T I U C H U N Q U C G I A TCVN 8376 : 2010

TIU CHUN QUC GIATCVN 8376:2010 TM V SN PHM TM PHT HIN VIRUT GY HI CHNG TAURA (TSV) BNG K THUT PHN NG CHUI TRNG HP - PHIN M NGC (RT-PCR)

Shrimp and shrimp products Detection of taura syndrome virus (TSV) by reverse transcription - polymerase chain reaction (RT-PCR)Li ni uTCVN 8376 : 2010 do Cc Qun l Cht lng Nng Lm sn v Thu sn bin son, B Nng nghip v Pht trin Nng thn ngh, Tng cc Tiu chun o lng Cht lng thm nh, B Khoa hc v Cng ngh cng b.

CNH BO m bo an ton cho nhn vin, cc thao tc phn tch ch c thc hin trong cc phng th nghim c trang b thch hp, di s kim sot ca cn b th nghim c kinh nghim. Etidi bromua (EB) l cht c kh nng gy ung th. V vy phi mang gng tay, eo knh v mc qun o bo h trnh tip xc. Nn nh du r rng thit b v dng c tip xc vi EB v khng di chuyn chng ra khi khu vc quy nh. cht thi c cha EB trong vt cha v c phng php loi b thch hp. 1. Phm vi p dng Tiu chun ny quy nh phng php pht hin virut gy hi chng taura (TSV) trn tm b m, tm ging, tm nui thng phm ca cc loi tm thuc chi Tm he (Penaeus) bng k thut phn ng chui trng hp - phin m ngc (RT-PCR).

2. Ti liu vin dnCc ti liu vin dn sau l rt cn thit cho vic p dng tiu chun ny. i vi cc ti liu vin dn ghi nm cng b th p dng phin bn c nu. i vi cc ti liu vin dn khng ghi nm cng b th p dng phin bn mi nht, bao gm c cc sa i, b sung (nu c).ISO 22174 : 2005 Microbiology of food and animal feeding stuffs Polymerase chain reaction (PCR) for the detection of food-borne pathogens General requirements and definitions (Vi sinh vt trong thc phm v thc n chn nui Phn ng chui trng hp pht hin sinh vt gy bnh t thc phm Yu cu chung v nh ngha).3. Thut ng v nh nghaTrong tiu chun ny p dng cc thut ng v nh ngha nu trong ISO 22174 v cc thut ng, nh ngha sau y:3.1 Virut gy hi chng taura (TSV) [Taura syndrome virus (TSV)]Virut thuc chi Picornavirus, h Picornaviridae, dng hnh cu 20 mt, kch thc t 30 nm n 32 nm, vt cht di truyn l RNA c kch thc khong 10,2 Kb.3.2 Pht hin virut gy hi chng taura (Detection of taura syndrome virus)

Vic xc nh s c mt hay khng c mt ca TSV trong mt khi lng c th ca sn phm khuch i khi tin hnh th nghim theo tiu chun ny.

4. Nguyn tc4.1 Phng php phn tch nh tnh, s dng k thut RT-PCR mt bc pht hin TSV da vo vic xc nh on RNA ch c c khuch i hay khng. Qu trnh xc nh c thc hin bng cch in di sn phm sau khuch i trn thch agaroza, nhum mu cDNA v quan st di nh sng UV c bc sng 302 nm.

4.2 Quy trnh pht hin TSV bng k thut RT-PCR qua 4 giai on k tip nhau (xem Ph lc A): tch chit RNA; phin m ngc RNA thnh cDNA; khuch i cDNA; in di pht hin cDNA v c kt qu.5. Thuc th v vt liu thTrong mi trng hp, ch s dng cc thuc th tinh khit phn tch thch hp v nc ct loi dng cho sinh hc phn t. Vic pha ch, bo qun v s dng thuc th phi tun th theo hng dn ca nh sn xut. Nhn chung, nn ly lng cn thit va cc dung dch phn ng cho php th PCR v bo qun chng trong iu kin thch hp.5.1 Mi5.1.1 Mi c hiu ca TSV s dng trong k thut RT-PCR mt bc gm c cp mi 7171F/ 7511R. Trnh t mi c th nh sau:MiTrnh tSn phm

7171 F5'- CGA CAG TTG GAC ATC TAG TG -3'341 bp

7511 R5'- GAG CTT CAG ACT GCA AGT TC -3'

5.1.2 Dung dch miDng dung dch m TE (5.1.3) pha long mi n nng 25 mM.

5.1.3 Dung dch m TE (Tris - EDTA)Chun b dung dch cha Tris [tris (hydroxymetyl) aminometan] 10 mM v EDTA 1 mM, dng HCl chnh pH 7,6.

5.2 Thuc th tch chit RNA dng h thng ct ly tm 5.2.1 Dung dch m ly giiChun b 25 ml dung dch gm: guanidin-HCl 4,5 M, natri phosphat 100 mM, pH 6,6. Bo qun 25 oC.5.2.2 Dung dch m I (DNAaza I) Chun b 500 l dung dch DNAaza I gm enzym DNAaza 5 U/l v dung dch m ra gii (5.2.6), trn k, bo qun 15 oC n 25 oC. 5.2.3 Dung dch m Chun b 10 ml dung dch gm NaCl 1 M, Tris-HCl 20 mM, MnCl2 10 mM, c pH 7,0. Bo qun 25 oC.5.2.4 Dung dch m ra IChun b 33 ml dung dch gm guanidin-HCl 5 M, Tris-HCl 20 mM, c pH 6,6. Thm 20 ml etanol tinh khit. Bo qun 25 oC. 5.2.5 Dung dch m ra IIChun b 10 ml dung dch gm NaCl 20 mM, Tris-HCl 2 mM, c pH 7,5. Thm 40 ml etanol tinh khit. Bo qun 25 oC.

5.2.6 Dung dch m ra giiChun b 30 ml nc ct hai ln v trng v c kh enzym nucleaza.CH THCH Tch chit RNA c thc hin bi nhiu phng php khc nhau, nhng t c kt qu mong mun c th s dng mt s kit tch chit RNA thng mi c bn sn. Nn s dng thuc th v cc sn phm dng cho phn ng khuch i PCR c tng hp thnh b ch phm ang c lu hnh trn th trng c thnh phn ph hp nh di y. 5.3 Hn hp phn ng RT-PCR C th s dng hn hp phn ng RT-PCR c bn sn hoc c pha ch t cc thnh phn ring l nh sau:Thnh phn25 l/ng phn ngNng cui

Nc thm vo6,5 l

Dung dch m EZ m c 5 ln 5,0 l1X

Hn hp dNTP (10 mM mi loi)3,0 l300 M mi loi

Mi ngu nhin pd (T)12-181,0 l0,62 M

Mi 7171 F1,0 l0,62 M

Mi 7511 R1,0 l0,62 M

Mn(OAc)22,5 l2,5 mM

Enzym rTth DNA polymeraza1,0 l2,5 U

DNA ch4,0 l

5.4 Dung dch m TBE m c 10 ln (Tris - axit boric - EDTA 10X)Ha tan 108 g Tris v 55 g axit boric trong 600 ml nc. Thm 40 ml EDTA 0,5 M (5.5) v thm nc cho 1 l. Hp v trng 121 oC trong 15 min. Bo qun nhit phng. Khi s dng, pha long dung dch vi nc thnh dung dch 1X.

5.5 Dung dch EDTA (etylen diamin tetra axetic) 0,5 MHa tan 93,05 g EDTA trong 350 ml nc, chnh pH 8,0 bng dung dch NaOH nng 4 M. Thm nc cho 500 ml. Hp v trng 121 oC trong 15 min. Bo qun nhit phng.5.6 Dung dch m ti mu DNA m c 6 ln Chun b dung dch gm Tris-HCl 10 mM (pH 7,6); bromophenol blue 0,03 %; xylen xyanol FF 0,03 %; glycerol 60 % v EDTA 60 mM [s dng dung dch EDTA 0,5 M (5.5)]. Bo qun nhit 20 (C. 5.7 Dung dch DEPC Dung dch di-etypyrocacbonat 0,1 % th tch, lc trn u khong 10 min. Sau qua m trong bnh kn, ni lng np v hp kh trng 121 (C trong 15 min. Bo qun nhit 20 (C.

5.8 Thch agaroza 1,5 %Cn 1,5 g agaroza, ho tan trong 100 ml dung dch m TBE 1X, un nng lm tan hon ton agaroza. ngui n khong 55 (C n 60 (C, lc u, trnh to bt kh. 5.9 Bng thch agarozaChun b khun, cn bng mt khun bng git nc, gn lc vo khun, thch agaroza (5.8) vo khun vi b dy t 3 mm n 5 mm. ngui v ng t nhin nhit phng. G lc, t thch agaroza vo trong mng in di, cc ging ca bng thch phi pha gn in cc m. dung dch TBE 1X cho ngp mt thch agaroza trc khi cho sn phm khuch i vo in di.5.10 Dung dch etidi bromua (ethidium bromide)Ht 5 l dung dch etidi bromua (10 mg/ml) ho tan trong 100 ml nc. Bo qun nhit khng ln hn 20 (C.5.11 Thang DNAS dng thang o ph hp c th c lng c on khuch i 341 bp. C th s dng sn phm khuch i c kch thc 341 bp bit trc.6. Thit b v dng cS dng cc thit b, dng c ca phng th nghim vi sinh thng thng v c th nh sau:6.1 My chu trnh nhit, c th thc hin chnh xc v lp li chu trnh thi gian v nhit c quy nh trong 7.4.5. 6.2 My lc Vortex.6.3 My ly tm lnh, c th hot ng vi tc ti a trn 13 000 r/min.6.4 H thng pht hin cc sn phm PCR, bao gm:a) b in di; b) khun, khay, lc dng to bng thch agaroza;c) bn c kt qu, vi tia UV bc sng 302 nm;d) b phn chp nh lu kt qu, v d: my c Gel- Doc (nu c).6.5 T ng, c th hot ng nhit khng ln hn 20 oC. 6.6 T lnh, c th hot ng nhit t 2 oC n 8 oC.6.7 T thao tc v trng.6.8 Cn phn tch, c th cn chnh xc n 0,001 g.6.9 Micropipet, dung tch 10 l, 20 l, 100 l v 1000 l; c u tp (vi dung tch khng ln hn 10 l phi s dng u tp c lc).6.10 ng phn ng, chuyn dng cho PCR, dung tch 1,5 ml v 0,2 ml.6.11 ng lc tinh, dng ng nha c 2 lp mng lc polypropylen, cha dch mu.6.12 ng gp, dng bc ngoi ng lc tinh.7. Cch tin hnh7.1 Chun b mu7.1.1 i vi mu tm hu u trng (postlarvae)Lng mu dng cho mi phn ng t 10 c th n 15 c th; ly phn ui, khng ly gan tu.7.1.2 i vi mu tm b mLng mu dng cho mi phn ng t 100 mg n 200 mg, ly cung mt hoc 1 phn chn bi.7.1.3 i vi mu tm thng phmLng mu cn dng cho mi phn ng t 100 mg n 200 mg, ly phin mang tm, hoc chn bi, chn b hoc phn c hoc dch bch huyt ca tm khong 100 l. 7.2 Tch chit RNA dng h thng ct ly tm Cho mu (7.1) vo cc ng phn ng dung tch 1,5 ml, thm 400 l dung dch m ly gii (5.2.1), nghin mu bng chy nghin v trng.Ly tm lnh vi tc 13 000 r/min trong 2 min, nhm loi b m cha thy phn. Chuyn 200 l phn dch ni sang cc ng phn ng dung tch 1,5 ml cha 200 l etanol tinh khit, trn u.Gn ng lc tinh (6.11) vo ng gp (6.12), chuyn ton b dung dch trn vo ng lc tinh. Ly tm lnh vi tc 13 000 r/min trong 30 s. Loi b phn nc cha trong ng gp, gn ng gp mi vo ng lc tinh.Thm 90 l dung dch m DNA polymeraza (5.2.3) v 10 l dung dch m DNA polymeraza I (5.2.2) vo ng lc tinh, 15 min nhit phng.Thm 500 l dung dch m ra I (5.2.4) vo ng lc tinh, ly tm vi tc 8 000 r/min trong 15 s, loi b dch trong ng gp.Thm 500 l dung dch m ra II (5.2.5) vo ng lc tinh, ly tm vi tc 8 000 r/min trong 15 s, loi b dch trong ng gp.Thm 300 l dung dch m ra II (5.2.5) vo ng lc tinh, ly tm vi tc 13 000 r/min trong 2 min, loi b dch trong ng gp. Ly tm cc ng gp cha ng lc tinh tc cao nht (c th) trong 20 s loi b hon ton dung dch m ra.Gn ng lc tinh sang mt ng phn ng mi, dung tch 1,5 ml. Thm 100 l dung dch m ra gii (5.2.6) vo ng lc tinh, ly tm vi tc 8 000 r/min trong 1 min. Loi b ng lc tinh, sn phm RNA c cha trong ng phn ng 1,5 ml. Dch tch chit RNA s dng ngay cho phn ng khuch i hoc bo qun nhit 20 oC.7.3 Chun b cc kim chng cho phn ng RT-PCR7.3.1 Mu kim sotNc ct hai ln hoc dung dch DEPC.

7.3.2 Mu kim chng m tnhMu tm khng b bnh bit trc.

7.3.3 Mu kim chng dng tnhMu tm nhim TSV bit trc hoc plasmid ca TSV.

7.4 Tng hp cDNA v khuch i cDNA mt bc7.4.1 Mu thHt 5 l dch RNA ca mu vo ng hn hp phn ng RT-PCR (5.3).

7.4.2 Mu kim sotHt 5 l dung dch nc ct hai ln hoc dung dch DEPC vo ng hn hp phn ng RT-PCR (5.3).

7.4.3 Mu kim chng m tnhHt 5 l dch RNA ca mu tm bit trc khng b bnh vo ng hn hp phn ng RT-PCR (5.3).

7.4.4 Mu kim chng dng tnhHt 5 l dch RNA ca mu tm nhim TSV bit trc hoc plasmid ca TSV vo ng hn hp phn ng RT-PCR (5.3).

CH THCH nh s cho cc ng v y cht np ng. Ly tm nh trc khi cho cc ng vo my chu trnh nhit. Mu kim sot dng kim tra tinh sch ca mi, hn hp phn ng RT-PCR v cc thuc th.

7.4.5 t cc ng vo my chu trnh nhit thc hin phn ng chui trng hp - phin m ngc (RT-PCR) theo chu k lun nhit nh sau:S chu kNhit , oCThi gianBc

14215 minTng hp cDNA

942 minBin tnh ban u

399445 sBin tnh

6045 sLai/ bt cp

7245 sKo di mch

1721 minKo di mch

4Gi n nh

7.4.6 Gi cc sn phm khuch i 4,0 0C cho n khi in di.

7.5 in di7.5.1 Cho t 5 l n 7 l dung dch m ti mu (5.6) vo cc ng sn phm khuch i (7.4.6) trn u.

7.5.2 Ht t 5 l n 7 l dch tng ng t cc ng (7.5.1), theo th t: thang DNA, cc mu th, mu kim chng m tnh v mu kim chng dng tnh, nh vo tng ging trn bng thch agaroza (5.9).

7.5.3 in di vi hiu in th t 70 V n 120 V. Kim tra bng cch quan st cc bng kh ni ln t hai pha in cc ca my in di sau khi ni in.CH THCH Dung dch m ti mu gm hai thnh phn mu: mu xanh m ca xanh bromophenol; mu xanh nht ca xylen xyanol. Khi quan st thy mu xanh m ca thuc nhum cch ging khong 2/3 chiu di bng thch agaroza, dng qu trnh in di. Sau , ly thch agaroza t bung in di ra nhum vi etidi bromua.

7.5.4 Nhum etidi bromua: t bng thch agaroza va in di vo khay nha sch, dung dch etidi bromua (5.10) ngp bng thch. 7.5.5 Lc nh trong khong 20 min. Vt bng thch ra khi dung dch nhum, ra bng nc ct trong 10 min loi b dung dch nhum cn d trn b mt bng thch.7.5.6 t bng thch trn bn c kt qu vi tia UV bc sng 302 nm hoc chp hnh nh bng my Gel- Doc (nu c).7.6 c kt qu7.6.1 Di nh sng ca tia UV, cc on cDNA c gn etidi bromua pht quang to thnh vch sng. 7.6.2 i chiu cc vch sng ca mu vi cc vch sng t thang DNA, mu kim chng dng tnh v mu kim chng m tnh a ra kt lun.7.6.3 Cch c kt quGingVch 341 bpKt qu

Thang DNA Cc vch sng r rngin di tt

Mu kim chng m tnh KhngKhng ngoi nhim

CB ngoi nhim

Mu kim chng dng tnh C Hn hp phn ng RT-PCR tt

KhngMu kim chng dng tnh hng hoc enzym hng

Mu thCDng tnh vi TSV

Khngm tnh vi TSV

8. Din gii kt qu8.1 Kt qu m tnh vi TSVKt qu c coi l m tnh khi:a) mu th khng c vch sng kch thc 341 bp hoc vch sng kch thc khc vi mu kim chng dng tnh; b) c vch sng ca mu kim chng dng tnh;c) khng c vch sng ca mu kim chng m tnh;d) thang DNA phn vch r rng theo kch thc s dng.8.2 Kt qu dng tnh vi TSVKt qu c coi l dng tnh khi:a) mu th c vch sng kch thc 341 bp;b) c vch sng ca mu kim chng dng tnh; c) khng c vch sng ca mu kim chng m tnh;d) thang DNA phn vch r rng theo kch thc s dng.9 Bo co th nghimBo co th nghim phi ghi r: mi thng tin cn thit nhn bit y v mu th; phng php ly mu s dng (nu c); phng php th s dng v vin dn tiu chun ny; mi thao tc khng quy nh trong tiu chun ny, hoc nhng iu kin c coi l t chn v bt k chi tit no c th nh hng ti kt qu (nu c); kt qu th nghim thu c.