Nghiên cứu tạo que thử phát hiện nhanh độc tố tụ cầu (SEA SEE) trong sữa bằng kỹ thuật sắc ký miễn dịch cạnh tranh

Lun Vn Thc S Nguyn Th Hng Nhung LI CM N Trc tiờn, tụi xin by t lũng bit n sõu sc nht ti TS Lờ Quang Hũa Phũng K thut Gen Trung tõm Nghiờn cu v Phỏt Trin CNSH & CNTP, Vin CNSH & CNTP Trng HBK H Ni, ngi ó hng dn tn tỡnh, chu ỏo sut quỏ trỡnh nghiờn cu v thc hin ti phũng thớ nghim v luụn sn sng giỳp , h tr tụi nghiờn cu tụi hon thnh lun tt nghip ny Tụi cng xin gi li cm n n cỏc thy, cụ thuc Vin CNSH & CNTP Trng HBK H Ni ó tn tỡnh dy bo v giỳp sut quỏ trỡnh tụi hc ti trng V nhõn dp ny, tụi mun gi li cm n ti tt c cỏc cỏn b, nhõn viờn thuc Trung tõm Nghiờn cu v Phỏt trin CNSH & CNTP, Vin CNSH & CNTP Trng HBK H Ni cựng cỏc bn sinh viờn c bit em l Nguyn Hong Hi, hc viờn, nghiờn cu sinh c bit l ch Trn Th Sao Mai thc ti õy ó nhit tỡnh giỳp tụi sut quỏ trỡnh tin hnh thớ nghim Cui cựng, tụi xin gi li cm n gia ỡnh tụi, nhng ngi thõn v bn bố quan tõm, ng h v ng viờn sut quỏ trỡnh hc v hon thnh lun Do iu kin, kh nng v thi gian thc hin cú gii hn nờn ti khụng trỏnh nhng sai sút Tụi rt mong nhn c s úng gúp ca cỏc thy cụ v cỏc bn ti ca tụi hon thin hn Tụi xin chõn thnh cm n! H Ni, thỏng nm 2012 Hc viờn Nguyn Th Hng Nhung Lun Vn Thc S Nguyn Th Hng Nhung LI CAM OAN Tụi xin cam oan, nhng ni dung c trỡnh by lun ny l s tỡm tũi, nghiờn cu ca chớnh bn thõn Cỏc kt qu nghiờn cu l trung thc v cha tng c cụng b bt k lun no ca cỏc tỏc gi khỏc Tụi xin chu trỏch nhin v nhng ni dung cam oan trờn H Ni, ngy 25 thỏng nm 2012 Tỏc gi Nguyn Th Hng Nhung Lun Vn Thc S Nguyn Th Hng Nhung MC LC LI CM N LI CAM OAN MC LC DANH MC CC Kí HIU, CC CH VIT TT DANH MC CC HèNH DANH MC CC BNG M U 10 PHN I: TNG QUAN TI LIU 12 I.1 Tỡnh hỡnh ng c thc phm 12 I.1.1 Tỡnh hỡnh ng c thc phm trờn th gii 12 I.1.2 Ng c thc phm c t rut ca t cu gõy trờn th gii 13 I.1.3 Tỡnh hỡnh ng c thc phm Vit Nam 15 I.2 T cu khun (Staphylococcus) 16 I.2.1 T cu vng 17 I.2.1.1 c im sinh hc 17 I.2.1.2 Phõn loi 19 I.2.1.3 c t v cỏc yu t c lc 20 I.2.1.4 Kh nng gõy bnh 23 I.2.1.5 Min dch 24 I.2.1.6 Chun oỏn vi khun hc 24 I.2.1.7 Kh nng gõy ng c v triu chng ng c thc phm t cu 25 I.2.1.8 Nhng yu t nh hng ti s sn xut c t rut ca t cu 26 I.2.1.8 Nhng yu t nh hng ti s sn xut c t rut ca t cu 26 I.2.1.9 Tn sut xut cỏc chng mang gen mó húa c t rut 27 I.2.1.10 Nguyờn tc phũng v iu tr bnh t cu vng gõy 28 I.3 c t rut ca t cu 29 Lun Vn Thc S Nguyn Th Hng Nhung I.3.1 c im ca c t rut t cu 29 I.3.2 C ch hot ng ca nhng c t rut ca t cu 30 I.3.3 Cu trỳc phõn t ca c t rut 33 I.4 Cỏc phng phỏp phỏt hin SE 34 I.4.1 Phộp th sinh hc 35 I.4.2 Phng phỏp sinh hc phõn t 35 I.4.3 Phng phỏp dch 35 I.4.3.1 K thut cht hp ph dch gn enzym (ELISA) 35 I.4.3.2 K thut sc ký dch 36 PHN II VT LIU V PHNG PHP NGHIấN CU 45 II.1 Vt liu nghiờn cu 45 II.1.1 i tng v húa cht nghiờn cu 45 II.1.4 Thit b s dng nghiờn cu 45 II.2 Phng phỏp nghiờn cu 46 II.2.1 Phng phỏp bin np 46 II.2.2 Phng phỏp nuụi cy t bo E coli v chun b dch protein thụ 46 II.2.3 Phng phỏp ELISA (Enzyme-Linked Immuno Sorbent Assay) 47 II.2.4 Phng phỏp in di bin tớnh protein (SDS-PAGE) 50 II.2.5 Chun b phc hp phỏt hin khỏng nguyờn - nano carbon 51 II.2.6 C nh khỏng th vch th nghim lờn mng nitrocellulose 52 II.2.7 Phõn tớch mu bng que th 52 PHN III KT QU V THO LUN 53 III.1 Bin np plasmid pGEX-6p-1-sea vo t bo E.coli kh bin 54 III.2 Sng lc kh nng sinh SEA cỏc dũng t bo chuyn gen 54 III.3 Ti u húa iu kin sinh tng hp SEA 56 III.4 Tinh sch SEA t dch chit protein thụ 58 III.5 ng dng SEA tỏi t hp to qua th 61 III.5.1 Xỏc nh lng khỏng th cn c nh lờn vch th nghim 61 III.5.2 Xỏc nh lng khỏng nguyờn cng hp cn s dng 63 Lun Vn Thc S Nguyn Th Hng Nhung III.5.3 Xỏc nh nhy phỏt hin ca que th vi loi c t 64 III.5.3.1 Xỏc nh nhy phỏt hin ca que th vi SEA 64 III.5.3.2 Xỏc nh nhy phỏt hin ca que th vi SEB 65 III.5.3.4 Xỏc nh nhy phỏt hin ca que th vi SED 67 III.5.3.5 Xỏc nh nhy ca que th vi SEE 68 III.6 ng dng que th phỏt hin SEA-SEE sa 69 III.6.1 Quỏ trỡnh chun b mu 69 III.6.2 Xỏc nh nhy phỏt hin ca ton b quỏ trỡnh phõn tớch 69 III.6.2.1 Xỏc nh nhy phỏt hin SEA sa 69 III.6.2.2 Xỏc nh nhy phỏt hin SEB sa 71 III.6.2.3 Xỏc nh nhy phỏt hin SEC1 sa 72 III.6.2.4 Xỏc nh nhy phỏt hin SED sa 73 III.6.2.5 Xỏc nh nhy phỏt hin SEE sa 74 III.6.2.6 nhy ca ton b quỏ trỡnh phõn tớch 75 PHN IV KT LUN V NGH 76 IV.1 Kt lun 76 IV.2 Kin ngh 76 TI LIU THAM KHO 77 Lun Vn Thc S Nguyn Th Hng Nhung DANH MC CC Kí HIU, CC CH VIT TT STT Ký hiu Tờn y APC Antigen Presenting Cell (T bo trỡnh din khỏng nguyờn) BSA Bovine Serum Albumin CDC Centers for Disease Control and Prevention (Trung tõm kim soỏt v ngn nga bnh) CFU Colony forming unit (S n v hỡnh thnh khun lc) CNS Coagulase negative staphylococci DNA Axit Deoxyribonucleic dNTP Deoxynucleotide ELISA Enzyme-Linked Immunosorbent Assay GST Glutathione-S-Transferase 10 IPTG IsoPropyl--D-ThioGalactopyranoside 11 kDa Kilo Dalton 12 LAMP Loop-Mediated Isothermal Amplification of DNA 13 MHC-II Major histocompatibility complex (Phc hp tng thớch mụ chớnh) 14 NTP Ng c Thc Phm 15 PBS Phosphate Buffered Saline- mui m phosphate 16 PBST Phosphate Buffered Saline + 0,1% Tween - mui m phosphate + 0,1% Tween Lun Vn Thc S Nguyn Th Hng Nhung 17 PCR Polymerase Chain Reaction 18 PEG Polyetylen Glycol 19 SEs Staphylococcal Enterotoxins 20 TCR T-Cell antigen Receptor (Th th ca t bo T) 21 TNF Tumor Necrosis Factor (Yu t hoi t u) 22 TSST Toxic Shock Syndrome Toxin 23 TSST-1 Toxic Shock Syndrome Toxin-1 24 VSATTP V Sinh An Ton Thc Phm Lun Vn Thc S Nguyn Th Hng Nhung DANH MC CC HèNH Hỡnh 1.1 T cu vng di kớnh hin vi quang hc v trờn mụi trng mch mỏu 17 Hỡnh 1.2 So sỏnh gia s liờn kt ca mt khỏng nguyờn bỡnh thng v s liờn kt ca mt siờu khỏng nguyờn vi th th t bo T v vi phc hp MHC lp II 32 Hỡnh 1.3 Cu trỳc khụng gian ca c t rut SEC, SEB, SEC2, SED 33 Hỡnh 1.4 Sc ký dch kp ụi 38 Hỡnh 1.5 Sc ký dch cnh tranh 39 Hỡnh 1.6 Cỏc hp phn h thng sc ký dch 40 Hỡnh 2.1 Nguyờn tc k thut ELISA kp ụi 47 Hỡnh 2.2 Nguyờn tc k thut sc ký ỏo lc giỏn on 49 Hỡnh 2.3 Cỏc hp phn ca que th 52 Hỡnh 3.1 Plasmid pGEX-6P-1-sea 53 Hỡnh 3.2 Kt qu bin np 54 Hỡnh 3.3 Kt qu sng lc ln mt bng ELISA 55 Hỡnh 3.4 Kt qu sng lc ln hai bng ELISA 56 Hỡnh 3.5 Kt qu ti u húa 58 Hỡnh 3.6 Protein sau tinh sch em in di 59 Hỡnh 3.7 Protein sau tinh sch em in di 60 Hỡnh 3.8 La chn lng khỏng th c nh lờn mng 62 Hỡnh 3.9 La chn khỏng nguyờn cng hp cn s dng 63 Hỡnh 3.10 Th nhy ca que th vi SEA 64 Hỡnh 3.11 Th nhy ca que th vi SEB 65 Hỡnh 3.12 Th nhy ca que th vi SEC1 66 Hỡnh 3.13 Th nhy ca que th vi SED 67 Hỡnh 3.14 Th nhy ca que th vi SEE 68 Hỡnh 3.15 Th nhy ca que th vi SEA sa 70 Hỡnh 3.16 Th nhy ca que th vi SEB sa 71 Hỡnh 3.17 Th nhy ca que th vi SEC1 sa 72 Hỡnh 3.18 Th nhy ca que th vi SED sa 73 Hỡnh 3.19 Th nhy ca que th vi SEE sa 74 Lun Vn Thc S Nguyn Th Hng Nhung DANH MC CC BNG Bng 1.1 Tỏc nhõn gõy ng c thc phm nm 1999 v nm 2000 Phỏp (tn s mi loi tỏc nhõn c a di dng %) 13 Bng 1.2 Cỏc tuýp dung huyt t ca t cu vng 22 Bng 2.1 Thnh phn gel tỏch 50 Bng 2.2 Thnh phn gel cụ 51 Lun Vn Thc S Nguyn Th Hng Nhung M U V sinh an ton thc phm l mt ang c c xó hi quan tõm bi vỡ nú nh hng trc tip ti sc khe ca ngi Nhng nm gn õy ng c thc phm xy khỏ thng xuyờn Trong ú vi sinh vt gõy bnh l mt nhng nguyờn nhõn hng u gõy ng c l thc phm (tỏc nhõn gõy 2/3 cỏc v ng c thc phm) Vỡ vy, vic phỏt trin cỏc phng phỏp phỏt hin nhanh cỏc vi sinh vt gõy bnh v c t ca chỳng to thc phm luụn l mt yờu cu cp thit v thng xuyờn m bo trỏnh c nhng thit hi v sc khe, tớnh mng ca ngi tiờu dựng v thit hi kinh t cho nh sn xut Trong s ú, Staphylococcus aureus hay t cu vng l mt nhng tỏc nhõn gõy ng c ph bin nht Ng c thc phm t cu c nh ngha l s tiờu th cỏc c t ng rut, ó tn ti sn thc phm, cỏc chng t cu cú coagulase dng tớnh, c bit l S aureus tng hp nờn Theo cỏc s liu thng kờ ti M, S aureus l tỏc nhõn vi khun gõy ng c thc phm ng hng th nm) c tớnh hng nm ti quc gia ny cú khong trờn 180000 trng hp b ng c thc phm S aureus gõy Cỏc thng kờ v dch t hc ti Phỏp hai nm 1999-2000 li ch rng S aureus l tỏc nhõn gõy ng c thc phm ng hng th hai, ch sau Salmonella Cỏc s liu thng kờ gn õy ti Chõu u ch rng cỏc c t ng rut ca t cu l nguyờn nhõn gõy 5,5% tng s cỏc v ng c thc phm Khi cỏc chng vi khun t cu mang gen mó húa cỏc c t ng rut SE (Staphylococcal Enterotoxins) phỏt trin n mt s lng nht nh thc phm (khong 104-5 CFU/g, ml), chỳng cú th tit mt lng c t gõy hin tng ng c cho ngi tiờu dựng Cỏc triu chng thng gp b ng c cỏc c t ng rut t cu l bun nụn, nụn, au d di vựng bng, gõy kit sc v ụi i kốm vi hin tng tiờu chy Cỏc triu chng ny thng biu hin 10 Lun Vn Thc S Nguyn Th Hng Nhung III.6 ng dng que th phỏt hin SEA-SEE sa Mc tiờu ca nghiờn cu ny l phỏt trin mt b sinh phm da trờn k thut sc ký dch phỏt hin nhanh cỏc SE (t SEA n SEE) sn phm sa trờn hin trng t c mc tiờu ny, chỳng tụi ó tin hnh quy trỡnh chun b mu v phõn tớch mu bng que th c trỡnh by di õy: III.6.1 Quỏ trỡnh chun b mu - Sa ti tit trựng khụng ng ca Vinamilk sn xut ngy 18/5/2012 c pha loóng ln dung dch m chy - Vỡ chỳng tụi ó tin hnh cỏc thớ nghim th nhy ca ca que th vi cỏc c t rut ca t cu t SEA SEE nờn s dng que th phỏt hin cỏc c t ny sa chỳng tụi ch lm ngng phỏt hin ca que th - Tin hnh nhim ch ng cỏc nng khỏng nguyờn SEA SEE tinh khit ó c pha loóng vo sa v sau ú phõn tớch mu bng que th III.6.2 Xỏc nh nhy phỏt hin ca ton b quỏ trỡnh phõn tớch III.6.2.1 Xỏc nh nhy phỏt hin SEA sa Khi phõn tớch que th phỏt hin SEA tinh khit dung dch m chy, chỳng tụi thy ngng phỏt hin ca que th khong 10 ng/ml Vỡ vy chỳng tụi tin hnh nhim ch ng SEA vo sa vi nng l 10, 30, 100 ng/ml Kt qu phõn tớch cho kt qu Hỡnh 3.15, nhn thy ch cú mu õm tớnh m v mu sa khụng nhim SEA thỡ cú xut hin vch v vch xut hin mu õm tớnh m thỡ rừ hn so vi vch mu õm tớnh sa Vy chỳng tụi cú th kt lun rng, nhy phỏt hin SEA ca que th sn phm sa khong 10 ng/ml v cht nn õy l sa (sau ó c pha loóng ln bng dung dch m chy) khụng nh hng n kt qu phõn tớch ca que th 69 Lun Vn Thc S Nguyn Th Hng Nhung Hỡnh 0.15 Th nhy ca que th vi SEA sa 1: Mu õm tớnh m khụng cha SEA ; 2: Mu õm tớnh sa khụng cha SEA 3: Mu cha 10 ng SEA 4: Mu cha 30 ng SEA 5: Mu cha 100 ng SEA 70 Lun Vn Thc S Nguyn Th Hng Nhung III.6.2.2 Xỏc nh nhy phỏt hin SEB sa Tng t nh vi SEA, cỏc kt qu phõn tớch vi khỏng nguyờn SEB tinh khit nhim ch ng vo sa Hỡnh 3.16 cho kt qu l: nhy phỏt hin ca que th l khong 10 ng/ml Hỡnh 0.16 Th nhy ca que th vi SEB sa 1: Mu õm tớnh sa khụng cha SEB 2: Mu cha 10 ng SEB 3: Mu cha 30 ng SEB 4: Mu cha 100 ng SEB 71 Lun Vn Thc S Nguyn Th Hng Nhung III.6.2.3 Xỏc nh nhy phỏt hin SEC1 sa Khi phõn tớch que th phỏt hin SEC1 tinh khit dung dch m chy, chỳng tụi thy ngng phỏt hin ca que th khong 100 ng/ml Vỡ vy chỳng tụi tin hnh nhim ch ng SEC1 vo sa vi nng l 100, 300, 1000 ng/ml Kt qu phõn tớch cho kt qu Hỡnh 3.17, nhn thy ch cú mu sa khụng nhim SEC thỡ cú xut hin vch Vy chỳng tụi cú th kt lun rng, nhy phỏt hin SEC1 ca que th sn phm sa khong 100 ng/ml Hỡnh 0.17 Th nhy ca que th vi SEC1 sa 1: Mu õm tớnh sa khụng cha SEC1 2: Mu cha 100 ng SEC1 3: Mu cha 300 ng SEC1 4: Mu cha 1000 ng SEC1 72 Lun Vn Thc S Nguyn Th Hng Nhung III.6.2.4 Xỏc nh nhy phỏt hin SED sa Mt cỏc tng t nh phõn tớch vi khỏng nguyờn SEC1, cỏc kt qu phõn tớch vi khỏng nguyờn SED tinh khit nhim ch ng vo sa Hỡnh 3.17 cho kt qu l: nhy phỏt hin ca que th l khong 100 ng/ml Hỡnh 0.18 Th nhy ca que th vi SED sa 1: Mu õm tớnh sa khụng cha SED 2: Mu cha 100 ng SED 3: Mu cha 300 ng SED 4: Mu cha 1000 ng SED 73 Lun Vn Thc S Nguyn Th Hng Nhung III.6.2.5 Xỏc nh nhy phỏt hin SEE sa Mt cỏch tng t nh phõn tớch vi khỏng nguyờn SEC1 v SED, cỏc kt qu phõn tớch vi khỏng nguyờn SEE tinh khit nhim ch ng vo sa Hỡnh 3.18 cho kt qu l: nhy phỏt hin ca que th l khong 100 ng/ml Hỡnh 0.19 Th nhy ca que th vi SEE sa 1: Mu õm tớnh sa khụng cha SEE 2: Mu cha 100 ng SEE 3: Mu cha 300 ng SEE 4: Mu cha 1000 ng SEE 74 Lun Vn Thc S Nguyn Th Hng Nhung III.6.2.6 nhy ca ton b quỏ trỡnh phõn tớch T cỏc kt qu sau phõn tớch bng que th vi loi c t rut ca t cu (SEA-SEE) phõn tớch m chy v s dng cht nn l sa, chỳng tụi cú th kt lun rng nhy phỏt hin ca ton b quỏ trỡnh phõn tớch nh sau : - Vi SEA v SEB: nhy phỏt hin ca que th khong 10 ng/ml - Vi SEC1, SED v SEE: nhy phỏt hin ca que th khong 100 ng/ml Khi so sỏnh vi cỏc phng phỏp ELISA hin ang c lu hnh trờn th trng hin cú nhy vi c t (SEA) khong ng/ml, nhy ca que th c phỏt trin ti cũn khỏ thp Tuy nhiờn, nu kt hp vi cỏc mỏy quột cm tay, nhy ca que th cú th c nõng cao 75 Lun Vn Thc S Nguyn Th Hng Nhung PHN IV KT LUN V KIN NGH IV.1 Kt lun Trong nghiờn cu ny, chỳng tụi ó t c cỏc kt qu nghiờn cu sau: ó bin np thnh cụng plasmid pGEX-6P-1-sea mang gen mó húa SEA vo t bo E coli BL21 ó sng lc c dũng t bo E coli cú kh nng sinh SEA cao ó xỏc nh c iu kin nuụi cy phự hp cho sinh tng hp SEA: nhit 30oC; nng cht cm ng IPTG 0,5 mM; thi gian cm ng: 5h Lng SEA c to iu kin ti u l 44 mg/l canh trng, gp khong 2000 ln so vi s dng S aureus ó tinh sch SEA tinh khit dng dung hp vi GST ó to thnh cụng que th vi nhy phỏt hin vi SEA v SEB khong 10 ng/ml, vi SEC1, SED v SEE khong 100 ng/ml IV.2 Kin ngh Tip tc nghiờn cu nhm ti u húa que th tng nhy phỏt hin vi loi c t rut ca t cu (SEA-SEE), n gin húa quy trỡnh phõn tớch cú th i vo sn xut, ng dng phỏt hin cỏc loi c t ny nhiu loi thc phm trờn hin trng ng dng mỏt quột cm tay phõn tớch bỏn nh lng cỏc c t ny 76 Lun Vn Thc S Nguyn Th Hng Nhung TI LIU THAM KHO Ting Vit on Th Nguyn (2009), Vi sinh vt hc, NXB Giỏo dc Vit Nam Lờ Huy Chớnh (2007), Vi sinh vt y hc, NXB Y hc, H Ni Nguyn Phỳc v cỏc cng s (2008), To khỏng th a dũng v xõy dng quy trỡnh ELISA phỏt hin c t rut nhúm A ca Staphylococcus aureus, Y hc TP H Chớ Minh 12:272-277 Nguyn Hong Minh v cỏc cng s (2008), A newly developed sandwich ELISA for possible rapid detection of Staphylococcal enterotoxin type A in milk product, Hi ngh khoa hc ton quc ln th IV Húa sinh v Sinh hc phõn t phc v nụng, sinh, y hc v cụng nghip thc phm, 15-17/10/2008 Ting Anh Akineden, O., Annemỹller, C., Hassan, A.A., Lọmmler, C., Wolter, W and Zschửck, M (2001), Toxin genes and other characteristics of Staphylococcus aureus isolates from milk of cows with mastitis, Clin Diagn Lab Immunol 8: 959-964 Balaban N, Rasooly A (2000), Staphylococcal enterotoxins, Int J Food Microbiol 61(1): 1-10 Bennet, R.W (1992), The biomolecular temperaments of staphylococcal enterotoxin in thermally processed foods, J Assoc Off Anal Chem Int 75: 612 Bergdoll, MS (1989), Production of a toxic shock syndrome toxin variant by Staphylococcus aureus strains associated with sheep, goats, and cows, J Clin Microbiol ;27(9):19461948 Bergdoll, MS (1989), Staphylococcus aureus, In Doyle, M.P (ed) Foodborne bacterial pathogens Marcel Dekker, Inc, New York, Basel, pp 463-523 10 Bergdoll, M.S (1983), Enterotoxins, In: Staphylococci and Staphylococcal 77 Lun Vn Thc S Nguyn Th Hng Nhung Infections (Easman, C.S.F and Adlam, C., eds.) 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