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Báo cáo " RAPID DETECTION AND QUANTIFICATION OF THE FOODBORNE PATHOGENES BY REAL-TIME PCR "

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RAPID DETECTION AND QUANTIFICATION OF THE FOODBORNE PATHOGENES BY REAL-TIME PCR

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  1. Tgp chi Cdng nghe Sinh hgc 7(4): 501-511, 2009 P H A T H I E N V A D I N H LlTONG N H A N H M Q T S O VI SINH V A T G A Y B E N H T R O N G THlTC P H A M B A N G R E A L - T I M E P C R Le Thi Chi, Nguyin QuIc Binh Trung tdm Cdng nghi sinh hgc Thdnh phd Hd Chi Minh TOM TAT Chimg toi da nghien cim va sir dung Real-Time PCR de phat hien va dinh lugng nhanh mot so vi sinh vat gay benh duong mot chinh a nguai trong thuc pham. Quy trinh da sir dung mot moi tnrong tang sinh chung va mgt chuong trinh PCR sir dung true tiep dich tang sinh voi 5 cap primer dac hieu cho 5 loai vi khuan gay benh frong thuc pham la Escherichia coli. Salmonella spp., Shigella spp., Listeria monocytogenes va Bacillus cereus. Cac primer dugc chgn dac hieu 100% voi loai dich va kliong xay ra phan img cheo voi cac loai khac. Phuong phap nay don gian, khong can ly trich, tinh sach DNA de thuc hien, co do nhay cao, phat hien dugc tir 1-10 ban sao DNA/phan irng khi dugc kiem tra voi vi khuan thuan. Do nhay phat hien ciia phuong phap la 1 - 10 CFU/25 g mau thirc pham va sau khi tang sinh 24 h thi mau nhiem co ham lugng vi sinh vat tir lO' -10^ CFU/ml (R^ = 0,998) ket qua dinh lugng nay khong chenh lech nhieu so voi phuong phap dem khuan lac (p = 0,90). Do chinh xac, do nhay va do dac hieu cao so voi phuong phap tmyen thong. Cac gia tri tmng binh nay la 97,99; 98,05 va 95,60%. Toan bo qua trinh chi mat 4 - 30 h so voi thoi gian 5 - 7 ngay khi sir dung phuong phap tmyen thong. Tie khoa: Bacillus cereus, Escherichia coli. Listeria monocytogenes, Real-Time PCR, Salmonella, Shigella, vi sinh vgt gdy benh trong thuc phdm GIOI THIEU - 7 ngay), cong siic, tien ciia, nguy hiem va kho khan cho ky thuat vien (Lampel et al, 1990). Ngg dgc thuc pham da va dang la moi de dga doi Hien nay, co rat nhieu nghien ciiu img dung PCR vai sire khoe cgng dong. Theo thong ke trong nhiing truyen thong va PCR cai tien nhu Nested PCR va nam gan day, binh quan bang nam c6 khoang 76 trieu Real-Time PCR de phat hien nhanh cac vi sinb vat nguoi nhiem benh va 5000 nguai chet (Mead et al, gay benh frong thuc pham. Cac nghien ciiu nay co the 1999). Theo thong ke ciia Trung tam Kiem dich va dimg de phat hien mgt bay nhieu chimg dich. Phat Phong benh My (CDC) frr nam 1993 din nam 1997 hien Salmonella frong cac loai thuc pham khac nhau xay ra 2751 vu ngg dgc thuc pham o My. Nguyen ciing da dugc cong bo (Waage et al, 1999; nhan dugc xac dinh cao nhat la do vi sinh vat gay nen Uyttendaele et al, 2003; Malomy et al, 2004; Wolffs chiem tir 75 - 86%). Theo theo thong ke ciia Bg Y te, et al, 2006; Josefsen et al, 2007; O'Regan et al, frong nam 2008, Viet Nam co 205 vu ngg dgc thuc 2008), Shigella (Lampel et al, 1990; Lindqvist 1999); pham lam 2258 truong hgp nhap vien va tap trung chu L. monocytogenes (Thomas et al, 1991; Filter et al, yeu a cac khu cong nghiep, khu che xuat. 1992; Wang et al, 1992), B. cereus (Fricker et al, 2007), hay phat hien dong thai mgt so chiing dich Cac vi sinh vat gay benh trong thuc pham theo (Wang et al, 1997; Fukushima et al, 2003; Nguyen et moi quan tam cua co quan Quan ly Thuc pham va al, 2004; Wang et al, 2007). Nhin chung, cac quy Dugc pham My (FDA) la: Escherichia coli. trinh de phat hien cac vi sinh vat frong thuc pham Salmonella, Shigella, Campylobacter, Yersinia bang PCR kha khac nhau. Khac nhau ve buac chuan enterocolitica, Y. pseudotuberculosis, Vibrio bi DNA cho phan irng (DNA khuon), chuang frinh cholerae, V. parahaemolyticus, V. vulnificus. PCR, thiet bi PCR va be dem. Hau het cac nghien cim Listeria monocytogenes. Staphylococcus aureus. noi fren co do nhay phat bien tir 1 - 10 CFU/g thuc Bacillus cereus, Clostridium perfringens va C. pham va do nhay phan img tir 10 ban sao DNA/phan botulinum. ling fra len. Cac tieu chuan ve an toan thuc pham quy Viec phat hien cac vi sinh vat theo phuang phap dinh doi voi thuc pham phai khong bien dien mgt so nuoi cay truyin thing (tang sinh, pban lap va khing loai vi khuan nhu Salmonella, Shigella, L. dinh sinb hoa, huylt thanh) vira ton kem thai gian (5 monocytogenes, E. coli 0157:H7 frong 25 g mau thi 501
  2. Le Thi Chi & Nguyin QuIc Binh cac phuang phap PCR fren chua dap img dugc do liet ke theo bang 1. Mgt s l moi truang sir dung de nhay can thiet theo phuang phap chuan (phuang phap chgn ra moi truang lam giau chung cho 5 chimg vi nuoi cay truyen thong). Vi vay, can thiet co mgt quy khuan can phat hien bao gIm: Buffer peptone water trinh phat hien vi sinh vat gay benh mai vira dap img (Oxoid, UK), Luria Bertani ((peptone 10 g/l (Biorad, dugc do nhay tuong img voi phuang phap nuoi cay USA), yeast exfract 5 g/l (Biorad, USA), NaCI 5 g/l truyen thong, vira co do chinh xac cao, don gian va co (Merck, Diic)), Tryptic Soya Broth Yeast Extract thai gian xet nghiem nhanh ban phuang phap nuoi ((TSBYE: TSB 30 g/l (Merck, Diic), yeast extract 6 cay truyen thong. g/l)) va Listeria cai bien (Merck, Dire). Mgt so moi truang agar chgn lgc: Hectoen enteric (HE), Xylose Dual day, nghien ciru cua chiing toi dua ra mgt Lysine Desoxycholate citrate (XLD), Desoxycholate quy frinh Real-Time PCR thong qua mgt buoc nuoi cifrate (DC), MacConkey, Mannitol Egg York cay lam giau cho cac vi khuan dich va phuang phap Polymycine (MYP), Oxford Agar va Violet Red Bile xir ly te bao chung cho 5 loai vi sinh vat gay benh Agar (VRB) (Merck, Diic). pho bien trong thuc pham (E. coli. Salmonella spp., Shigella spp., L. monocytogenes va B. cereus) nham Tat ca cac chiing deu dugc nuoi cay frong dieu phat hien va xac dinh dugc nong do vi sinh vat sau kien vi hieu khi a 37 °C trong thai gian 20 - 24 h. khi lam giau trong mau thuc pham. Rieng doi voi chitng Vibrio moi truang TSBYE dugc dilu chinh pH den 8,5 va bo sung muoi NaCI den nong do 3%. Tat ca cac chung dugc bao quan a VAT LIEU v A PHUONG PHAP -70°C vai nong do glycerol 10%. Cac chung vi khuan, moi tru-cmg va dieu kien Moi truang phan lap cac chimg dich: HE va nuoi cay XLD {Salmonella), DC va MacConkey {Shigella), VRB {E. coli), MYP {B. cereus) va Oxford Agar {L. Cac chiing vi khuan dung trong nghien citu dugc monocytogenes). Bang 1. Cac chiing vi khuan sir dung 6k kiem tra < 6 dac hieu cac cap primer. 3 Ten chung Nguon goc Ky hieu Salmonella spp. Trung tam phan tich TPHCM Sal si Salmonella spp. Vien Pasteur TPHCM Sals2 Salmonella typhimurium Vien Pasteur TPHCM SA-106 Shigella spp. VTCC Shil Shigella spp. Vien Pasteur TPHCM (100102) Shi 2 L monocytogenes Vien Pasteur TPHCM Lis mono V. parahaemolyticus Truong Dai hgc Bach khoa TPHCM Vpa 1 V. parahaemolyticus Vien Pasteur TPHCM Vpa2 E. coli Truong Dai hgc Bach khoa TPHCM E coli 1 E. coli ATCC 25922 ATCC 25922 B. cereus Vien Pasteur TPHCM B cer Staphylococcus aureus Vien Pasteur TPHCM S. au B. thunngiensis VTCC BUN B. thu Corynebactenum glutamicum VTCC B656 B656 Xir ly te bao loai bo dich noi, bo sung 50 pi nuac cat vo trimg, vortex deu va ly tam cimg dieu kien nhu tren thu Cac chiing vi khuan thuan dugc nuoi cay qua nhan sinh khoi (lap lai 3 ian). Xir ly tl bao bSng dem frong moi truang TSBYE. Riit 500 pi vao phuang phap dun soi va phuang phap phan giai eppendorf 1,5 ml, ly tam 10000 rpm frong 10 phiit. kilm. Doi vai phuang phap dun soi, sinh khii vi 502
  3. Tgp chi Cdng nghi Sinh hgc 7(4): 501-511, 2009 sinh vat sau khi nia, bl sung 50 pi nuac cat vo triing, Do nhay phat hien trong mau thye pham vortex deu trong 2 phut, dun soi 10 phiit va lam lanh De kiem tra kha nang phat hien cac vi khuan ngay de pha va te bao, dun soi lai 5 phut. Doi vai hien dien trong mau thuc pham, tien hanh cac thi phuang phap phan giai kiem, sinh khoi dugc bo sung nghiem nhiem nhan tao fren mgt so co chat thuc 10 pi nuac cat vo triing, vortex trong 2 pbiit, sau do pham (bim tuoi, cha lua, ngheu va thit bam). Cac bo sung 20 pi dung dich 0,1 N NaOH, vortex trong 2 mau thuc pham dugc chuan bi bang each can 25 g phut, tiep theo trung hoa kiem bang 20 pi dung dich mau vao tiii nilong (20 x 30 cm) c6 vai lgc, bo sung 1 M Tris-HCl pH 6,0. them 225 ml TSBYE. Tat ca dugc hap tiet triing o Primer va chu'O'ng trinh Real-Time PCR 121°C trong 20 pbiit. Sau khi hap de ngugi hoan toan. Mgt dung dich vi khuan thuan biet truoc nong • cac gen dich dugc tham khao tren GenBank do bo sung vao mau thuc pham vo khuan, dong nhat (http://www.ncbi.nlm.nih.gov/Genbank/) va so sanh mau bang may dap mau (stomacher 400, UK) trong cac gen bang chuang trinh clustalW 2 phut. Dem u a 37°C trong thai gian 20 - 24 h. Lay (http://www.ebi.ac.uk/Tools/clustalw2/index.html). 1 ml dich tang sinh ly tam a 2000 rpm/3 phut, hiit Primer dugc thiet ke voi chuang trinh primer3 \ 500 pi dich noi de tien hanh xir ly te bao theo hai 0.4.0 (http://frodo.wi.mit.edu/) va cac primer dugc phuang phap dugc mo ta. Dong thai vai xir ly te bao tong hgp boi IDT (USA). 18 cap primer %ii dung cho phuang phap phan tich Real-Time PCR, tien frong nghien ciiu dugc thiet ke mai 12 cap v.i 6 cap banh kiem fra nong do te bao vi khuan trong mau tham khao tit mgt so ket qua nghien cim liioc bang phuang phap dem khuan lac tren dia doi vai (Thomas et al, 1991; Wang et al, 1997; Fukushima timg chiing vi khuan fren moi truang chgn lgc tuang et al, 2003; Croci et al, 2004). Chuang trinh Real- ling vai tirng chiing dich. Time PCR dugc tien hanh nhu sau: Chuan bi mau xay dyng ducmg chuan Chu ky dau: 95°C frong 5 phiit, 40 chu ky tiep theo: lap lai cac buoc: 95°C trong 30 giay, 55°C Duong chuan xay dung tir 4 phan iing co ham frong 30 giay, 72°C frong 30 giay. Chu ky cuoi: 72°C lugng te bao dugc xac dinh bang phuang phap dem frong 5 phut. BChoang gia nhiet At = 0,5°C. khuan lac. Ham lugng DNA frong 4 mau chuan c6 ham lugng tuang iing la 10^; IO'*; 10^; 10^ ban sao Phan img Real-Time PCR dugc thuc hien vai DNA/pi. may Cycler iQ^'^5 (BioRad, USA) frong mgt hin hgp C tong the tich 25 pi bao gom: nuac cat vo Chuan bi mau thirc pham O triing 10,5 pi, MasterMix 2X (Fermentas, Canada) Muoi ba mau thuc pham bao gom 9 mau hai san 12,5 pi, primer 10 pM rieng cho tirng chiing 1 pi va va 4 mau sua tuoi dugc thu thap ngau nhien tai sieu DNA khuon 1 pi. thi Big C mien Dong (To Hien Thanh, tbanh pho Ho Kiem tra do dac hieu cua primer Chi Minh), cac mau dugc trii lanh a 4°C frong 1 ngay. Chuan bi mau cho kiem fra cac vi khuan dich bang Cac cap primer dugc kiem fra vai cac chung Real-Time PCR nhu sau: 25 g (ml) -\- 225 ml TSBYE, thuan theo chuang frinh Real-Time PCR noi fren. tang sinh frong 24 h a 37°C. Song song voi buac Ket qua phan iing dugc dien di tren gel 1,2% agarose chimn bi de phan tich bang Real-Time PCR, chuan bi nhuom vai ethium bromide (50 pg/ml) 20 phut, hien mau cho phan tich bang phuang phap truyen thong mau voi UV bang thilt bi Gel Doc (BioRad, USA). (FDA, 1998). BChi kiem fra bang phuang phap truyen Kiem tra do nhay cua phan ung Real-Time PCR thong chiing toi lua chgn mgt so khuan lac dac trung khang dinh bang PCR thay vi khang dinh bang cac Cac chung thuan dugc nuoi cay qua dem trong phan ling sinh hoa va huyet thanh. moi truang TSBYE, kiem tra nong do te bao fren moi truang chgn lgc tuong iing (Salmonella/UE, Te bao dugc dugc xit ly bang theo phuang phap ShigellafMacConkcy, Listeria monocytogenes/ dun soi. Oxford Agar, E. coli/NRB, B. cereusMYV). Xir ly tl bao bang phuang phap dun soi va phuang phap phan Tinh toan do chinh xac, do dac hieu va do nhay giai kilm nhu da mieu ta. Cac dimg dich sau khi da cua phuo'ng phap Real-Time PCR (O'Regan et al, xu ly dugc pha loang din lO"; 10^ lO'; lO' va 10° 2008) ban sao DNA/pl (xem 1 tl bao tuong duong 1 ban . , PA + NA sao DNA) va sir dung lam DNA khuon. Do chinh xac (AC): AC = x 100% 503
  4. Le Thi Chi & Nguyin QuIc Binh NA phap Real-Time PCR; Do dac hieu (SP): SP = x 100% N": NA + PD; PA N"": ND -h PA. Do nhay (SE): SE= X 100% N^ KETQUA Trong do: PA: sl mlu cho kit qua duong a ca hai phuang Kiem tra do dac hieu ciia cac primer pbap (Real-Time PCR va truyen thong); Tir 18 cap primer chgn dugc 5 cap primer dac NA: s l mlu cho kit qua am tinh o ca bai hieu cho 5 loai dich (Bang 2; 4 va Hinh 1) bao gom phuang phap (Real-Time PCR va truyen thong); primer iai cho Shigella, primer invA cho Salmonella, N: Tong so mau phan tich; primer BC cho B. cereus, primer LL cho L monocytogenes va primer eae2 cho E. coli. Tat ca 5 PD: s l mau cho kit qua duong tinb gia a Real- cap primer nay khong cho phan iing cheo voi cac Time PCR; loai khac. San pham sau khi PCR dugc dien di tren ND: s l mau cho kit qua am tinh gia voi phuang gel 1,1% agarose (ket qua khong trinb bay o day). Bang 2. Trinh tg cac primer dac hieu. Primer va trinh ty (5' - 3') Gen dich/protein Tham khao invA 139, GTG/\AATTATCGCCACGTTCGGGC/\A invA Fukushima ef al, 2003 invA 141, TCATCGCACCGTC/V\AGG/VKCC iaI-F, GCTGTACTTTCGGTGG/\AGC lai Nghien CLPU nay iaI-R, ACGTCATGCCAGCTATTTCC eae2-F, CACCACCACCATCACCATTA eae2 Nghien CLPU nay eae2-R, ATGTACCGCCG/\ACTTCAAC LL5,AACCTATCCAGGTGCTC hlyA Thomas etal, 195 LL6, CTGTAAGCCATTTCGTC BC-1, CTGTTAGCG/\ATCGTAC hemolysine Wang eta/., 1997 BC-2, TACTGCTCCAGCCACATTAC Bang 3. So sanh kha nang tang tru-ang cua cac loai vi khuan tren mpt so moi tru'O'ng tiln tang sinh. Cac chung Mdi tryang BPW LB TSBYE Listeria cai bien Salmonella 1,00 1,17 28,04 Khong kiem tra Shigella 0,45 0,65 1,00 Khong kiem tra L monocytogenes 0,12 1,49 2,38 1,00 E. coli 1,00' 1,08 58,11 Khong kiem tra B. cereus 1,00' 0,97 0,89 Khong kilm tra Ghi chu:": khong co trong quy trinh FDA's BAM. 504
  5. Tgp chi Cdng nghi Sinh hgc 7(4): 501-511, 2009 Bang 4. Kit qua kilm tra cac primer dac hieu. Chung VSV Primer invA la! LL eae2 BC Sal si + Sals2 + SA-106 + Shil - Shi 2 - Lis mono - Vpal - Vpa 2 - E coli 1 - ATCC 25922 - B cer - S. au - B. thu - B656 - Kich thLPOc san 284 107 267 185 251 pham (bp) Tm ("C) 82,3 + 0,7 78,5 ±0,4 74,5 ± 0,7 80,6 ± 0,5 77,0 ± 0,4 Ghi chu: -i-: Du-ong tinh; -: Am tinh; Tm: dipgc tinh trung binh tu 20 - 25 phan Cpng. Moi trirong tang sinh chung cho cac chiing vi sinh vat ket qua duang tinh khi sir dung phuong phap nuoi cay truyen thong va phuang phap Real-Time PCR te Bon loai moi truang dung de kiem tra su tang bao xir ly bang each dun soi va ket qua dinh lugng truong cua cac chiing Salmonella, Shigella, E. coli, giira 2 phuang pbap nay khong dang ke (p = 0,90). B. cereus va L. monocytogenes. Neu quy dinh moi Trong khi do voi phuong phap Real-Time PCR te truang tien tang sinh quy trinh FDA's BAM (ngoai bao xu ly bang kiem thi co 14/175 mau cho ket qua tni' E. coli va B. cereus tu quy uoc) cho timg chiing am tinh va ket qua djnh lugng dao dgng tir 10 10' vi khuan la 1,00 thi ket qua kiem fra cac moi truang ban sao DNA/ml. dugc the hien trong bang 3, cho thay rang moi truang TSBYE la moi truang ma cac vi khuan dich Cac gia tri AC, SE, SP dugc tinh toan tir 30 - tang sinh tot nhat. 40 mau nhiem nhan tao, trung binh 97,99; 98,05 va 95,6%o (khi sir dung phuong phap dun soi) va 86,44; Tir ket qua nay chung toi da sir dung moi truong 89,97 va 89,33%) (khi sir dung phuong phap phan TSBYE de lam giau chung cho cac vi khuan dich. giai kiem) Do nhay phan irng Real-Time PCR Do vay khi tien hanh phan tich hien trang nhilm khuan cua thuc pham, chiing toi sir dung phuong Do nhay phan irng Real-Time PCR ciia cac cap phap dun soi de xu ly mau. primer dugc kiem tra trong dung dich chira DNA tir vi khuan thuan, co kha nang phat bien khi su hien Kiem tra mau thuc pham dien cua duai 10 ban sao DNA /mgt phan img. Khong C mau nao phat bien Listeria O Do nhay phat hien monocytogenes. Cac mau bi nhiem vi sinh vat dich sau khi tang sinh, ket qua dinh Iugng dao dgng hi Tat ca cac mau thuc pham dugc gay nhiem (35 10 "10 ban sao DNA/ml. Toan bg kit qua phan mlu/ chiing dich) sau khi tang sinh 20 - 24 h deu cho ticb the hien trong bang 5 va hinh 2. 505
  6. Le Thi Chi & Nguyin Quoc Binh lUMf.A.l-U'lt t A W J ^ l l I I ULta.! 1 1 , 1 i ^ V / • " " _^"" 1 r ,/ / / • •j / / 1 / i M1 A ^ /' lit- ^' .' »jeS?! • ll • " • • • -7^=" . 1 1 . 1 yi ? primer iiivA / Salniottella B J ^ »l^*n(h»t.t N»»ft/ftl'IHlAw} iw w Om. c* aMi-nitttirt 1 1 1 '~\ .^ f it) 1! £ / / « —f- f ll' / 1 *» 1 * / ! 3 /' / « / / /" __^ ^_ _^ ; _ f 1 r ii;H • ^ A r- ? r.*i t ' ^ VM s s C S n J t,C«n 1 e f primer BC / Bacillus cereus B u% . « ( M , , H.M114H.>M. fw letivacrirt [•«:a!iir.^iii» 1 * R » 1 B.s.ir h i 71! : | 3 ^W j f 1 ;i 1 1 / / ^inl 1 ^ ^ , i / i ,J«* •«ateiw • N M M H 1 i ;M. • ^ - - - . ^, - ^ .^_ . - , ,....,, \ * * ' "-7"—¥ • •! li\* fc»?^3 a.' ; 1 1 i.,< 1 \ « 1 4J i e J 3 primer eae2 E ro// B 506
  7. Tgp chi Cdng nghi Sinh hgc 7(4): 501-511, 2009 ft COS33BJSJIJ1Z5MU *tK*'Do. cojjnwiuciaj 1 1 C?-?il.l 1 :i ~im j; 'J ^ (^ ;• • •ii 5 ^ ,'';' il'^**'' ******K*^!t i!"**^* **'.* • 1 .' • 1 * " v . . , . , ..^ ,..._„ -. r~ F TJP * . ' ,.„ . ^,r.rt * - . , «t: E;m3i^[£^:st :si3U mvfst'39t:imi!s,wz\'maA 1 1 i < [ fi.Sfffir 1 X' 1 • ^ ^ i| t '' » 1 1 ' t 1 < , • ' 1 * > i 5 Sl * 11 1 . i J 1 r tr.« ^-' .. , j • 1 * lt*^«"'»l "";hK:;s*e' ,J i !'"^^1 t t *-» -i^- ,-^» . : '. • i • t ••-*-i J * '• ^s 5 1 II T G 3 X S prinier LL / L. inonocytogerws B Hinh 1. Phan tich k i t qua Reai-Time PCR vai cac cap primer dac hieu tirang Cpng vai chiing dich. A. Bieu di§n chu ky phat hien (Ct); B. Bieu dien gia tri Tm cua san pham PCR. Unknov.Ti 40; 351 25; ZD; is: 0 2 4 6 Log Starting Quantity,copv number -^SYBRl E - Sr.SSf R A 2 _ o . 9 9 S s 1 o p e - - 3 . 6 6 2 y--in-t=42.- PCR Standard Curve : Data 2 0 0 9 - 0 4 - 1 7 12S9.opd Hinh 2. D u a n g chuan de djnh lu'gng ban sao DNA trong phan tich bang Real-Time PCR. 507
  8. Le Thi Chi & Nguyin QuIc Binh Bang 5. Phan tfch hien trang nhiim khuan cua thge pham sau khi Iam giau bang Real-Time PCR va bang phuang phap truyen th6ng. Miu thiPC pham Salmonella Shigella L. monocytogenes B. cereus £. coli Tom the -1- 8,6.10'/-i- -/- -h -1+ So lua 4,88.10'/-!- 2,75.10V -/- -/- 1,35.10'% Ca vien basa -/- 1,75.10^°/-!- -/- 2,26.10% 2,26.10% Mge ong nguyen -/- 4,04.10'/-i- -/- 9,86.10% 8,22.10% con Muc 6ng cat —I— -/+ -/- 3,74.10% 3,45.10% khoanh Ngheu lua dong —1— 8,85.10% -/- 6,35.10% 1,14.10'% lanh Sua tLPai huang —j— -/- -/- 2,44.10'% 2,84.10% dau Sira tu'al khong —I— 1,09.10'/-H -/- 1,93.10% 7,86.10% du'ong So long luge -1- -/+ -/- -/-•- -h DIU mge -/- -/+ -/- 4,70.10% 2,45.10% C6i so diep -/- 1,43.10% -/- 3,52.10°/- 1,97.10% Sira tLPai co -/- -/- -/- 2,08.10% -/- duang SiJ'a dau nanh 1,37.10% Ghi chu: /- hay l-t-: Ket qua phan tfch bang phipang phap truyin th6ng (/-: Am tinh, h: Duang tinh). THAO LUAN thuan, ket qua dinh lugng ciia Real-Time PCR vai te bao vi khuan pha vo bang phuong phap dun soi va Thuc pham, khi bi nhiem vi sinh vat, no co the phuang phap phan giai kiem khong chenh lech so bi nhiem nhieu loai vi sinh vat khac nhau va o mat voi phuang phap dem khuan lac (p = 0,90). Trong do thap, khong nhu doi vai mau benb pham. Do vay, khi cac phan tich Real-Time PCR doi voi vi khuan tir hau bet cac nghien cira de phat bien vi sinh vat trong mau thuc pham thi DNA khuon tir phuong phap dun thuc pbam deu co giai doan tang sinh tren moi soi cho ket qua dinh tinh va dinh lugng khong chenh trucrng khong chgn lgc de khoi phuc cac te bao bi ton lech nhieu so vai phuang phap truyen thong nhung thuong trong qua trinh che bien va bao quan, moi DNA khuon tir phuang phap pban giai kiem it on loai dugc tang sinh tren moi truang khac nhau. Dieu dinh ban so voi phuang phap truyen thong. Ket qua nay vira iam mat thai gian va cong sue chuan bi mau nay cho thay khi sir dung phuang phap phan giai cho phan tich. Vi vay, chung toi da tien hanh thir kiem de pha vo te bao vi khuan trong thuc pham gay nghiem tit 4 loai moi truang rieng biet BPW, ire che phan irng PCR vi su hien dien ciia hon hgp TSBYE, LB va Listeria cai bien de chgn ra mgt moi cac chat phirc tap trong thuc pham, khi xir ly bang truang tang sinh chung cho 5 loai vi khuan can phat NaOH C the tao ra mgt so chat moi ire che phan O hien la TSBYE. Moi truang nay ciing da dugc sir img, con doi voi phuang phap dun soi kha nang hinh dung trong nghien cira truoc (Wang et al, 1997; thanh cac chat mai sau khi xit ly thap. Do vay, DNA 2007). Trong nghien cira nay, chung toi da thiet ke khuon ebo phan ung Real-Time PCR tir dich tl bao tbanh cong hai cap primer moi cho E. coli vk xir ly bang phuong phap dun soi cho ket qua on dinh Shigella dua tren hai doan gen eaeA cua E. coli va hon phuo'ng pbap phan giai kiem. Nen chimg toi da iai (mgt locus tren plasmide xam nhiem) cua sir dung phuang phap dun soi de xir ly te bao vi Shigella. Chimg toi da sir dung hai phuo'ng phap pha khuan trong mau thuc pham. Phuang phap chuan bi vo te bao la phuang phap dun soi va phuong phap DNA khuon dugc chgn -vita don gian trong thao tac, phan giai kiem de chgn ra phuong phap chuan bi vita it ton kem so voi mg so phuang phap ly trich DNA toi U'U cho phan irng. Doi voi mau vi khuan DNA khac nhu sii' dung proteinase K, kit ly trich 508
  9. Tgp chi Cdng nghe Sinh hgc 7(4): 501-511, 2009 DNA (Fukushima et al, 2003). Chiing toi sit dung ket qua phan tich trung thuc ban cac phuang phap thi nghiem gay nhilm nhan tao dl so sanh hai PCR thuang. Vi moi chiing dich dugc phan tich phuang phap xir ly tl bao vi chimg gay nhiem la trong moi phan itng khac nhau, do vay phuang phap chimg thuan, c6 thl xac dinh nong do te bao trong chimg toi co do nhay cao tir 1 - 10 te bao/phan img mau sau gay nhiem bang phuang phap dem khuan va c6 kha nang phat bien va dinh lugng hien dien cua lac. vi sinh vat dich tir 1 - 10 te bao/25 g mau thuc pham sau khi tang sinh, ket qua dugc phan tich ngay trong Mgt so nghien ciiu truac day phat hien dong thai qua trinh phan irng nen ket qua phan tich trang thuc hai hay nhieu chimg dich trong cimg 1 phan img, co ban phuang phap PCR thuang. Do chinh xac, do do nhay 10^ te bao/g (Fukushima et al, 2003), phat nhay va do dac hieu ciia phuang phap chiing toi dua hien dong thai E. coli 0157:H7, Salmonella va Shigella bang multiplex Real-Time PCR co do nhay ra so vai phuang phap trayen thong cao, cac gia tri phan ling tir 10 - 100 te bao/phan img va c6 kha nang tuang img 97,99; 98,05 va 95,6%. phat Men doi vai E. coli 0157: H7, Salmonella, Mat khac, trong nhiing traang hgp ngg dgc thuc Shigella \in lugt la 10^ 10^ va IO"* tl bao/g (Wang et pham khi tieu thu nhiing thuc pham da qua nau chin al, 2007), hay phat hien dong thai L. monocytogenes ma vai phuang phap phan tich trayen thong khong va E. coli 0157:H7 co do nhay phat hien tuong ung the xac dinh dugc nguyen nhan thi phuang phap la 1 - 6 va 1 - 3 te bao/g thit bo sau khi tang sinh 30 h Real-Time PCR hoan toan co the vi phuang phap tren moi truang BHI (Nguyen et al, 2004), 5.10^ tl dua tren phan tich DNA. bao Shigella/phan irng (Wang et al, 1997), 10 CFU/g (thuc pham hay phan) E. coli va Salmonella Vai phuang phap chung toi dua ra, neu phan sau khi tang sinh 18 h tren moi truang GNTSB (moi tich mau benh pham co nong do vi sinh vat ban dau truang Gram Negative -i- TSB) (Sharma et al, 2000). cao C the phan tich true tiep tir mau ma khong trai O Mgt so nghien ciru dimg PCR doi vai tirng chiing qua giai doan tang sinh, ket qua phan tich chi trong dich C kha nang phat hien tir 1 - 10 te bao/g O khoang 4 h. (Lindqvist, 1999; Eyigor et al, 2002; Croci et al, 2004; Josefsen et al, 2007), 1 0 - 1 0 0 CFU/g (Filter KET LUAN et al, 1992), 1 - 10 te bao/25 g mau nhung phai qua 24 - 48 h tang sinh tren BPW (Uyttendaele et al, Tir ket qua nghien ciiu nay, chiing toi ket luan 2003; O'Regan et al, 2008), 5 - 50 tl bao/25 g (Wang et al, 1997; Cheung et al, 2004). Do nhay rang phuang phap Real-Time PCR voi DNA khuon ciia phan iing tir 1 - 10 te bao/phan irng (Lindqvist, sir dung trong phan tich tir te bao dugc xir ly bang 1999; Uyttendaele et al, 2003; De Medici et al, phuang phap dun soi do chiing toi xay dung nen 2003; Wolffs et al, 2006), 5 - 50 tl bao/phan iing hoan toan co the thay the phuang phap nuoi cay co (Malomy et al, 2004), 50 - 500 tl bao/phan img dien de dinh tinh va dinh lugng vi sinh vat gay benh (Filter et al, 1992), 10 - lO^tl bao/phan iing (Lampel trong mau thuc pham tren phuang dien do nhay va et al, 1990). Nhin chung cac nghien ciiu trade day do chinh xac. Mat khac, phuang phap cua chimg toi khi phat hien dong thai cac chung trong cimg 1 phan CO thai gian thuc hien ngan ban nhieu (4 - 30 h so ling tuy nhanh nhung co do nhay thap va kha nang voi 5 - 7 ngay) va thao tac don gian ban. Phuang phat hien trong thuc pham chua cao so vai phuang phap nay nen dugc irng dung cho cac trang tam phan phap truyen thong, trong khi do cac phan tich PCR tich ve viec danh gia an toan ve sinh thuc pham, dac cho timg chiing rieng le co kha nang phat hien su biet hiiu ich trong cac traang hgp chan doan nhanh hien dien cua chiing vi khuan dich cao (Uyttendaele cac ca ngg dgc thuc pham. et al, 2003; O'Regan et al, 2008), nhung dl phat hien moi chiing dich phai sir dung mgt moi truang Lcri cam an: Nhdm nghiin cieu cua chiing tdi xin tang sinh khac nhau va san pham dugc phan tich chdn thdnh cdm an Trung tdm phdn tich thi nghiem trong buoc hau PCR bang phuang phap dien di hay Thdnh phd Hd Chi Minh, truang Dgi hgc Bdeh khoa lai DNA, dilu nay de xay ra hien tugng lay nhiem Thdnh phd Hd Chi Minh da tgo diiu kien thudn lgi cheo, cho kit qua duang tinh gia. Tong hgp mgt so vd giup do chiing tdi trong nghien cieu. uu dilm cua cac nghien cira ciia cac tac gia traoc day chiing toi da nghien cira va dua ra mgt quy trinh T A I L I E U THAM K H A O dl phat hien nhanh, du do nhay, dan gian va tilt kiem dugc thai gian chuan bi DNA cho phan tieh va Cheung FY, Chan CW, Wong W, Cheung TL, Kam KM (2004) Evaluation of two real time polymerase chain 509
  10. Le Thi Chi & Nguyin QuIc Binh reaction pathogen detection kits for Salmonella spp. in detection of Salmonella in food. Appl Environ Microbiol food. Lett Appl Microbiol 39: 509-515. 1.0: 7046-7052. Croci L, Delibato E, Volpe G, De IVledici D, Palleschi G Mead PS, Slutsker L, Dietz V, MeCaig LF, Bresee JS, (2004) Comparison of PCR, Electrochemical Enzyme- Shapiro C, Griffin PM, Tauxe RV (1999) Food-related Linked Immunosorbent Assays, and the Standard Culture illness and death in the United States. J Emerg Infect Dis Method for detecting Salmonella in meat products. Appl 5: 607-625. Environ Microbiol 70: 1393-1396. O'Regan E, McCabe E, Burgess C, McGuinness S, Barry De Medici D, Croci L, Delibato E, Di Pasquale S, Filetici T, Duffy G, Whyte P, Fanning S (2008) Development of a E, Toti L (2003) Evaluation of DNA extraction methods real time multiplex PCR assay for the detection of multiple for use in combination with SYBR Green I Real-Time Salmonella serotypes in chicken samples. BMC Microbiol PCR to detect Salmonella enterica Serotype Enteritidis in 8: 156. poultry. Appl Environ Microbiol 69: 3456-3461. Sharma VK, Calson SA (2000) Simultaneous detection of Eyigor A, Carli KT, Unal CB (2002) Implementation of Salmonella strains and Escherichia coli 0157:H7 with Real-Time PCR to tetrathionate broth enrichment step of Fluorogenic PCR and single-enrichment-broth culture. Salmonella detection in poultry. Lett Appl Microbiol 34: Appl Environ Microbiol 66: 5472-547. 37-41. Thomas EJ, King RK, Burchark J, Gannon VP (1991) FDA (1998) FDA's Bacteriological Analatycal Manual Sensitive and specific detection of Listeria monocytogenes (BAM), 8* ed. in milk and ground beef with the Polymerase Chain Reaction. Appl Environ Microbiol 57: 2576-2580. Filter S, Heuzenroede M, Thomas CJ (1992) A combined PCR and selective enrichment method for rapid detection Uyttendaele M, Vanwildemeersch K, Debevere J (2003) of Listeria monocytogenes. J Appl Bacterial 13: 53-59. Evaluation of Real-Time PCR vs automated ELISA and a conventional culture method using a semi-solid medium Fricker M, MesselhauBer U, Busch U, Scherer S, Ehling- for detection of Salmonella. Lett Appl Microbiol 37: 386- Schulz M (2007) Diagnostic Real-Time PCR assays for the 391. detection of emetic Bacillus cereus strains in foods and recent foodbome outbreaks. Appl Environ Microbiol 73: Waage AS, Vardund TLV, Kapperud G (1999) Detection 1892-1898. of low numbers of Salmonella in environmental water, sewage and food samples by a nested polymerase chain Fukushima H, Tsunomori Y, Seki R (2003) Duplex Real- reaction assay. J Appl Microbiol 87: 418-428. Time SYBR Green PCR assays for detection of 17 Species of food- or waterbome pathogens in stools. J Clin Wang L, Li Y, Mustaphai A (2007) Rapid and Microbiol41: 5\34-5\46. simultaneous quantitation of Escherichia coli 0I57:H7, Salmonella, and Shigella in ground beef by multiplex Josefsen MH, Krause M, Hansen F, Hoorfar J (2007) Real lime PCR and immunomagnetic separation. J Food Optimization of a 12-hour TaqMan PCR-based method for Pr, ^0: 1366-1372. detection of bacteria in meat. Appl Environ Microbiol 73: 3040-3048. Wang RF, Cao WW, Cemiglia CE (1997) A universal protocol for PCR detection of 13 species of foodbome Lampel KA, Jagow JA, Tracsess M, Hill WE (1990) pathogens in foods. J Appl Microbiol 83: 727-736. Polymerase Chain Reaction for detection of invasive Shigella flexneri in food. Appl Environ Microbiol 56: Wang RF, Cao WW, Johnson MG (1992) 16S rRNA- 1536-1540. Based probes and Polymerase Chain Reaction method to detect Listeria monocytogenes cells added to foods. Appl Lindqvist R (1999) Detection of Shigella spp. in food with Environ Microbiol 58: 2827-2831. a nested PCR method sensitivity and performance compared with a conventional culture method. J Appl Wolffs PF, Glencross K, Thibaudeau R, Griffiths MS Microbiol %6: 911-91%. (2006) Direct quantitation and detection of Salmonellae in biological samples without enrichment, using two-step Malomy B, Paccassoni E, Fach P, Bunge C, Martin A, filtration and real-time-PCR. Appl Environ Microbiol 72: Helmuth R (2004) Diagnostic Real-Time PCR for 3896-3900. 510
  11. Tgp chi Cdng nghi Sinh hgcl{A): 501-511,2009 RAPID DETECTION AND QUANTIFICATION OF THE FOODBORNE PATHOGENES BY REAL-TIME PCR Le Thi Chi, Nguyen Quoc Binh' Biotechnology Center of Ho Chi Minh City SUMMARY We developed and assessed Real-Time PCR method for the rapid detection and quantification of infected in foodstuff pathogens, which are major bacteria in the intestinal tracts of human. The protocol used a universal culture preenrichment medium and PCR program that used direct enrichment culture broth with 5 sets of specific primers for 5 species of foodbome pathogenes were Escherichia coli. Salmonella spp., Shigella spp., Listeria monocytogenes and Bacillus cereus. The primers 100% specific as determined target species and no interference reaction with non-target species was observed. This method was simple that unnecessary DNA extraction, purification and had highly sensitive, detection limits from 1 to 10 copies/reaction as pure bacteria and 1-10 CFU/25 g food. After enrichment in TSBYE medium in 24 hours, samples infected microbial were determined the amount of cells from lO'to lO'cells/ml (R^ = 0.998) and the same colony counting method (p = 0.90). The accuracy, sensitivity and specificity is high, these averange values are 97.99, 98.05, and 95,60%. All the time to finish diagnosis only spent 4 - 3 0 hours in comparison with 5 - 7 days when using conventional culture method. Keywords: Bacillus cereus, Escherichia coli. Listeria monocytogenes, Real-Time PCR, Salmonella, Shigella, infected in foodstuff pathogens * Author for correspondence: Tel: 84-8-38222481 ;E-mail:binhns(a).hcmbiotech.com.vn 511
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