GUO Yun -xia,et al: Authentication of Shark Derived Material in Food Using SYBR Green PCR 1,2 *1,, 1, 1, 3, 2, 3 (1., 200135; 2., 201306 ; 3., 100025) :, SYBR Green PCR SYBR Green PCR 9 48,9, (84±1.5) ;, (84±1.5) 0.000 1 ng/μl DNA 0.01% 20 PCR,,, : SYBR Green PCR; ; :Q 789 :A :1673 1689(2012)12 1300 07 Authentication of Shark Derived Material in Food Using GUO Yun-xia 1,2, ZHANG Shu-ya *1, CHEN Hong-chao 1, REN Shuo 1, HUANG Wen-sheng 3, BAO Jian-qiang 2, CHEN Ying 3 (1.Shanghai Entry -Exit Inspection and Quarantine,Shanghai 200135,China;2.College of Food Science and Technology,Shanghai Ocean University,Shanghai 201306,China; 3.Chinese Academy of Inspection and Quarantine,Beijing 100025,China) Abstract: A specific and sensitive SYBR Green real-time fluorescence PCR method was developed to determine of the shark derived material in food. The specificity and sentivity of this method was examined. The results showed 9 kinds of shark samples displayed theamplified curves and the highest point of melt curves at (84±1.5),whereas other 48 kinds of common plants and animals displayed no amplified curves and the highest point of melt curves at temperatures out of (84±1.5). The detection limit of the method was 0.000 1 ng/μl DNA and 0.01% shark meat powder. The 20 commercial shark products samples were analysized by SYBR Green PCR and all the products : 2012-03-06 : (2010IK191) * : (1978 ),,,,, E-mail:zhangshuya@shciq.gov.cn 1300
, : SYBR Green PCR indicated existence of shark DNA except the artifical shark fin and shark liver oil. The results indicated that the detection method is highly specific and sensitive,and can be used to the identification of shark derived material in food. Keywords: SYBR green real-time fluorescence PCR, food, shark derived material, authentication, [1] DNA: [2-6],, [7],,, [8], EPA 48 [9] DHA :,, 5 :,,, ;2 : SYBR Green(SG), ;4 : DNA, PCR DNA SYBR Green, DNA, ; 1 000, : PCR DNA [10] PCR, 60, 95, ; :, [11] 1.2, SYBR Green PCR DNA, 80 48 h [12-13] GenBank PCR, 10% 1% 0.1% 0.01% SYBR Green PCR, DNA 1.3 DNA 1 1.1 ; : ; : ; : :, :DP324; DNA : : DP323; DNA 2012 31 12 1301
GUO Yun -xia,et al: Authentication of Shark Derived Material in Food Using : :DP305; DNA 40 s 72 45 s 35 ;95 15 s 64 30 s BioPhotometer plus 99 15 s 64 (Eppendorf) DNA, -20 1.4, Blast GenBank, 2 g/dl Primer5 PCR, :Primer1:5 - CACCTTTAGGCAAACCAGCAC -3 ; Primer2:5 - GGGCCAGGGTCGAATCTCT-3 1.5 PCR 18S rrna, DNA PCR, DNA PCR, DNA 18S rrna :5 -TCTGCCCTATCA ACT TTCGATGGTA-3 5 -AATTTGCGCGCCTGC TGCCTTCCTT-3 PCR :1 PCR,2.5 mmol/l Mg 2 +,1 U Taq,200 μmol/l 2.2 dntps, 100 nmol/l, 50~100 ng, SYBR Green PCR 9 25 μl :94 3 min,94 20 s,54 40 s,72 40 s,40 SYBR Green PCR DNA (ABI 7300 PCR ), :12.5 μl SYBY Green Pre Mix,1 μl Primer1 (5 μmol/l),1 μl Primer2 (5 μmol/l), 8.5 μl,2 μl DNA,, SYBR Green PCR 25 μl :95 10 min 95 30 s 64 1.6 PCR 10 μl 18S rrna, 1 μl 10, 2 2.1 18SrRNA 18S rrna 137 bp, DNA, DNA PCR DNA,, 1 (84±1.5), 2 48 DNA PCR,, 3 (84± 1.5), 4 9 Fig.1 1 PCR Specific detection of real-time PCR for shark 1302
, : SYBR Green PCR Fig.2 2 DNA SG-PCR SG-PCR melting curves of PCR product from shark Fig.4 3 SYBR Green PCR,10% 1% 3 48 PCR 0.1% 0.01% DNA Fig.3 Specific detection of real-time PCR for non-shark materials,0.001%, 6 4 48 SG-PCR SG-PCR melting curves of PCR product from nonshark materials 2.3 1 TE DNA 10 1 10-1 10-2 10-3 10-4 10-5 ng/μl, SYBR Green PCR, 3, :10 1 10-1 10-2 10-3 10-4 ng/μl DNA, 10-4 ng/μl, 5 10% 1% 0.1% 0.01% 0.001%, DNA Fig.5 5 DNA PCR Amplification curves generated by serial dilution of shark DNA 2012 31 12 1303
GUO Yun -xia,et al: Authentication of Shark Derived Material in Food Using Fig.6 6 (W/W) PCR Amplification curves generated by different shark percentage content 2.4 7, (84± : 1.5) ( 8), ; DNA,, (84±1.5), DNA Fig.7 7 PCR Amplification curves generated by commercial shark products 1304
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