TAP CHI KHOA HOC TRUONG DAI HOC MO TP.HCM - SO (37) 2014 BUOC DAU xAy DUNG QUY TRINH PCR NHAM PHAT HIEN THANH PHAN ONG VAT TRONG THUt PHAM CHAY DI A TREN VUNG 16S rDNA TY THE Ngiy nhin bai: 10/04/2014 Ngiy nhin lai: 10/06/2014 Ngay duy't ding: 07/07/2014 Lao Dirc Thui)n, Nguyin Thi Than/i Nhan, Nguyen Thi Thien Throng, Trim Ki'Jn Vo Phi Phi Nguyen, Phan Thi Tram' Le Huy'en Afi Thity2 TOM TAT Hien nhu eau su dung thuc pham chay ,igay tang tang,cao chY den "tinh thulin chay", nghia khong lan bat ki phiin nao.co nguon c ter clang vat Day mot dac tinh quan trong the biens va san xueit thuc phdm chay muc dich kiem tra c6 hoac khong su hien di en ctia phiin dOng vat thtrc phcim chay, chthng toi tien hanh xdy dung quy trinh phat hien sir hien dien cua phein dOng vat dva tren ki thudt PCR khuech dui vitng 16S rDNA ty the bang cap moi TP1 va TP2 co tinh pho.quat va dac hieu voi 16S rDNA hdu het cac loci Ong vat Ket qua.cho thdy, buac dau xdy dung tong quy trinh phat hien DNA ddng vat Min thuc pheim chay Quy ,trinh Avec hinh so bO ctli &roc thin nghiem tren 11 mdu duac clan nhan la thuc pheim chay thu nhan ter cha va mot s6 tong ty che bien thuc pheim chay tren thi &yang; Ket qua ghi nhan 4/11 meiu thus philm chay nhiem phan co nguon goc Ong vat (chiem 36,36%) Qua d6, quy trinh co the khong dinh dit ca khoa hoc de' Olen khai tren mot so hcvng mait low how thus te Tir khoa: 16S rDNA ty the, thgc phi'rn chay, PCR, Ong vat, th‘rc vat ABSTRACT The demandfbr using vegetarian foods more and more increase nowadays with focusing to the veganism that mean don't contain any ingredients have origin from animals that is an important feature in the processing and production of vegetarian foods For this purpose, we check whether have or not presence of animal ingredients in vegetarian food PCR assay that are specific to detect the presence of ingredients animal were designed basing on 16S rDNA gene of the mitochondrial DNA genome by primer TP1 and TP2 Oligonucleotide primers that are universal and specific for 16S rDNA gene in most of animals As the results, assay was initially successfully established for detecting animal- origin components in vegetarian foods We experimentally tested and detected animal ingredients in 11 samples vegetarian food from the market and vegetarian food companies, as the results, 4/11 samples (counting for 36.36%) contain the animal ingredients In addition, we conducted sequencing and indentified successfully this ingredient originating from Sus scrota and Gallus gallus According to this sequencing result that are completely accordant, we affirm primer-specific amplification in this study can be apply to experiment on the large number of samples in the reality Keywords: mitochondria' 16S rDNA, vegetarian food, PCR, animal, plant I Truang Doi hoc Ma TP.HCM PGS.TS, Truang Doi hoc Ma TP.HCM Email: lhathuy@gmail.com CONG NGHE V4t lieu va phuang phi') nghien Gieri thqu Thuc pham chay dugc hieu thkrc pham kh6ng china cac thAnh phan c6 nguen g6c tir Ong vatlll Thuat ngir "an chay" dugc biet den nhieu a dao Phat va nhieu ton gido kit& dtrai chay" khac nhauil l ve xu nhieu hinh thirc huang An chay hien cang gia tang a nhieu quec gia tren the giai ding nhu a Viet Nam LY dm skr gia tang IA an chay IA met phuang thirc viec phong den tim cac benh tat nguy hiem lien pan den mach, cao huyet Ap ung thu, E21131141 Nam bat dugc xu the Oat trien, thi tnrang cang xuat hien nhieu san pham chay vai mh ma da dang, phong pha nhu: thit ga chay, heo chay, bo vien chay, Tuy nhien, "tinh thuan chay" lai khong dugc dam bao, dac biet chua c6 met ca quan tharn quyen nAo dam bao thuc pham chay khong IL bat kY met phan c6 nguen goc dong vat Do do, met so cong ty thgc pham chay vai muc dich de dam bac) "thtrang hieu" phai giri matt thgc pham (san pham va/hoac nguyen lieu san xuat) sang nu& ngoAi de kiem dinh Xuat Oat tir nhu cau thuc to teen, viec nghien ciru va phat tri6n mot quy trinh nham kiem dinh sg hien dien co thAnh phan c6 nguen gee tir dOng vat thkrc pham chay IA can thiet phuang phap PCR Teen the (polymerase chain reaction) IA phuang phap thong dung dugc nhi6u tac giA sir dung de cac phan khuech.dai trinh DNA dich co nguOn gec tir Ong vat Met so cac trinh to dich dugc sir dung nhu gen Cytochrome b cua be gen ty the151161, 12S rDNA caa ty the f 71i83, 16S rDNA ty the [9][10] Trong nghien ciru nay, 16S rDNA ty the dugc chon lam trinh to DNA dich cho phan img PCR, ly cho six lkra chen (1) 16S rDNA c6 tinh phO quat, tinh bao ton cao ding met loci, chang thay dOi cham theo thoi OW111111121; (2) So lugng nhieu ban 16S rDNA to baoi ll i Do de, muc dich cifa nghien cuv bao gem xay dkrng quy trinh phat hien phan c6 nguon gee tir Ong vat thut pham chay va buoc dau quy trinh dugc kiem tra tinh thuan chay tren cac mau dux pham chay thu nhart tren thuc te cum Veit fieu Mau chi:mg throng bao gem mL Ong vat thit ga va thit heo Mau chirng am mh thkrc vat Mau thuc to cac mau thgc pham chay thu mua ngh nhien tir cac cong ty san xuat thgc pham chay tir cong ty Au Lac, tir chg Thu Dau Met NIA sieu thi Thiit ke ni'di Trinh tkr mei xuoi va moi ngtrgc dugc thiet ke dga teen 16S rDNA ty the ten Ngan hang Genbank (NCBI: http://ncbi.nlm.nih.gov/) cifa cac loAi dong vat bao gem ga (KF908854, AB489247, ), heo (JN714132, KC208030, KF799977 ), bo (KF799979, ) bang cac phan mem tnrc tuyen nhu Primer3(v.0.4.0) (http://bioinfo.ut.ee/primer3-0.4.0/) Mei sau thiet ke dugc clanh giA cac thong so vat ly nhu dAi, nhiet de nong chay, phan tram GC, nang lugng hinh cac cau true bac hai bang phan mem trgc tuyen IDT (http://sg.idtdna.com/analyzer/Applications/Oli goAnalyzer/) Deng thoi tinh dac hieu dugc kiem tra bang chuang trinh BLAST (NCBI: http://blast.ncbi.nlm.nih.gov) v.v Tdch chili DNA DNA dugc tach chit theo phuang phap Phenol-Chloroformi131[14) Tat ca cac nth dung de tach DNA deu dugc lan loot rira sach bang ntrac, 70° va dung dich PBS (Phosphate buffer saline) Sau rir a sach, cac mh dugc cat Nth gia nhuyen met matt dong nhat 2,0 g mL dich huyen phit vai ml dung dich dong nhat rnAu (NaC1 5M, Tris-HC1 1M, EDTA 0.5M ddH2O) Sau dong nhat hat 750 pl dich chuyen vao eng eppendorf mai be sung 20 pl SDS 10% va 20 pl proteinase K (1 mg/ml), u qua dem a nhiet de 65°C Sau de 250 p.1 NaC1 bao hoa dugc be sung, U matt a 4°C 30 phat, ly tam 10.000 vOng/phat 15 phat Chuyen 500 p.1 dich vao Ong eppendorf khac va be sung vai 500 pl hen hop Phenol:chloroform:isoamylalkohol (25:41:1) dao nhe Ong, ly tam 13.000 vong/phat 10 phut (lap,lai 2-3 lan), them 500 pl Chloroform, dao nhe eng va ly tam 13.000 vong/phitt TAP CH[ KHOA HOC TRUONG DAI HOC MO TP HCM - SO (37) 2014 10 phut Qua trinh ttia dugc thuc hien vai NH4OAc va Isopropanol a nhiet dO -20°C Yong gia Sau do, tien hanh ly tam thu tug va luu gift DNA dung dich TE cho nhang thi nghiem sau Nong d8 DNA dirge xac dinh thong qua phtrcmg phap OD va kiem tra dO tinh sach th6ng qua tri so A260/A280 Phan sing PCR Phan img PCR duce thirc hien vai chu trinh nhiet sau: chu ky vai nhiet dO 95°C phitt; 35 chu kS7 vai nhiet dO 95°C 30 giay, 67°C phtlt, 72°C phUt; chu 1(5/ 72°C phut Thanh phan phan Ung PCR dugc the hien a Bang San pham PCR dugc dien di tren gel agarose 1,5% va giai trinh tu tai ding ty Nam Khoa Bing Thanh phAn phin tin PCR Lung Thanh plan Master mix (2X) 7,5 ul MOi xuoi (1012M) 0,5 ul MOi ngtrgc (101AM) 0,5 tl DNA mach khuort 1,0 pi ddH2O 5,5 gl Tiing the tich 15,0 p1 Ket qui NI thin luOn dank giti ctic tinh cut: in'di Trinh tu mOi de khuech dai 16S rRNA ty the có trinh tu: moi xuoi (TP1) 5'CCYAGGGATAACAGCGCAATC-3' va mOi 5'(TP2) ngugc TCCGGTCTGAACTCAGATCAC-3' (Trong do, Y thay the cho C va T) Cac dac tinh vat ly m8i dupe danh gia bang phan mem IDT the hien ar Bang Bing Cic dac tinh vat ly ciia mOi TP1 va TP2 INI4i Chien dai (bp) %GC T., (°C) (1) (2) TP1 21 54,7 56,9 0,81 -11,53 TP2 21 52,4 56,2 -2,00 -9,75 (3) -4,64 Ghi chzi: T,„: nhiet ming chay: (I) AG cua frac hp tot: (hairpin-loop) (kcaLmole-i ); (2) AG caa true self-dimer (kcaInzolil); (3) AG clia can ink hetero-dimer (kcaLniole.1) Dip tren ket qua Bang cac gia tri vat 1), ye chieu dai, %GC, nhiet nong chay va six chenh lech, nhiet nong chay de deft ,thoa man cac yeti cau thiet ke moil''' Ve AG phan Ian deu thoa man bam hcrn -9 kcal.mor l, ngoai trir rang lixgng hinh cau true tu bat cap dm TP1, TP2 Tuy nhien, tien hanh kiem tra bang phan mem true tuyen, cau true tu bat cap xay a dau 5' va gia tri khong chenh ,lech qua nhieu so yen yeu cau Do cap mai Van dugc sir dung va kiem tra tinh dac hieu Tinh dac hieu dugc tien hanh kiem tra bang chtrang trinh, BLAST va Annhyb deu cho thay kha nang bat cap dac hieu tren 16S rDNA a cac loci dOng vat vai mire dO worn CONG NGHE d6ng dat 100% (Hinh 1) va khOng bat cap teen cac trinh ttr I 6S rDNA thdc vat nhtx bap cai, ngo, rau den, dau ht : (Dir lieu khong trinh bay) Dong thai kiern ,tra bang phan, mem ClustalX, ket qua cho thay cap moi bat cap chuyen biet d6i vt7i cac trinh tu 16S rDNA tir d6ng vat (Dil lieu khong trinh bay) Dda tren nhiing ket qua thu nhan dtrgc, m6i TP1 va TP2 van throe chon de su dung cho cac thdc nghiem sau Hinh (a) Su• bat cap cua TP1 va TP2 len trinh tu 16S rRNA cua Gallus Gallus (Ma so truy cap teen Genbank: AP003321) Vi tri Y cap moi TP1 tuang thich yeri C trinh tu bat cap; Ket qua BLAST (b) cap mcii TP1 va (c) TP2 the hien tinh dac hieu hi lef4 I ottermetts 1343 133 fourtl 4490 now 91.9$ 1441 1463 fool Mr wee: 140 56%.*: 1101 TTIAAAATCA CGACCACCTT ACAACCTTAC ACAGCCCCAC TGGGTCCACC 1151 CACACATAAA CCCCTGGTCG ACATTITTCG GTTGGGGCGA CCTTGGAGAA 1201 AAAAAAATCC TCCAAACCCA CAGACCACAA CTCTTCACTA AGACCAACTC 1251 CTCAAAGTAC CAACAGTAAC CAGACCCAAT ATAATTGAGC AATGGACCAA 1301 GCTACES IMETCCT CCAAGAGCCC ATATCGACAA 1351 GGAGGTTTAC GACCTCGATG TTGGATCAGG ACAACCTAAT GGIGCAACCG 1401 CTATTAAGGG TTCGITTGIT CAACGATTAA CAGICCTACG TGATCTGAGt 1451 #AGACCGGA GCAATCCAGG TCGGTTTCTA TCTATGAACA CACTCCTCCT 1501 AGTACGAAAG GACCGGAGAA GTGGGGTCAA TACCACTGAG CACACCCCAA moms (a) Desoption Max Tail Ovary E Mot AcrAssam Kos sort caw aloe O c s hum hamtre i4vi Co 13 fritothoocort earOS otnnt 394 394 100% 023 95% 1F342417 • %s , reffixosus ernathocvw celcott ceetortt 394 39.4 100% 023 95% KF92s379 t Calls Iustis trnhans ritocnonton conYee cenome 394 39 100% 023 95% K192i378 I O Fos tams ribtborienon ornItIt 2enrit 39 39 100% 023 95% 0925377 t • ~ylum:anlamcoonwlen ameet ctmemt 39 39 100% 23 95% 0925375 o 39.4 39 100% 023 95%