... DNA verviclfiilligL Anschlieflend wurde die gebildete DNA in Ejnzelstrimge uber- ... Reagent ki[ for PCR: PCR Master @oc!~ringcr-Mannheim), Ihc final ...
209
lnf. Fischwimch. 42(4), 1995
Schreikn des B G W : Scnsorische Qualit21sbccinu'dchrigungbei Secfischen durch K o n h n a t i o n mi1 Chemikalien in Verbindung mil dem Seetmnsport fliissiger Massengdter (ausgenornmcr, Mjneralolj und verpacbcr tiiiter. Bcrlin, 6.9.1995. S~ccg,I;.; Swarczyna, H . ; Specr, K.: M a s s e n s p c k ~ o m c ~ s c hUnlcrsuchung e von Fchlaromen in cincm Ormgennekrar uod i n einem Fischmarinierungsbad. Dtsch. Lebensmittcl Rdsch. 86( I), 6-9, I 990. Whifield, F.B.; Shaw. K J., Walker, D. I.: Thc source of 2.6-dibron~ophenol.h e cause of the idoEorm taint in Aoslralian prawns. Watcr Sci. Tcchnol. 25(2), 13 1 - t38, 1992. Whitfield, F.B.: Flavour of prawns and lobstcrs Fnnd Rcv. Int. 6, 505-5 19, 1990.
Fish species identification in Canned Tuna by DNA Analysis (PCR-SSCP) Bcslimmung dcr Fischarl in Tl~u n[iscWronservcn durch D N A - h a l y s e ('PCRSSCP)
Reh bcin, H., Insti tut fiir Biochcmic und Tecbnologie, 13uadesforschungsanshlt Tiir Fiscl~crei, Hanrburg Mackie, 1.M. and S.Pryde, Central Science Laboratory-Torry, Aberdeen Gonzales-Sofelo, C. and R. Perez-Martin, hstituto de Investigaciones Marinas, Vigo Quintciro, J. and M. Rey-Mcndez, Universidad de Santiago de Compostcla, Santiago
Abstract
DNA in canned iuna is degraded into short fragments of a few hundred base pairs. The polymerase chain reaction (PCR) was used to amplify short sequences of nlitochondrinl DNA, whiclr wcrc dcnatured and analysed by poly~crylamide gel electrophoresis (native PAGE) for detection of single skand conformalion polymorphisms. Species specific pallerns o r DNA bands were obtained far a number of tuna and bonito species.
I n Thun~~chkonserven liegt die DNA in Form kurzkettiger Fragmcn~evon wenigcn Hundcrl Basenpaaren Lange vor. h 3 l HiKe der Polymerase-Ketfenreaktion (PCR) wwden k u n e Sequenzen der mitochondrialen DNA verviclfiilligL Anschlieflend wurde die gebildete DNA in Ejnzelstrimge uberfiihrt, die durch eine native Polyacrylamidgel-Elektropl~orese(PAGE) nufgelrennt wurden. Fiir cine Reihe von Thunfischen und Boniten ergaben die Einzelstriinge artspezitische Bandenmuster, die auf unterschiedlichc Konformationen der DNA-Stringc der einzelnen Fischarlcn zuriickzufiihren sind.
Introduction
Species ident ificalion of raw or processed fish is mostly pcrforrned by protein separation techniques, like isoelecrric focusing (EQor high performance liquid chromatography (I-IPLC). Due to the severe protein denaluration i n consequcrice of heating, these merhods are of lilnired utility in the case o f canned fish (Mackie el aI., 1992).
DNA analysis has k e n applied
i~lt,ntf~cafion of pork in autoclaved meat products (bleyer et al., 1994), and to the dctennination o f species idth~)ticy in canned salmorl (Bartletl and Davidson, LO ( l i t .
Inf. Fischwirtsch. 42(4), 1995
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1992) and tuna (Unseld et al., 1995). PCR was used to amplify shon fragments of DNA. which were then sequenced ( B d e t t and Davidson, 1992; Unseld et al., 1995) For routine analysis in food contol laboratories DNA sequencing bas {.he disadvmtage of being technically demanding and time consuming.
PCR-SSCPhas proved to
be a rapid and sensitive method for detect.ion of point mutations and
DNA polyniorpb.isms (Fujita and Silver. 1994). md recenlly it has heen successhlly applied for fish species idenrificarion,too ('Hara et a]., 1994).
Here we present n prelimi.nary report about our experience in using PCR-SSCPfor the idenrificat.ion of canned tuna.
kIaterids and Methods Origin of Satrrples Deep-frozen raw muscle aad canned muscle of runas and bonitos had been prepared by the hstituto de Invesrigaciones Marinas in Viyo. Commercial cans were obrained From a wholesale distributor.
DNA was isolated following the procedure described by Meyer e[ a!. (1994) using the WIZARD DNA Clean U p sysrem (SEKVA Promega). The purified DNA was stored in TE buffer ( 10 mM Tris-HCI. 1 m M EDTA, pH 8.0) a[ 4 "C or 31 -25 "C.
PCR Conditions Two different regions of the mitochondria1 cy(oclrrome b gene were selected for ampIificalion. The method of Unseld et al. (1995) delivered a product of 123 base pairs (bp; primers 59-3159-5 included). The primers for amplification of he second DNA region (148 bp. primers included) were construcred from sequences of anotber par1 of the cytockrome b gene (Finnerty and Block, 1995). TI-le PCR wac performed as follow i: Primer FB 349: 23 mer 5'- GTC GAA TGA ATC TGA GGA GGC TT -3' Primcr FB 496: 5'-CCR A'TT GGG TTG TTT GAC CCT GIT TC -3' 26 mer Preheating step: 5 tnir ill 94 "C;cycling parmeten: 1 min at 94 "C.2 min at 50 "C.2 min ct 72 OC: 35 cycle; f i n d extension step: 7 min nl 72 OC. Apparatus: Persoilal Cyclcr (3iomerra, Go~ingcn),eq~ippedwid1 0.5ml rubes.
Reagent ki[ for PCR: PCR Master @oc!~ringcr-Mannheim),Ihc final concenlration of MgCi2 in h e assay raised to 2 rnM.
Thc PCR mixturc (assay volume 50 $) conlained 100 og o f DNA and 25 pmoles o f each primer.
WE,
Lnf. Fischwificch. 42(4), 1995
21 1
Preparation of Single S t r d DNA After completion of PCR,5 pl of t h e assay werz mixed with 15 pl of denaturing solution (95 % (wlv) . formamide, 0.05 % bromphenol blue, 0.05 % xylene cyanol, iil 10 mM NaOH), heated for 5 min at 95 O C , and placed immediately in iced water. The samples then were loaded onto the polyacrylamide gel without delay.
Gel Electrophoresis
ClemGel 10 % 48s or CleanGel 7.5 % 25s (F'harrnacja, Freiburg) were used for native PAGE, generally following the operating instructions given by P b m c i a . DNA bands were visualised by si.lver staining. Rehydration buffer: 112 rnM Tris acetate pH 6.4; electrode buffer: 0.2 M Tricine, 0.2 M Tris, 0.55 % SDS. pH 8.3. Silver staining: 1 . Fix:
30 min
200 ml
1 0 % (vlv) acetic acid
2. Wash:
3 x 2n~in
200 ml
3. Silvcr:
30 min
200 ml
3 x 200 nrl distilled water 0.1 96 (wlv) AgN03 + 200 pI formaldehyde (37 %)
4. Wash gcl. film hacking and way thoroughly w i ~ hdisrilled water (squeeze bottle). 5 . Devclop;
+ 2 0 0 ~ 1Na-
30 scc
200 rnl
3 % (wlv) Na2C03 diiosulfatc (2 %, wlv).
2-5 mjn
200 ml
3 0/n Na2C03 + 100 p1 forrnaldchyde + 200 pl thiosulfate Tl~eNa2C03 solution should be precooled in rhe refrigoraror
-t-
100 pl formaldehyde
(lo- 1s "C)
10 % (vlv) acetic acid 10 % acctic acid / I0 % ( v l v ) glycerol
6. Stop: 7. Impregnate:
10 min 10 min
8. Dry:
overnight at ambient ~cmpera~ure
2W ml 200
ml
Results and Discussion
Both of the two sets of primers used for PCR were suit.able for amplifying DNA of canned tuna (Figure 1). The patterns of ssDNA of skipjack and albacore were clurl y different and a1lowed identification of the commercial cannecl tuna as produccd From skipjack. Tlle primers 59-3159-5 have been used recently for identification of canned tunas by PCR and sequencing of the amplified DNA fragrnet~t(Unseld et al., 1995). Figure 2 demonstrates that SSCP with these primers gave species specific patterns for a number of tunas and boniros. Blue fin and albacore couId not be distinguished; this had to be expected, because the sequence of ltie DNA (ragmen1 is identical for both species (Unseld et a]., 1995).
Tnf. Fischwirtsch. 42(4), 1995
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Fig. I : SSCP of canried tuna (commodity. declared to contain skipjack) and reference samples o f carined yellow fin and skipjack. CleanGel 7.5% 25s. Lane I : 100 bp ladders (Pham~acia);lane 2: no sample applied; lane 3-7: PCR with primers 593159-5; lane 8-1 1: PCR with primers FB 349/FB 496. ss: single slranded DNA, ds: double sfr-andedD N A . Lane 3, 4, 8. 9: Cominerci;ll can; lane 5, 10: skipjack, Knuuwotrlrs p e l a ~ ~ l i s ;lane 6 , 1 1 : Yellow fin, rhutrt~rrs albocnres, Isne 7 : PCR control withoui D N A .
Figure 2: SSCP of canned reference samples. PCR was performed with pnmers 59-3159-5. CleanGel 10% 48s. Sequence of samples, from left to right: Blue fin, Thunrrlrs rliy~inus;yellow fin, T. nlbacores; albacore, T. ahlunga; bigeye. T.obesus; skipjack, Kalsl~wonrls peh1tni.s; little luna, Eulhynnus allet~erotus;Frigate mackerel, ~~~~is~~~a~urd;Atlantrcb~n~to,Sardasarda. ss: single stranded DNA, ds: double stranded DNA.
In comparison with other methods suitable for fish identification by DNA analysis, SSCP offers several advantages. The technique is very sensitive in the detection of single base changes. and it is fast and easy to perform. The disadvantages are (i) the necessity to tun references and samples side by side on the same gel, and (ii) the lower content of information of the SSCP pattern compared to the sequence of the respective DNA strand. Comparison of SSCP patterns and DNA sequences for differ- , ent mitochondnal DNA fragments of a number of runas and bonitos is under way, to prove the reliabjIity of the method and to evaluate the intra-species variability of the SSCP patterns. Acknowlcdgemcnls: 'fic
work was supported by thc EU projcct NR 2 CT 94-1 126. Wc thank G.K r a s Tor skilful Iechnical
assistance.
References Mackie, l.M., Chalmers, M. Reece, P., Scohhie, A.E., Ritchie, A.H.: The application of clcctrophoretic tcchniques 10 h e identification of species of canned tuna and bonito. In: Pelagic Fish - The Resource and its
Exploira~ion(eds.: BWL, J.R., Hardy, R., Whitcle, K.J.), Fishing News Books, Oxford, 200-207, 1992. Meyer, R.. Candrian, U. L M I ~ J. , : Dctccrion of park in heated mcat products by Lhe polymerase chain rcacfion. Journal of AOAC International 7 7 , 6 17-6?.2, 1994. Bnrtleu, S.E., Davidson, W.S. : FINS (Forensically informative nucleotidz sequencing): a procedure for identifying h e nnimal origin of biological specimens. BioTwhniques 12, 408-4 1 I , 1992. Unseld. M . , Beyermann, B.;Brandt, P.. Hiesel, R:ITdendlication of he species origin of highly proccsscd meal products by mitochondrial DNA sequences. PCR Methods and Applications 4.24 1-243. 1995.
Fujira, K., Silver, J. : Single-strand conformaliortel polymorphism. PCR Mehods and Applicalions 4, SI37S139, 1994. Haro, M., Noguchi, M., Naira, E., Dewa, K., Yarnanouchi, H. : Bull. Jap. Sea Natl. Fish. Res. Inst. 44, 13 1 - 1 38. 1994.
Finncrty, J.R., Block. B.A. : Evolurion of cytochromc b in h e Scombroidei (Teleostei): moleclur insights inlo billfish (Isriophoridac and Xiphiidac) rclalionships. 17ish. Bull. 93.78-96, 1995.
