BO NaNG NGHIBpvA pHAT TRlEN NaNG THaN

CVC THU Y

S6: 529 IQD-TY-IS

CONG HoA xX HOI CHU NGHiA VIET NAM. . .DQc l~p - Tl}'do - H~nh plnic

Ha N(Ji, ngay ~3 thang 9 ndm 2017

QUYETD!NHBan hanh Tieu chu§n co so

Can cir Luat Tieu chuan va Quy ehu~n ky thuat ngay 29/6/2006;Can cir Nghi dinh s6 127/2007/NI)-CP ngay 01/8/2007 cua Chinh phu quy

dinh chi tiSt thi hanh mot s6 diSu cua Luat tieu chuan va quy chuan ky thuat;Can cir Thong tu s6 2112007/TT-BKHCN ngay 28/9/2007 cua B9 tnrong

BQKhoa hoc va Cong nghe vS viec huang d~n xay dung va ap dung tieu chuan;Can cir Quyet dinh s6 1399/QD-BNN-TCCB ngay 13/4/2014 cua BQ

tnrong BQNong nghiep va Phat trien nong thon quy dinh chirc nang, nhiem vu,quyen han va co efrut6 chirc cua Cue Thu y;

Theo dS nghi cua Truong phong Thu y Thuy san,

QUYETDJNH:

Di~u 1. Ban hanh Tieu chuan co s6 xet nghi~m phat hi~n Tilapia lake vi ruttren ea ro phi b~ng ky thu~t Semi-nested Reverse Transeriptase PCR (Semi-nestedRTPCR).

Di~u 2. D6i tm;mgva ph~m vi ap dvng:1. Ap dVng ~i tfrt ea cae phong thli nghi~m eua cae dan vi tf\l'e thuQe Cve

Thuy.2. Chi e\le Thu y/Chi eve Chan nuoi va Thu y, cae dan vi lien quan can eu

diSu ki~n phong thli nghi~m eua dan vi tham khao, ap dVng Tieu ehu~n co s6 nayeho phli hqp, hi~u qua.

Di~u 3. QuySt dinh nay co hi~u h.rethi hanh kS til' ngay kY.Di~u 4. TruOng phong Thu y Thuy san va Thu truOng cae don vi lien quan

ehiu traeh nhi~m thi hanh quySt dinh nay.!.NO'i nhan:- Nhu DiSu 4;- B<)tmang NguySn Xuan Cuemg (dS hie);- Thu twang VGViin Tam (d~ hie);- Cl,letmc.l11g(d~ hie);- T6ng el,leThuy san;- CCTY, CN&TY cae tinh, thimh ph6;- Luu VT, TS.

TIEU CHuAN co saTCCS

,.. ;: ?

TIEU CHUANco sa

TCCS 05:2017/TY-TS

QUY TRINH XET NGHI¥M PHAT HI¥N TILAPIA LAKE VI RUTTREN cA RO PHI BANG KY THU~T SEMI-NESTED REVERSE

TRANSCRIPTASE-PCR (SEMI-NESTED RT PCR)

Ha NQi, ngay 13/9/2017

Ml)C Ll)C

, , ..LaI NOI DAD 2.. , ,PHAN KHAI QUAT 3

1. TEN GOI 3

2. PRAM VI Ap D1)NG 3

3. GIGI THI$U 3.. - -PHAN KY THU ~ T ' 4

1.TIEUCHUANKYTRUAT 4

2. M1)C BICR 4

3.NOI DUNG 4

3.1. Nguyen t&c 4

3.2. Nguyen lieu hoa h9Cva sinh h9C .4

3.2.1. Nguyen lieu hoa h9C 4

3.2.2. Nguyen lieu sinh h9C 5

3.3. Thiit bi va dung C1:l 5

3.4. Cach tiin hanh 63.4.1. Chudn bi mdu 6

3.4.2. Chiit tach RNA 6

3.4.3. Thuc hien ph an ung Semi-nested RT PCR 7

3.4.4. Dien di 7

3.4.5. D9C kit qua 8,- ?

TAl LI~U TRAM KHAO 10z ,

PHV LVC A: CHIET TACH RNA e ••••••••••••••••••••••••••••••••••••••••••••••• 11

PHU LUC B: TIEU TON VAT TU CHO MOT MAU xET NGHIEM 14. . . . .

1

LiJINOIDAu

TCCS .0.5.. :20 17ITY -TS phat hien Tilapia lake vi rut tren ca ro phi bangkY thuat Semi-nested Reverse Transcriptase PCR (Semi-nested RT PCR) do C\lCThu y bien soan va ban hanh, Tieu chuan nay duoc ap dung tai tftt ca cac phongthir nghiem cua cac don vi true thuoc C\lCThu y. Cac phong thir nghiem benhthuy san khac co thS tham khao dS ap dung cho phu hQ'P,hieu qua.

Co quan bien soan: C\lCThu y.

Quyet dinh ban hanh s6 529 ngay.{3 thang 9 nam 2017 cua C\lCtnrongC\lCThu y.

2

PHAN KHAI QUAT

1. Ten gQiTCCS 05:2017/TY-TS xet nghiem phat hien Tilapia lake vi rut tren ca ro

phi bang ky thuat Semi-nested RT PCR.

2. Pham vi ap dung

Tieu chuan nay ap dung d@xet nghiem phat hien Tilapia lake vi rut tren caro phi b~ng ky thuat Semi-nested RT PCR tai cac phong thir nghiem cua dan vitrue thuoc Cue Thu y.

3. Gi6'i thi~uTilapia lake vi rut (goi t~t la TiLV), la mot vi rut moi, RNA soi dan am,

nrong d6ng voi Orthomyxovirus. Vi rut TiLV gay benh & ca ro nuoi bao g6m caro phi v~n (0. niloticus), ca ro phi lai tao (0. niloticus x 0. aureus), ca ro phidol ca dieu hong (Oreochromis sp). Cac loai ca ro phi hoang da:nhu ca ro phixanh (0. aureus), ca ro phi Mango (Sarotherodon galilaeus), Tilapia zilli vaTristamellasimonis intermedia cling m~n cam voi vi rut nay.

Ty l~ chct trong cac 6 dich tir 09-90%.B~nh gay ty l~ chet cao trong danca nuoi, d~e bi~t & ca nho. Ca m~c b~nh co bi@uhi~n chan an, mau s~c co th@bi~n d6i (s~m mau); thay d6i t~p tinh boi lQi(nhu t~p trung & tren bS m~t, boi 10do), ngirng keo dan, hon me truac khi eh~t. Da ca bi an mon 1&106t tiI d?ngdi@md~n mang; mang tai nhqt; m~t bi teo l?i ho~c 16i ra, co hi~n tUQ'llgdvc thuytinh th@;v~y d\fllg, co th@bong troc; duoi bi an mon (Dong va cs, 2017a; Fathiva es 2017).

Phuong phap Semi-nested RT PCR duQ'cap dvng d\l'a tren k~t qua nghienctm eua Dong va es (2017a,b).

3

PHAN KY THUAT.1. Tieu chuan kY thu~t

TCCS xet nghiem, phat hien TiLY tren ca ro phi bang kY thuat Semi­nested RT PCR.

2. Ml}.cdichPhat hien virut Tilapia lake (TiLY) tren ca ro phi bang ky thuat Semi­

nested RT PCR nham phuc vu cong tac chan doan xet nghiem va phong chongdich benh tren ca ro phi.

3. Nqi dung

3.1. Nguyen t~c

TiLY la mot vi rut moi co acid nhan la RNA soi dan am, co 10 doangene. Vi rut duoc phat hien bang ky thuat Semi-nested RT PCR co san phamPCR thuoc gene dich la doan gene s6 3 (segment 3).

3.2. Nguyen Ii~u hoa hQCva sinh hoc

3.2.1. Nguyen Ii~u hoa hoc

- Dung dich Phosphate Buffered Saline (PBS):

Thanh phan S6 hrong (gram)

NaCI

KCl

Na2HP04

KH2P04

8,0

0,2

1,15

0,2

Luong hoa ch~t tren duoc cho vao binh thuy tinh, cho mroc c~t 2 l§.nvaovira dung 1 lit, khuay tan hoan toan, dieu chinh pH 7,2 ± 0,2 va dern h~p votrung (Autoclave) 121°C, 15 phut,

- C6n (Ethanol) 90% va 70%;

- Thach Agarose ;

- Nuoc TBE IX;

- Loading dye 6X (Thermo Cat .No: R0611);

- Ladder (Thang chuan DNA) (Thermo Cat. No: SM0242);

- Sybr safe (Invitrogen Cat. No: S33102);

- Kit chiSt tach InviMAG®Yirus DNA/RNA Mini KitIKF96, Cat. No:7441050100 ho?ckit Invitrogen PureLink Viral RNAIDNA Mini Kit, Cat. No:12280-050 ho?c Kit TACO RNA/DNA Extraction ho?c kit tuang duang;

- Kit nhan gen SuperScript® III One-Step RT-PCR with Platinum®Taq.Catalogue Number: 12574-026 ho?c kit tuang duang;

4

- Kit Thermo Scienetific Dream Taq PCR Master Mix (2X), Cat. No: K1071 hoac kit tuong duong.

3.2.2. Nguyen li~u sinh hoc- Doan mBi chophiin tmg Semi-nested RT peRBuoc 1RT-PCR :

Ten m6i Trinh tir Kich thiroc san pham

Nested ext-1 5'TATGCAGTACTTTCCCTGCC3'(m6i xuoi) 415 bp

ME 1, 5'GTTGGGCACAAGGCATCCTA3'(moi nguoc)

Buoc 2 nested PCR:T" A. Trinh tv Kich thuoc san phamen mot

ME2 5'TATCACGTGCGTACTCGTTCAGT3'(m6i xuoi) 250bp

MEl(mdi nguoc) 5'GTTGGGCACAAGGCATCCTA3'

- H~ thong d6i chirng:+ D6i clnrng am la mftu mroc khong chua enzyme pha huy RNA, DNA

dung dS pha loang cac ch~t phan irng.+Mftu d6i clnrng dirong la mftu co chua RNA cua TilV duoc chiet tach tir

mftu duong ehucln.Mftu dirong chuan la mftu ca thuc dia co biSu hien lam sangva benh tich d~c tnrng ella benh do TiLV gay ra. D6ng thai, trinh tu gene ellasan pham PCR va segment 3 d6ng nh~t voi trinh tir gen cua segment 3 cua cacTiLV trong gene bank.

Co thS sir dung mftu duong chuan la plasmid (pGEM-415_bp) chua doangene dich (415 bp san pham PCR cua biroc 1) diroc Trung tam Sinh hoc phan tuva cong ngh~ sinh hQc tom, Truang D~i hQcMahitol Thai Lan cung e~p (Dongva cs 2017).

3.3. Thi~t hi va dl]ng Cl]

- May trQnmftu (Vortex);

- May ly tam nh6 (Centrifuge Spin);

- May l~c va IInhi~t HLC-MHR23 ho~e tuang duang;- May ehiSt tach RNA/DNA t\1"dQng Thermo Scientifie™ KingFisher™

Flex; Thermo Scientifie™ KingFisher™ mL ho~c may chiSt tach RNAIDNA t\1"dQngTACO ho~c tuang duang;

c ::.' May ly' tam l~nh cho 6ng eppendorf 1,5 ml MIKRO 200R ho~c tuangduang;

- May h~p khu trung Autoclave;

5

- MayPCR;

- BS dien di;

- May d9C gel dien di;

- Tu an toan sinh h9C c~p II;

- Tu mat va am sau: + 4°C; -20°C va _SO°C;

- Micropipettes va dftu tip co mang 19Ctuong irng l~y duoc cac thS tich:0,5 - 10 Ill; 10 -100 III va 100 - lOOOIlI;

- Cac rna nhua 96 gieng hoac 6ng nhua chua dung dich tach chiet, phuhop voi may chiet tach RNAIDNA tv dong;

- Ong PCR 200 IIIhoac ilia PCR 200 III96 gieng;

- Ong eppendorf the tich 0,2 ml va 1,5 ml;

- Ong ly tam nhua thS tich 5 ml, 10 ml va 50 ml;

- But viet, d6ng h6 chuyen dung trong phong thi nghiern va cac dung cucftn thiet khac,

3.4. Cach ti~n hanh3.4.1. Chuan bi m§u.Thu m~u ca co d~u hieu benh Iy, con song hoac vira moi chet- V6i ca nho (~u trung, ca bot, ca giong) thu nguyen con, tuy thea kich cO'

ca, co thS thu tir 10-30 con/ao, bS; ca giong nho, co thS gop 05 con thanh motm~u xet nghi~m.

- V6i ca co kich thu6c 16n (ca truang thanh, ca b6 m((), co thS thu nguyencon ho?c gan, th~n, nao va mango

Lo~i m~u: duQ'c su d\lng dS xet nghi~m v6i quy trinh nay co thS la m~utuai ho?c mftu duQ'cc6 dinh trong c6n 90% thea ti l~ mftu:c6n (1: 10).

Dung panh, keo vo trung dS thvc hi~n cac thao tac: tach, c~t l~y mftu. MftuduQ'cchia thfmh 2 phftn: 1 phftn cho thvc hi~n xet nghi~m va 1phftn luu tru.

Mftu duQ'c nghiSn nhuySn v6i ty l~ 1 thS tich mftu trong 9 thS tich dungdjch mU6i d~m PBS (Phosphate Buffered Saline), dS t~o thanh huySn djch 10%(su d\lng can phan tich can tr9ng luqng mftu duQ'c nghiSn dS diSu chinh luqngdung djch PBS nh~m dam bao t~o duQ'chuySn djch 10%). Thu h6i huySn djch10% van 6ng n~p v?n vo trung, r6i chuySn van tu am sau (-SOoC)trong thai giant6i thiSu 1 gia tru6c khi tiSn hanh chiSt tach DNA.

3.4.2. Chi~t tach RNA

Quy trinh chiSt tach RNA duQ'cthvc hi~n thea huang d~n cua Cong ty sanxu~t kit chiSt tach InviMAG®Yirus DNAIRNA Mini Kit/ KF96 ho~c TacoRNA/DNA kit ho?c Invitrogen PureLink Viral RNA/DNA Mini kit (Ph\l l\lcA).

JI

6

Hoac co thS sir dung cac loai kit chiet tach khac tuong dirong, phil hQ'Pcho chiSt tach RNA.

3.4.3. Thuc hi~n phan tmg Semi-nested RT peRKy thuat Semi-nested RT PCR bao g6m 02 giai doan (hoac 2 buoc): Giai

doan 1 (biroc 1), thuc hien phan irng RT-PCR Slr dung C?P moi Nested-extl vaMEl; Giai doan 2 (buoc 2), thuc hien phan irng nested PCR Slr dung C?P m6iMEl va ME2.Chu~n bi Master Mix va chu ky luan nhiet diroc thuc hien theaBfmg 1.

Bang l: Chuan bi MasterMix cho phan irng RT-PCR theo kit SuperScript" III One­Step RT-PCR with Platinum" Taq. Cat: 12574-026 va Master Mix cho phan irngnested-PCR thea kit Thermo Scienetific Dream Taq PCR Master Mix (2X), Cat. No:K 1071

Burrc Thanh phan Chu trinh nhi~tLuong(ul)

Niroc kh6ng c6RNAIDNA

2X Reaction Mix

M6i Nested - ext 1 (10

RT-PCR IlM)M6i ME 1 (10 IlM)

Enzyme Taq Mix

MfruRNA

T6ng th~ tich

Nuoc kh6ng c6RNAIDNA

Master Mix 2X

Nested PCRM6i ME 1 (10 IlM)

M6i ME 2 (10 IlM)

San pham RT-PCR

T6ng th~ tich

8.5 50°C - 30 phut12.5 94oC- 2 phut1 25 vong cua:

1 • 94°C - 30 giay

0.5 • 60°C - 30 giay

1.5 • 68°C - 30 giay25 lvong:

• 68oC- 5 phut

8 94°C - 2 phut

10 25 vong cua:

0.5 • 94°C - 30 giay

0.5 • 60°C - 30 giay

1 • 72°C - 30 giay20 lvong:

• 72oC- 5 phut

8?t 6ng PCR hoac dia peR 96 gieng chua thanh phan phan irng Semi­nested RT PCR (Master Mix) va mfru vao may PCR. V~n hanh may thea huangdfrn va chQn chuong trinh luan nhi~t da:duqc cai d?t.

3.4.4. Dien di.3.4.4.1. Chu~n bi ban gel

Pha th?ch vai n6ng dQ agarose tu l,5 % dSn 2 % b~ng dung dich d~mTBE IX ho?c TAE IX vao binh thuy tinh 250 mI, l~c dSu r6i dun soi. Khi nhi~t

7

dQ giam xuong khoang 40°C dSn 50°C thi bE>sung 10 III chat nhuorn mauDNA (SYBR green,GelRed hoac GelGreen ...) vao m6i 100 ml thach. L~c nhedS ch~t nhuom mau tan deu va tranh tao bot,

Chuan bi khuon db thach, d~t luoc vao khuon rbi db thach vao khuon,khong nen db ban thach day qua 0,8 cm. Khi ban thach dong lai thi tiSn hanh goluoc khoi ban thach, Chuyen ban thach vao bS chay dien di , dE>dung dich d~mcung loai voi dung dich pha thach agarose da dun vao bS dien di cho toi khingap ban thach,

CHU THICH: C6 thS dung cac san pham c6 s£n ch~t nhuom ADN dS pha ch~ thachagarose (vi du: Sybr safe ADN gel stain') va sir dung thea quy dinh cua nha san xuat,

3.4.4.2. Chay dien di

Hut 2 /Jl chat dem tai m&u(Loading dye 6X) vao 8 ul san pham PCR trondSu va cho vao cac giSng tren ban th~ch.

Thvc hi~n di~n di trong bS di~n di, ch~y kern thea thang chufrn ADN(Ladder) dS dV doan kich thuac san phfrm khuSch d~i. Hut 10 /Jl thang chufrnADN vao mQt giSng tren ban th~ch.

8i~n di a hi~u di~n thS 100 V trong thai gian 20 - 30 phUt.

3.4.5. DQc k~tqua

- KSt qua m&uxet nghi~m am tinh khi:

+ T~i giSng cua m&uthu khong xu~t hi~n v~ch sang (khong co san phfrmkhuSch d~i).

+ 86i chung am co kSt qua am tinh (khong co san phfrmkhuSch d~i)

+ 86i chUng duong co kSt qua duong tinh dugc thS hi~n b~ng v~t sang r5co kich thuac 415 bp va 250 bp so vai thang chufrn. (Co thS co v~ch san phAmphv, khong d~c hi~u, kich thuac khoang 800 bp ho~c 300 bp)

- KSt qua m&uxet nghi~m duong tinh khi:

+ T~i giSng cua m&uthu co san phfrm khuSch d~i co kich thuac 415 bp va250 bp va co thS co v~ch san phfrm phv kich thuac khoang 800 bp ho~c 300 bp;ho~c chi co san phfrm khuSch d~i co kich thuac 250 bp. Trang truimg h9'Pm6uduang tinh yiu, co thi kh6ng phdt hi¢n iJu9'escmphdm eua bUGe1RT peR (415bpj.

+ 86i chung am co kSt qua am tinh (khong co san phfrmkhuSch d~i).

+ 86i chung duong co kSt qua duong tinh dugc thS hi~n b~ng v~ch sangr5 co kich thuac 415 bp va 250 bp so vai thang chufrn. (Co thS co v~ch sanphAmphv kich thuac khoang 800 bp ho~c 300 bp).

Giai thich kSt qua sau khi d9Cdi~n di thea bang sau:

1)Thong tin nay dua ra ~o di€u kien thu~ tien eho ngum Slr d\lng tieu ehu§n va khong dn dinh Slr d\lng san phfun nay.C6 the Slr

d\lng cae san phfun tuang tJ,r neu cho cae ket qua tuang duang.

8

TrU'O'ng 415 bp 250 bp K~t quahop

1 - + Mftu ~ism nhe TiLV (+)

2 + + Mftu ~ism nang TiLV (++)

3 - - Mftu ~m tinh

Bang 2 : Giai thich k~t qua di~n di Semi i nested RT peR

9

TAl LI~U THAM KHAo

1. Emergence of tilapia lake virus in Thailand and an alternative semi­nested RT-PCR for detection. (2017a). H.T. Dong, S. Siriroob, W. Meemetta ,W. Santimanawong , W. Gangnonngiw, N. Piraratd , P. Khunraea , T.Rattanarojponga R. Vanichviriyakitb , S. Senapin.Aquaculture. (476),111-118.

2. A warning and an improved PCR detection method for tilapia lakevirus (TiLV) disease in Thai tilapia farms. (2017b) HT Dong, , S Siriroob , WMeemetta, W Santimanawong , W Gangnonngiw, N Piraratd , P Khunraea , TRattanarojponga, R Vanichviriyakitb, S Senapin.https:llenaca.org/enclosure.php?id=858

3. Tilapia lake virus (TiLV): Literature review. (2017). Mona DverdalJansen and Chadag Vishnumurthy Mohan. CGIAR Research Program on FishAgri-Food Systems. Working Paper: FISH-2017-04.(https:llenaca.org/enclosure.php?id=914).

4. Fathi M, Dickson C, Dickson M, Leschen W, Baily J, Muir F, Ulrich Kand Weidmann M. 2017. Identification of tilapia lake virus in Egypt in Niletilapia affected by 'summer mortality' syndrome.Aquaculture 473:430-32.

10

PHU LUC A: CHIET TAcH RNA. .

I. Tach chi~t RNA b~ng b{}kit InviMAG®Virus DNAIRNA Mini KitlKF96

a. ChuAn b] hoa ch§tCac hoa chat c~n duoc chu~n bi va bao quan thea dung hirong d~n cua bQ

kit InviMAG®Yirus DNAIRNA Mini Kitl KF96, Cat. No: 74410S0100, voi thStich vira du cho so hrong m~u chiet tach, bao g6m:

(1) Dung dich d~m Lysis Buffer RY(2) Dung dich nra 1 (Wash 1)(3) Dung dich nra 2 (Wash 2)(4) H6n hop hat tir tinh (Bead Mix)(S) Dung dich thu h6i DNA/RNA Elution Buffer (EB)

NSu chist tach RNA bang may chiSt tach tir dQng Thermo Scientific™KingFisher™ Flex, c~n phai chuan bi tnroc cac dia chua dung dich chiet tachthea h~ thong cua may nhu sau:

+BTaWashing 1: 800jll dung dich Wash l/gieng;+ BTa Washing Wash 2 va Washing Wash 3: 800jll dung dich Wash

2/giSng;+BTaElution Buffer: l Ouul dung dich EB/giSng.b. Ti~n hanh- Huyen dich 10% cua m~u benh pham duoc ra dong, tron d~u b~ng may

vortex. Ly tam m~u trong may ly tam lanh voi hrc ly tam 2S00 g/lS phut,- Hut 200jll dich trong ben tren sau khi ly tam vao 6ng eppendorf I.Sml

vo hung co chua s~n 200 JlI dung dich d~m Lysis. Tron d~u m~u b~ng mayvortex va spin dS keo cac ph~n bam tren n~p 6ng xu6ng.

- ChuySn toan bQ dung dich trong 6ng eppendorf VilOrna chua m~u 96giSng (m6i giSng tuang trng vai mQtm~u chist tach). Dung gifty dan rna chuyend\lng dan kin m?t rna.

- B?t rna len may l~c, u nhi~t (HLC-MHR23). L~c ma vai gia t6c 7S0rpm/IS phut, nhi~t dQ6SoC.

- Lfty rna ra kh6i may l~c, dS nguQi trong S phut, thao b6 gifty dan ma. B6sung 420 jll dung dich Bead Mix (g6m co 400 jll Binding solution va 20 JlIMAP) VaGm6i giSng.

- ChuySn tftt ca cac rna bao g6m: rna Tip Combs, rna Elution Buffer, rnaWashing Wash 3, ma Washing Wash 2, ma Washing Wash 1 va ma chua m~uVaGtimg khay cua may chiSt tach tll dQngthea hu6ng d~n cua may.

- ChQn chuang trinh chist tach RNA da dugc cai d?t truac do thea hu6ngd~n cua hang san xuftt kit InviMAG®Yirus DNAIRNA Mini Kitl KF96.

- Sau 34 phut, qua trinh chist tach hoan tftt. Thu h6i RNA cua tung giSngVaG6ng eppendorfO.S ml tuang trng da ghi r5 kY hi~u m~u.

11

han.- Baa quan mftu RNA a 4°C trong vai gio va (-)80°C trong thai gian lau

II. Tach chi~t RNA bing bq kit Invitrogen PureLink Viral RNAIDNAMini Kit

a. Chu§n b] hoa ch§tCac hoa ch~t c§.ndiroc chu~n bi va bao quan thea dung huang dftn cua b9

kit Invitrogen PureLink Viral RNAIDNA Mini Kit, Cat. No: 12280-050, voi thStich vira du cho s6 hrong mftu chiet tach, bao gom:

- Proteinase K- Dung dich d~m Lysis Buffer- Dung dich Carrier RNA- Dung dich Wash buffer (W5)- Nuoc RNase-free (E3)- Cach pha h6n hop dung dich Carrier va Lysis BufferNxO.21 mL=AmLA mL x 28 JlL/mL = B JlLTrong do: N la s6 mftu c§.ntach chietA la tong s6 mL Lysis Buffer c§.ncho N mftu;B la tong s6 ul, Carrier RNA c§.ncho N mftu.b. Ti~n Hanh1. Cho 25 ul, Proteinase K van 6ng eppendorf 1,5 ml;2. Cho 200 ul, mftu van 6ng eppendorf chua Proteinase K;Note: nSu thS tich mftu <200 ul, thi co thS diSu chinh thS tich mftu cho du

200 ilL bang dung dich PBS hoac 0.9% NaCl.3. Cho h6n hop dung dich Lysis Buffer va Carrier RNA van 6ng chua

m~u; dong nAp6ng va vortex 15s;4. (Jmftu a 56°C trong 15 phut;5. Cho them 250 ul, c6n 96 - 100 % van 6ng mftu; dong nAp va vortex

15s',6. DS a nhiet d9 phang 5 philt;Note: mftu mo sau khi dS a nhi~t d9 phang 5 philt thi dem ly tam 12.000

vang trong 2 philt va l~y dich n6i ben tren.7. ChuySn toan b9 mftu tach chiSt van c9t lQc;8. Ly tam c9t lQCa 12.000 vang trong 2 - 3 philt; chuySn C9t lQc sang 6ng

thu moi;

9. Cha 500 ilL nuoc rira Wash buffer (W5) van C9t lQc; ly tam 12.000vang trong 2 - 3 philt; ChuySn c9t lQc sang 6ng thu moi;

10. L~p l;;tibuoc 9 voi 500 ilL nuoc rlla Wash buffer (W5) m9t l§.nnfra;11. Lo;;tib6 6ng thu va chuySn c9t lQc sang 6ng thu moi;12. Ly tam kho I2000v trong 1 philt cho kh6 s;;tchnuoc rna W5;13. ChuySn C9t lQcsang 6ng eppendorf 1,5 JlL;

12

14. Cho 10 - 50 JlLmroc RNase-free (E3) (co s~n) vao cQt loc;15. BS a nhiet dQ phong 1 phut; Ly tam cQt loc & 12000 yang trong 1-2

phut.16. L~y san pham duoi cQt lee; Bao quan RNA a nhiet dQ- 80°C.III. Tach chi~t RNA b~ng TACO RNAillNA extraction Kit1.Vat lieu. .BQ kit chiet tach TACO DNAIRNA extraction kit (GeneReach Cat. No.

atc-d/rna, 320 tests)C6n Ethanol 100% ( c6n tuyet d6i)2. Chusn b]- Pha dung dich d~m nra (washing buffer A) voi 135ml c6n tuyet d6i.- Pha dung dich dern nratwashing buffer Bjvoi 230ml con tuyet d6i.3. Ti~n hanh theo so dB

Reagent ul Reagent ul

Ethanol 250

Washing buffer A 750 Magnet beads 50

Washing buffer A 750

Washing buffer B 750

Washing buffer B 750

Elution buffer 100

Same as above

H G FED C B A

.... 1

.... 2

... 3

-+4-+5-+6

7

8

... 910

11

12

1. Huyen dich 10% cua m~u benh pham diroc ra dong, tron dSu bang mayvortex. Ly tam m~u trong may ly tam lanh voi hrc ly tam 12000 g/5 phut,

2. Hut 200JlI djch trong ben tren sau khi ly tam vao cac gieng & cot 1 hoac7 co chua s~n 500 JlI dung dich d~m Lysis va 250 Jll ethanol 95%

DU'amfiu vao may TACO1.B~t dia vao may TACO2. D~t hroc vao may TACO.3. SU dung may TACO thea huang d~n sir dung: May TACO tir dong

chiet tach trong khoang 50 phut.4. Thu 100JlL DNAIRNA tir cac gieng trong cQt 6 ho~c 12 sang cac 6ng

moi

13

PHU LUC B: TIEU TON VAT TV' CHO MOT MAU XET NGHIEM... . . .

DO'nvi I.

TT Ten V~t Tn' So hrQ'ng/ Di~n giaitinh 01 m§u

1 cai: chua m&u

2 cai: chuan bi m&ud6i clnrng.

1,8ml free1 cai: chua Proteinase K workingCryotube solution1 RNAIDNA (n~p v~n CM 7

ngoai, ttr dung) 1 cai: chua Carrier RNA workingsolution

1 cai : chua primer & probes1 cai : chuan bi master mix

Cryotube 5ml free 1 cai: chua Elution Buffer2 RNAIDNA (nap v~n Cai 2 1 cai: chua Binding Solution va

ngoai, tu dung) MAP solution

3 PCR tube 1,5 ml Cai 3 LUll gift m&uRNA/DNA

4 Trip PCR co n~p 0,2ml CM 2 Nhan gene qRT-PCR

InviMag® Virus5 DNAIRNA Mini Kit/ Kit 0,0069 Chi~t tach RNA hoac DNA

KF96( 480 test! kit)

6 Ethanol Absolute ml 7,15 B6 sung vao dung dich rita 1,nra 2

7 KF ml tube Cai 3 Chiet tach RNA hoac DNA

8 KF ml Tip Combs Cai 1 Chi~t tach RNA hoac DNA

1 cai: chua dung dich Lysis9 Tube ly tam nhua 50 ml CM 3 1 cai: chua dung dich nra 1

1 cai: chua dung dich nra 2

1 cai: cho dung dich rita 12 cai: eho dung dich rita 2

10 Finntip Mu1tistepp er CM 5 1 cai: cho Lysis Solution vao1500111 sterile eppendorf

1 cai: cho Elution Buffer vao rnachiet xudt

14

TT Ten V~t TO' Do'n v] S610'Q'ngiDi~n giaitinh 01 m~u

2 cai: Mix Proteinase K va CarrierRNA

3 cai: chuyen miu + Lysis Solutionvao rna chiet xu~t

11 Micropipet filter tip Ccii 8 1 cai: cho Binding Solution VaG1000J.ll 6ng cryotube 5ml

1 cai: cho MAP solution VaG 6ngcryotube 5 ml

1 cai: cho Binding Solution vaMAP solution VaG rna chiet xu~t

3 cai: cho miu va d6i clnrng VaG

eppendorf chua Lysis Solution

12 Micropipet filter tip Ccii 8 3 cai: cho thu hoach RNAIDNA2001-11 1 cai: chuan bi master mix

1 cai: phan ph6i Master mix vao rnaPCR

13 Micropipet filter tipCcii 3 Chuan bi master mix20J.ll

1 cai: chuan bi master mix

1 cai: dua miu RNA/DNA VaG rna

14 Micropipet filter tipCcii 6 PCR chua master mix

10ul 4 cai: dua mau RNAIDNA d6iclnrng VaG rna PCR chua mastermIX

15 Quan ao bao hQ dung 1BQ 2 Xu ly miuIfrn

2 doi: xu Iy miu

2 doi: chiet xua; RNA/DNA16 Gang tay Doi 8

2 doi: chuan bi master mix

2 doi: Di~n di miu PCR

17 Khfru trang Ccii 2 Xirly miu

Primers TiLV18 BQ 0,0028 Nhan gene RT-PCR

(2000 test/be)

15

Ten V~t TO' DO'n vi S610'(}'ngl Di~n giaiTTtinh 01m~u

Kit S . ® IIISuper cnptOne-Step RT-PCR with

19 Platinum" Taq.Kit 0,0275 Nhan gene RT-PCR: 25 flll reactionCatalogue Number:

12574-026 (100 reactwith 50fll/reaction)

Kit Thermo Scienetific

20 Dream Taq PCR MasterKit 0,0275 Nhan gene RT-PCR: 25 fll! reactionMix (2X), Cat. No: K

1071.

21 Agarose 16500 (500gram 0.35 Thach di~n digr/bag)

22 Sybr safe (400flI/6ng) flL 2 Chat nhuom man

TBE buffer lOXflL 5 Dung dich pha thach23 Solution (llit/chai)

1000 bp DNA LadderflL 10 Thang chuan24

(500fll!6ng

25 Loading dyeflL 14 Chc1tdem Utiml1u(500flI/6ng)

16