109169001 Protein Precipitation Solution Promega A7953 Isopropanol p.a. Serva 39559.02 NaOH p.a. BDH 102525P HCl 37% Merck 1.00317.2501 PCR-H2O18.2 MΩ cm Sartorius Arium
Table II-B. List of solutions
EDTA 0.5M pH 8.0
93g EDTA-Na2 dihydrate (Mw: 372.2) Add 350ml PCR-H2O, add 10N NaOH until pH 8.0 Add PCR-H2O until 500ml and sterilize for 20 minute at 121°C (1 bar)
NaOH 10N Add 40g NaOH with 100ml PCR-H2O 500ml and sterilize for 20 minute at 121°C (1 bar)
Tris-HCl 1M, pH 7.4 30.29g Tris Base (MM: 121.14) with 150ml PCR-H2O in a 250ml volumetric flask. Add HCl 37% until pH 7.4, add with H2O until 250ml and sterilize for 20 minute at 121°C (1 bar)