TIU CHUN VIT NAM TCVN 5157-90

THT V SN PHM CA THT PHNG PHP PHT HIN VI RT DCH T LN C quan bin son: Trung tm kim dch ng vt xut nhp khu H Ni C quan ngh ban hnh: B Nng nghip v Cng nghip thc phm C quan trnh duyt: Tng cc Tiu chun o lng Cht lng C quan xt duyt ban hnh: U ban Khoa hc Nh nc Quyt nh ban hnh s: 736/Q ngy 31 thng 12 nm 1990

TCVN 5157-90
THT V SN PHM CA THT PHNG PHP PHT HIN VI RT DCH T LN Meat and meat products Detection of Swine fever virus Tiu chun ny quy nh phng php pht hin vi - rt dch t ln bng khng th globulin dch t ln gn hunh quang iso - thyocyanat (Conjugat khng th hunh quang). 1. c tnh chung Vi rt dch t ln gy bnh cho ln cc la tui. Bnh tin trin nhanh di th bi huyt, c thn nhit cao, triu chng v bnh tch chnh l xut huyt, hoi t v lit thn kinh. Vi rt sinh trng c trn t bo thn ln, nhng thng khng gy c nhng bin i trn t bo. 2. Nguyn tc Khi khng nguyn dch t ln gp conjuget khng th hunh quang th kt hp thnh phc hp khng nguyn - khng th hunh quang. Phc hp ny qua knh hin vi hunh quang pht ra nhng m sng mu sc ring bit. 3. Ly mu 3.1 Tiu chun ny dng xc nh bnh dch t ln cho mt l ln. 3.2 L ln l s lng ln c cng ni xut pht (tri, phng, x) c vn chuyn trong mt thi gian, khi nhp v c nui mt khu ring (cch ly tn o) khng tip xc vi cc mi gii truyn bnh khc. 3.3 Mu th l amydan, lch, hch lm ba va cht hoc va git m. Bng thao tc v khun ly v ng mu vo hp (bnh, l) thu tinh hoc cht do, c np y kn trong phch lnh, c cha vn trn ln vi mui n (NaCl) v chuyn nhanh v c quan kim dch trong vng 24-48 gi. 3.4. Mt na mu kim nghim, phn cn li lu. Mu lu c bo qun nhit ti thiu 15oC khng qu 1 thng. Ch thch: a/ C quan kim dch ch nhn mu th theo ng quy nh trn (3.3), km theo bn khai bo v l ln nghi bnh (ngun gc, s lng, trng lng, ngy nhn, ngy pht bnh, s ln m, cht, triu chng, bnh tch). b/ nh bnh c chnh xc th sau khi nhn c mu v bo co, c quan kim dch phi c ngay bc s th y n ni xc minh tnh hnh dch, kt hp tnh hnh ti ch vi kt qu phng xt nghim v nu cn c php mi nhng c quan chuyn trch (trong v ngoi nc) cng nh bnh. 4. Thit b v dng c 4.1 Thit b. 4.1.1. Phng cy v khun, trong c t cy tay (thanh trng phng cy v t cy bng n cc tm 1,5-2,5 ot/m3 trong 3 gi, sau phun phenol 5% hoc xng formon ri phun phenol 5%. 4.1.2. T m c iu chnh nhit n nh 35-37-43 1oC.

4.1.3 T sy kh. 4.1.4 T hp t. 4.1.5. Ni ct nc 2 ln bng thu tinh. 4.1.6. T lnh 0-4oC. 4.1.7. T lnh -35oC. 4.1.8. Bnh cha nit lng. 4.1.9. My ct lnh c bnh kh CO2. 4.1.10. Ni un cch thu c iu chnh nhit n nh t 35-56oC. 4.1.11. Ly tm thng thng, 5.000 vng/pht v ng ly tm y nhn. 4.1.12. My xay tht. 4.1.13. My khuy t. 4.1.14. Cn phn tch c chnh xc khng ln hn 0,01g. 4.1.15. Cn thin bnh roberval) c nh (50-100g). 4.1.16. Bung m bch huyt cu. 4.1.17. Knh hin vi c th knh x 10, x 15. 4.1.18. Knh hin vi hunh quang v cc linh kin. 4.1.19. My o pH c chnh xc hiu chnh l 0,1 n v pH 25oC. 4.1.20. Phu thu tinh ng knh 3cm. 4.1.21. Phu lc vi khun: Seitz, EKS2, Sephadex G25, 50. 4.1.22. Dao bistouri, ko, pince cc loi. 4.1.23. n cn, bng, gc, vi lc. 4.2 Dng c. 4.2.1. Bnh tam gic v bnh cu y bng c dung tch 50, 150, 250ml. 4.2.2. Cc ong c chn cc c. 4.2.3. Bnh ong hnh tr cc c. 4.2.4. Pipet Pasteur v pipet c khc 1,5-10ml. 4.2.5. a Petri ng knh 150mm, chiu cao 15mm. 4.2.6. ng Leighton nui cy t bo c km l knh. 4.2.7. ng nghim 18 x 150mm. 5. Ho cht, thuc nhum, mi trng t bo thn ln. 5.1. Nguyn liu c bn: Ho cht dng trong mi trng pha ch dung dch phi s dng loi tinh khit A. R.; nc ct 2 ln trung tnh qua kim nghim khng c tp cht nh hng n t bo v vi rt sinh trng. Nn s dng nhng thnh phn c bn hoc thnh phn ch sn, nhng khi dng phi tun theo ch dn ca ni sn xut. 5.2. Ho cht, thuc nhum, mi trng t bo (ph lc). 5.2.1. Ra tay v nhum tiu bn. 5.2.1.1.Khng huyt thanh dch t ln ( nhum trc tip v gin tip). 5.2.1.2. Conjugat khng th hunh quang (nhum gin tip). 5.2.1.3.Conjugat khng th hunh quang (nhum gin tip). 5.2.1.4. Virt dch t ln cng c tiu chun. 5.2.1.5. Dung dch m mui phosphate pH 7,0. 5.2.1.6. Dung dch xanh evans. 5.2.1.7. Dung dch m Tris pH 8,6. 5.2.1.8. Axton. 5.2.2. Ch mi trng v x l mu th trc khi cy.

5.2.2.1. Clorofooc. 5.2.2.2. Natrihydro cacbonat, dung dch 1,4% v 7% (NaHCO3). 5.2.2.3. Dung dch phenol (phenol red). 5.2.2.4. Dung dch khng khun t Penicillin v Streptomycin. 5.2.2.5. Dung dch mun in gii Hanks. 5.2.2.6. Dung dch trypsin. 5.2.2.7. Dung dch hydro-lysat-lactabunin. 5.2.2.8. Dung dch Hanks x l mu th v ra t bo. 5.2.2.9. Dung dch Versen. 5.2.2.10. Huyt thanh b s sinh. 5.2.3. Mi trng t bo. 5.2.3.1. Mi trng Eagle cn thit cho t bo sinh trng (minimum essential medium Eagle-growth; MEM-G). 5.2.3.2. Mi trng Eagle cn thit bo qun t bo (minium essential medium Eagle- mainterance; MEM-M). 5.2.4. K thut sn xut t bo thn ln. 6. Cch tin hnh. 6.1. Tu theo tnh hnh v iu kin ca tng c s m tin hnh trc sau hoc cng mt lc c hai phng php: Nhum trc tip v gin tip. 6.2. Phng php nhum trc tip mu th ct lnh hoc p in. 6.2.1. Chun b mu th - Dng my ct lnh, ct mu th thnh nhng lt ct c dy t 5-6 micromt. t nh v dn u lt ct ln 2 phin knh A v B bng mi pince u nhn (mi phin knh t t 2-3 lt) hoc p in mu th thnh cc vt mng (thay cho lt ct). Hong kh tiu bn v c nh bng axton -25oC trong 60 pht. 6.2.2. Nhum. 6.2.2.1. Phin knh A ( xc nh virt dch t ln) t trn lp bng thm nc trong a petri, nh 0,20,3ml conjugat khng th hunh quang (thuc nhum trc tip) ln tiu bn, 37oC trong 30 pht. Ly ra ngm ra 3 ln, mi ln 10 pht bng dung dch in mui photphat pH 7,0; trng nc ct, hong kh c kt qu. 6.2.2.2. Phin knh B (i chng c ch c hiu) t trn lp bng thm nc trong a Petri, nh 0,20,3ml khng huyt thanh dch t ln ln tiu bn, 37oC trong 15 pht. Ly ra ra nc ct ri nh tip ln tiu bn 0,2-0,3ml conjugat khng th hunh quang (thuc nhum trc tip) v lm tip theo 5.3.2.1. 6.3.1. Chun b mu th: Cn 10g mu th (l lch), dng ko ct nh, nghin trc trong ci s, nghin tip trong my nghin, va nghin va ho long dung dch Hanksoc Penicillin v Streptomycin thnh huyn dch 1:5. Ly tm 2000 vng/pht trong 15 pht. Ly lng nc trong pha long tip bng dung dch Hanks -1 thnh huyn dch 10 ng thi lm thm mu th cng dch t ln i chng. 6.3.2. Cy mu th vo mi trng t bo thn ln. 6.3.2.1. T bo thn ln nui cy v ph kn trn mt l knh trong ng Leighton cha 2ml mi trng MEM-G sau 48-72 gi. Mi trng mu th phi nui cy t nht trong 2 ng mi trng, km theo 3 ng i chng cho mi t kim nghim. Cc ng mi trng c k hiu ring: A. xc nh virt dch t ln trong mu th. B. i chng c ch c hiu. C. i chng dng tnh. D. i chng m tnh (kim tra t bo thn ln). 6.3.2.2. Gn b lng mi trng c i v ra lp t bo bng mi trng MEM-M t 2-3 ln, cy vo ng A, B mi ng 0,2-0,3ml huyn dch mu th 10-1 vo ng C 0,2-0,3ml huyn dch virt dch t ln 10-1 vo ng D 0,2-0,3ml mi trng MEM-M, tt c 37oC t

1-2 gi. Gn b lng nc va cy v ra lp t bo bng mi trng MEM-M 37oC t 24-48 gi. 6.3.3. Nhum: Dng pince ly l knh, c nh bng axton -25oC trong 10 pht, hong kh. - Nhum cc l knh A, C, D theo 6.2.2.1. - Nhum l knh B theo 6.2.2.2. 6.4. Phng php nhum gin tip mu th (chung cho c 2 loi tiu bn). 6.4.1. Chun b mu th: Theo 6.2.1. (mu th ct lnh hoc p in); 6.3.1. v 6.3.2 (mu th cy trong mi trng t bo). 6.4.2. Nhum chung cho c hai loi tiu bn) c nh tiu bn bng axton -25oC trong 10 pht, hong kh, nh ln tiu bn 0,2-0,3ml khng huyt thanh dch t ln, 37oC trong 30 pht. Ly ra ngm ra 2 ln, mi ln 5 pht bng dung dch in mui photphat pH 7,0. Nh tip 0,2-0,3ml conjugat khng th hunh quang (loi thuc nhum gin tip pha long theo ch dn ca ni sn xut); 37oC trong 30 pht, sau ngm ra 2 ln mi ln 3 pht bng dung dch in mui photphat pH 7,0. Gn lam knh ln trn tiu bn, c kt qu. 6.5. Nhn nh mt s ch tiu gia 2 phng php nhum. Ch tiu Nhum trc tip Ct lnh in p Thi gian (gi) T l pht hin dng tnh (%) 7. Tnh ton kt qu 7.1. Dng knh hin vi hunh quang t trong bung ti, c kt qu cc mu i chng trc, mu kim nghim sau. Chung cho c 2 phng php: - Trn mu D (kim tra t bo) khng c hoc c rt t nhng im pht quang gi mu xanh nu. - Trn mu C (i chng dng tnh): C nhiu t bo hoc m t bo pht quang mu vng xanh, tri u trong nguyn cht, chung quanh t bo c mu sng, lng pht quang t l thun vi s lng virt, ring phng php nhum gin tip cho kt qu r hn (sng trn nn xanh Evans). - Trn mu B (i chng c ch c hiu) nh mu D. - Trn mu A: Nu kt qu c c nh mu C l dng tnh v ngc li l m tnh. 7.2. Kt lun chung cho c hai phng php. Mu th A + + Mu th B + Mu th C + + + Mu th D Kt lun C virt dch t ln Sai st k thut (lm li) Khng c virt dch t ln 2-3 80 Mi trng t bo 50-75 85 Nhum gin tip Ct lnh in p 2-3 90 Mi trng t bo 50-75 95

PH LC
Ho cht, thuc nhum, mi trng t bo thn ln. A. Dng ra tiu bn v pha long conjugat khng th hunh quang (nhum gin tip). A1. Dung dch m mui photphat pH 7,0. A1.1. Dung dch 1 - Natri dihydrophotphat (NaH2PO4) - Nc ct 2 ln (trung tnh) A1.2. Dung dch 2. - Natri dihydrophotphat (NaH2PO4.12H2O) - Nc ct hai ln (trung tnh) 35,85g 1.000ml 14g 1.000ml

A1.3 Dung dch hon chnh - Dung dch 1 - Dung dch 2 - Natri clorua (NaCl) - Nc ct 2 ln (trung tnh) Hp 121 C trong 30 pht. Bo qun 4 C. A2. Dung dch xanh evans. - Xanh evans - Nc ct 2 ln (trung tnh) Hp 113oC trong 10 - 15 pht. Bo qun 4oC. A3. Dung dch m Tris pH 8,6. - Tris, dung dch 2,42% trong nc - Axit clohydric, dung dch 0,2N (HCl) - Nc ct 2 ln (trung tnh) va Lc v khun. Bo qun 4oC. Ghi ch: Axit clohydric, dung dch 0,2N (HCl). - Axit clohydric m c - Nc ct 2 ln (trung tnh) Hp 121 C trng 15 pht. B. Dng ch mi trng v x l mu th trc khi cy. B1. Natri hydro cacbonat, dung dch 1,4% hoc 7% (NaHCO3). - Natri hydro cacbonat - Nc ct 2 ln (trung tnh) Lc Seitz. Bo qun 4oC. B2. Dung dch phenol (Phenol red). - phenol - Natri hydroxit, dung dch 0,1N trong nc (khong) - Nc ct 2 ln (trung tnh) va 0,4g 10-12ml 100ml 1,4g hoc 7g 100ml.
o o o

39ml 61ml 8,77g 900ml

0,25g 100ml

25ml 6,1ml 100ml

2ml 98ml

Nghin phenol trong ci s, nh dn dung dch Natri hydroxit vo cho n khi phenol tan hon ton, sau b sung nc ct hai ln va 100ml. Hp 121oC trong 30 pht (nu cn th chnh pH 7,0 bng Natri hydroxit dung dch 0,05N trc khi hp. Ghi ch: Natri hydroxit dung dch 0,1N hoc 0,05N. - Natri hydroxit - Nc ct 2 ln (trung tnh) - Penicillin (bt tim) - Streptomycin (bt tim) - Nc ct 2 ln (trung tnh) Bo qun -20 C. B4. Dung dch mui in gii Henks B4.1. Dung dch 1 - Natri clorua (NaCl) - Kali clorua (KCl) - Magi sulphat (MgSO4.7H2O) 160g 8g 4g
o

2g hoc 1g 100ml 2.000.000 n v. 2.000.000 n v. 100ml

B3. Khng khun t, dung dch c 20.000 n v Penicillin v Streptomycin trong 1ml nc.

- Canxi clorua (CaCl2) - Nc ct 2 ln (trung tnh) Cho thm clorofooc 2ml. Bo qun 4oC. B4.2. Dung dch 2. - Natri hydro photphat (NaH2PO4.12H2O) - Katri dihydro photphat (KH2PO4) - Clucose - Dung dch phenol (B2) - Nc ct 2 ln (trung tnh) Cho thm clorofooc 2ml. Bo qun 4 C. B4.3. Dung dch hon chnh. - Dung dch 1 - Dung dch 2 - Nc ct 2 ln (trung tnh) Hp 115 C trong 10 pht. Bo qun 4 C.
o o o

2,8g 1.000ml

3,04g 1,00g 20g 100ml 1.000ml 50ml 50ml 900ml

Khi dng cho thm Natri hydro cacbonat, dung dch 1,4% (B1): 2,5ml. B5. Dung dch Trypsin. - Trypsin - Dung dch Hanks (B4,3)
o

1g 100ml

Lc nh cho tan u. 4 C t 12-18 gi, tip trong ni chng cch thu 37oC trong 3 pht cho trypsin tan hon ton. Lc v khun bng phu thu tinh xp. Cho thm dung dch khng khun t (B3) 1ml. Chia vo cc l nh. Bo qun -20oC. Khi dng pha long tip thnh m 0,25%. B6. Dung dch hydrolysat lactabumin. - Hydrolysat lactabumin - Dung dch Hanks (B4.3) Hp 121oC trong 10 pht. Bo qun 4oC. B7. Dung dch Hanks x l, pha long mu th v ra t bo. - Dung dch Hanks (B4.3) - Dung dch khng khun t (B3) + x l mu th: + pha long mu th (10 ) v ra t bo B8. Dung dch m Versen; - Natri clorua - Kali clorua - Natri hydro photphat (NaHPO4.2H2O) - Versen - phenol - Nc ct 2 ln (trung tnh) Ho tan. Lc v khun. Bo qun 4 C. C. Mi trng nui t bo. C1. Mi trng Eagle cn thit cho cc t bo sinh trng (MEM-G). - Dung dch hydrolysat lactabumin (B6) - Dung dch khng khun t Penicillin v Streptomycin (B3) iu chnh pH 7,2 bng Natri hydro cacbonat, dung dch 7% (B1). - Huyt thnh b s sinh 10ml. 90ml. 1ml.
o -1

5g 1.000ml

100ml 2ml 1ml 8g 0,2g 0,41g 0,2g 0,01g 1.000ml

Bng thao tc v khun trn u, chia vo cc bnh tam gic 50ml. Dng nui cy t bo thn ln. 02. Mi trng Bagle cn thit bo qun t bo (MEM-M). Thnh phn nh C1, ch khc l dng 1ml huyt thanh b s sinh thay cho 10ml. Dng cy mu th (phn lp virt dch t ln). D. K thut sn xut t bo thn ln. D1. Chn t bo: Dng t bo thn ln s sinh, kho mnh, khng c bnh. D2. Tch t bo bng dung dch Trypsin (B5) di tc ng ca my khuy t. D3. Loi b Trypsin v thu gom t bo bng my ly tm. D4. Pha mu bng MEM-G. m s t bo bng bung m bch huyt cu di knh hin vi thng thng. D5. Tnh s lng t bo cn thit nh t l pha long bng MEM-G (yu cu 50.000 t bo/1ml mi trng). D6. Chia vo cc ng leighton c ghp l knh, mi ng 2ml, 37oC t 24-72 gi. Khi t bo sinh trng v ph kn trn b mt l knh th cy mu th (theo 6.3.2). Ghi ch: Nu s dng t bo dng th pha ch theo ch dn ca ni sn xut, nu chuyn tip t bo dng th dng dung dch versen (B8) lm bong lp t bo ri dng dng dch trypsin (B5) tch t bo v lm tip theo ph lc D2 n D6.