X A Y D U N G Q U Y T R I N H P H A N T I C H P H A T H I E N N H A N H D O C T O T U C A U V A N G D A N G A ( S E A ) T R O N G S A N P H A M T H I T
S U D U N G 6 0 K I T B K - S E T A
x L E QUANG HOA, NGUYEN HOANG MINH , NGUYEN T H I HUE, NGUYEN T H I KHANH TRAM, TRAN NGUYEN HOA CUONG, TO K I M ANH
Vien Cong nghe sinh hoc - Cong nghe thuc pham, Truang Dai hoc Bach khoa Ha Noi
Tom tat:
Bp sinh pham BK-SETA dupe xay dpng de phat hien nhanh doc to tu cau vang dang A
(SEA) trong san pham thit theo nguyen tac ELISA sandwich tich hpp cac hoa chat va thuo'c thd can thiet nham gian lupc thao tac va dam bao tinh dac hieu cua phep phan tich. Kit thCr dupe thiet ke hudng tPi cho trien khai phan tich tai cho hoac tai cac co sd phan tich tuyen dudi. Dpa tren bp kit, quy trinh phan tich phat hien SEA trong san pham thit (nem chua) dupe xay dpng va toi Uu hoa, bao gom ca khau chuan bi mlu de loai trp anh hudng thanh phan thpc pham tPi ke't qua phan tich. Bp kit co kha nang phat hien SEA tai ndng dp 0,3 ng/ml thpc pham trong thdi gian 3 gid. Bp kit va quy trinh phan tich dupe kiem dinh hai bUPc: noi bp va dpc lap. Viec kiem dinh khang djnh bp kit va quy trinh phan tich cho dp chinh xac va tinh dac hieu la 100%.
DEVELOPING T H E PROCEDURES FOR QUICKLY DETECTING THE STAPHYLOCOCCUS AUREUS (SEA) ON MEAT PRODUCTS BY BK-SETA
Abstract:
The biological product BK-SETA is established to quickly detect the A Staphylococcus aureus (SEA) on meat products following the principle ALISA sandwich integrate chemicals substances and necessary reagent in order to reduce manipulation and ensure the specific of analysis method. BK-SETA is designed to deploy analysis on the premises or at local analysis Labo. Base on BK-SETA, the process of Analysis SEA in meat products (fermented meat roll) is established and optimized, includes preparing samples phase to exclude effects of food ingredient on analysis result. BK-SETA can detect SEA at concentration 0.3 ng/ml food in 3 hours. BK-SETA and analysis process are verified two steps: internal and independent. The verification confirm BK-SETA and analysis process is 100% high fidelity and specifically.
I . D ^ T V A N D E
Staphylococcus aureus dupe coi la tac nhan gay ngp dpc dung thd ba tren the gidi.
Cac thdng ke dich td hoc da chi ra rang dpc td S. aureus (SE) la nguyen nhan gay ngp dpc thuc pham ddng hang thu hai chi sau Salmonella. Trong so cac SE cua 5. aureus, SEA la loai dpc to thong thudng nhat lien quan den cac vu ngp dpc thuc pham [3,4] .
Cac phuong phap phan tich SEA trong thuc p h i m hien nay con nhieu bat l o i trong sir dung do tinh phuc tap, gia thanh cung nhu yeu cau vd didu kien tdi thieu cho viec ap
469
dung. Tuy nhien Viet Nam hien nay chua co mpt don vi phan nao dam nhi£m nhiem vu phan tich nay, vi the SEA con n i m ngoai pham v i kiem soat cac nguyen nhan gay ngp dpc thuc p h i m . X u hudng hien nay tren the giPi la phat trien cac phuong phap phan tich sang lpc va co the ap dung phan tich tai hien trudng de dap Png vPi yeu cau thuc te' [2]. Nham dap ung nhu cau phan tich, bp kit phan tich SEA da dupe thiet ke dua tren nguyen ly phan ung ELISA sandwich va quy trinh phan tich SEA trong san p h i m sua sir dung bp kit da dupe tP'i uu hoa [ 1 ] . Trong nghien cdu nay, chung toi hoan thien bp kit va xay dung quy trinh phat hien SEA trong san p h i m thit, k i e m dinh quy trinh phan tich SEA khi su dung bp kit.
IL D6l TlTONG VA PHlTONG PHAP NGHIEN CUU 2.1. Mdu thuc pham
M i u thuc p h i m dung cho nghiSn cuu la cac m i u nem chua, sua thanh triing va sua tiet trung mua tai cac sieu thi tai Ha Npi.
Cac m i u thuc pham dupe nghien trong nude cit, cho nhiem chu dpng dpc td chuin SEA dd dat dupe ndng dp SEA cuoi ciing trong m i u td 0.1- 10 ng/ml. L y tam 2ml m i u da nhidm dpc td d lOOOOvp uong 5 phut. Thu dich ndi, bao quin d 4°C, su dung cho phan ung ELISA.
2.2. Hda chat
Co chit cho phan ung ELISA: Urea peroxide, hoac hydrogenperoxide; Chromogen:
T M B (3,3',5,5'-tetramethylbenzidine), Biorad. Cac dpc td tu c i u chuan cua Toxin. Coop, M% Khang the tdm b i t (Mouse monoclonal) va khang thd cpng hpp (Rabbit polyclonal HRP conjugated) Abeam, U K .
BP kit RIDASCREEN xac dinh SE cua r-biopharm A G , Damstadt, Germany va bp kit xac dinh SEA [ l j .
Ky thudt ELISA
- Pha loang khang thd bit cap vdi SEA d ndng dp thich hpp trong PBS, phu lOOpl
khang thd da pha loang vao mdi gieng ELISA, sau dd u qua dem d 4°C. Horn sau rua 3 l i n bang 300pl PBST (PBS chda 0,5% Tween 20).
- Phii bd sung mdi gieng bing 250pl GPBS (PBS cd chua 5% gelatin), u d 37°C trong 90'. Rua 3 lan bang PBST.
- Cho vao mdi gieng lOOpl dpc td SEA da pha loang o ndng dp thich hpp, u tai nhiet dp phong Uong l-2h. Rua cac gieng 5 l i n bing PBST.
- Bd sung lOOpl khang the cpng hop HRP khang SEA d dp pha loang thich hpp
theo khuyen cao ciia hang san x u i t vao mdi gieng, u tai nhiet do phong trong l h . Rua 5 l i n vdi PBST.
- Cho lOOpl dung dich TMB nhupm. De d nhiet dp phong trong 5 phut, luc nay mpt
sd gidng se chuyen sang mau xanh. Dung phan ung b i n g lOOpl HC1 I N , mau se chuydn sang vang.
- Do ket qua phin ung ELISA tai OD450 nm.
2.3. Phuong phap nghien cuu
Xdy dung quy trinh phdn tich SEA cho sdn phdm thit Xdc dinh dnh hudng cua cdc chat nen cd trong thuc phdm
M i u thit dupe x u ly khac nhau, bao gom: ly tam, dupe lpc hoac giu nguyen de thu nghiem anh huong cac thanh phan thit tdi phan dng ELISA. Mau thuc p h i m n h i l m dpc td chuin SEA dupe chuin bj nhu da neu d tren. Bd sung 100 p i djch thit nhidm SEA vao cac gieng ELISA. Thuc hien phan dng ELISA, danh gia i n h hudng cua each x u ly m i u tdi ket qua OD 450 nm do dupe. Cac thi nghiem dupe lap lai 31in.
Xac dinh do nhay cua phuong phap doi vdi mau thuc phdm
Ndng dp SEA su dung trong khao sat thay ddi td 0,3ng/ml; lng/ml; 3ng/ml;
lOng/ml. Cac thi nghiem dupe tien hanh song song vdi hai m i u ddi chung duong cd dpc td SEA d ndng dp cuoi cung la 0,3ng/ml va lOng/ml va mpt m i u am tinh (dung djch N a H C 03 hoac PBS). T i t ca cac thi nghiem deu dupe thuc hien song song vPi m i u am tinh (dung djch N a H C 03 hoac PBS) va m i u duong tinh. T h i nghiem dupe lap lai 3 l i n .
Toan bp cac thi nghiem xay dung kit ELISA va quy trinh phan tich dupe thuc hien vdi dpc td chuan va sau dd kiem tra vdi dpc to do de tai thu nhan dupe.
Kiem dinh bp kit
BP kit dupe kiem djnh tai phong thi nghiem va kiem dinh dpc lap bdi hai phong thi nghiem chdc nang:
- Phong Thi nghiem khoa Thuc phim - Ve sinh an toan thuc phim, Vien Dinh duong, Bp Y te.
- Phong Hda sinh - Thuc phim, Trung tam Ky thuat tieu chuin do ludng chat lupng 1, Tdng cue Tieu chuan chit lupng.
Viec kiem djnh dupe thuc hien song song tai hai phong thi nghiem, tren 20 m i u san phim thjt, bao gdm:
- 08 miu san phim thjt, mdi miu dupe nhidm SEA vdi ba ndng dp SEA khac nhau:
dudi nguong phat hien ( m i u am tinh), tai nguong phat hien 0,3 ng/ml va tren ngudng phat hien ( m i u duong tinh, lng/ml). Tdng cpng 18 m i u thjt.
- 02 m i u san p h i m thjt nhidm hdn hpp cac SE khac vdi SEA (bao gdm SEB, SEC, SED va SEE) tai cac ndng dp lng/ml mdi dpc td de kiem tra tinh dac hieu cua kit (kh6ng b i t cap vdi cac SE khac SEA).
HE KET QUA VA BAN LUAN
3.1. Xay dung quy trinh phat hien SEA trong san pham thjt su dung kit B K SetA Anh hudng cua cdc thanh phdn thit
5g m i u thjt da xay dupe trpn ddu vdi 10ml dung djch S6 dd tao thanh mpt hdn hpp ddng nhit. Ddi vdi viec chuin bj m i u b i n g cac thiet bj ddng hda m i u , 5g m i u thjt dupe ddng hda vdi 10ml dung djch S6 trong cac tui ddng hda chuyen dung vdi khoang thdi gian 5-10 phut. M i u thjt sau k h i ddng hda dupe x u ly theo cac each khac nhau nhu trinh bay trong p h i n phuong phap. lOOpl dung dich m i u thjt thu dupe su dung lam m i u phan tich.
Ket qua thu nghiem cho t h i y cac bien phap xu ly m l u khac nhau cho ket qua khac nhau, su khac biet giua cac m i u duong tinh va am tinh la ro ret tai tat ca cac m i u cd xu ly va khPng xd ly m i u . Neu vPi tieu chi la phuong phap djnh tinh, mpt trong cac phuong phap ddu cd the chpn de thuc hien. Tuy vay, mpt nhan xet t h i y ro la, gia tri O D4 5 0 n m thu dupe trong m i u nhidm SEA co gia t r j nhd hon khi so sanh vdi m i u SEA tinh khiet d cung
471
nong do SEA. Dieu nay co the do su anh huong cua nen dung dich thit so vPi dung dich chuin. So sanh cac phuong phap xu ly m l u , chung toi chon giai phap ly tam dich m i u cho viec phan tich SEA.
0.4 0.3 0.2 0.1 0
43i
<s> s$ it if It*
Xurly miu
Hinh 1. So sanh dnh hudng cdc phuong phap xurly mdu thit tdi ket c/ud phdn tich SEA
Ve do nhay
Nhu da noi d tren, cac chat nen trong m i u anh huong deh ket qua phan tich do vay ngudng xac dinh cua phuong phap ddi vdi doc td chuan pha trong dem PBS se cd the kh6ng gidng nhu khi ap dung vdi m i u thuc pham. C i n thiet khao sat nguong phat hien SEA trong m i u thit.
Ndng do SEA su dung trong khao sat dp nhay phat hien SEA trong m i u thit bang bp kit thay ddi td 0,3ng/mg; lng/mg; 3ng/mg; lOng/mg. Cac thi nghiem duoc tien hanh song song vdi hai m i u ddi chdng duong cd dpc td SEA duoc pha trong dung dich dem PBS d ndng do cuoi cung la 0,3ng/ml va 3ng/ml va mpt m i u am tinh PBS khong chda SEA. Ket qua trinh bay tren hinh 1. Ket qua thu nghiem cho thiy bp kit cd the phat hien SEA tai nguPng 0,3 ng/mg thit (hinh 2,3).
A
B
miu am am tinh 0,3 1 3 tinh PBS
SEA (ng/g)
Hinh 2. Kiem tra dd nhay phdt hien SEA ciia bd kit phdn tich tren nen mdu thit A. Hinh dnh ke't qua ELISA: 1. mdu thit 0,3 ng/ml, 2,3.mdu thit 1 ng SEA/mg; 4,5:
mau thit 3 ng SEA ng/ml; 6,7: man dm tinh, 8. ddi chung duong 3ng/ml B. Gid tri OD 4 5 0 i m ciia phdn ung ELISA
Hinh 3. Hinh dnh bd kit phdn tich phdt hien SEA
3.2. Kiem dinh bd kit
Bo kit BK SetA dirge kiem dinh bdi hai phong thi nghiem:
- Phong thi nghiem Khoa Thirc pham - Ve sinh an toan thirc phim, Vien Dinh duong, Bo Y te.
- Phong Hda sinh - Thuc pham, Trung tam Ky thuat tieu chuan do luong cha't lupng 1, Tdng cue tieu chuin cha't lupng.
- Viec kiem dinh dupe thuc hien song song tai hai phong thi nghiem nhu trinh bay trong phan phuong phap.
Bdng 1. Ke't qua kiem dinh (cho ngudng phdt hien SEA 0,3 ng/ml, mg) Ket qua duong tinh k h i Ke't qua am tinh k h i
phan tich bang bp k i t phan tich bang bp k i t
M i u duong tinh EA 12 0
Mau
M i u duong tinh EA
(PA) (PD)
Mau
M i u am tinh SEA 0 8
(ND) (NA)
Bang 2. Ket qud cdc tieu chi kiem dinh kit cho hai dang san phdm
M a u PA N A P D N D
PA+NA+PD+ND PA + PD ND + NA
Dp chinh xac tuong ddi
A C ( % )
Dp nhay tuong ddi
SE ( % )
T i n h dac hieu tuong
ddi SP ( % )
(PA+NA) x 100 PA+NA+PD+
N D
PA x 100 PA+PD
N A x 100 N D + N A
Thit 12 8 0 0 20 12 8 100 100 100
473
Nhu vay, bp kit thiet ke cho phan tich phat hien dpc to' tu c i u vang dang A da dupe kiem dinh co the phat hien dac hieu SEA trong mpt so' san p h i m sua va thit tai nguong 0.3 ng/ml,mg. Thdi gian kiem dinh 3h, bao gdm chuan bi m i u . Bp kit cd thd thuc hien tai hien trudng hoac trong phong thi nghiem.
T A I L I E U T H A M K H A O
1. Nguyen Hoang Minh, Pham Phuong Thao, Le Quang Hoa, To Kim Anh, 2008, A
newly developed sandwich ELISA for possible rapid detection of Staphylococcal enterotoxin type A in milk product. Hdi nghi Hda sinh toan quoc. Ha Ndi 2008.
2. Buscarlet et al, 2001, Performance of an immunoassay-based method for monitoring the staphylococcal enterotoxin production in food and culture supernatant. Prague, 2001.
3. Le Loir et al, 2003, Staphylococcus and food poisioning. Genetic and Molercular Re search.2: 63-76.
4. Tirado et al, 2001, WHO surveillance program for control of food-borne infection and intoxication: preliminary results and strends across greater Europe. Journal oflnfection.43: 80-84.
