S U D U N G K Y T H U A T P O L Y M E R A S E C H A I N R E A C T I O N ( P C R ) KHAO SAT THU NGHIEM NGUY CU NHIEM
LISTERIA MONOCYTOGENES TRONG MOT SO THUC PHAM TREN TH| TRUONG HA NOI
NGUYfeN MINH THl/C, TRAN MANH HUNG,
TRINH THU L f i , T 6 K I M ANH Vien Cong nghe sinh hoc - Cong nghe thuc phdm, Dai hoc Bach khoa Ha Noi
Tom tat:
Ky thuat PCR sii dung cap moi LM-F76, LM-R543 cho phan tich phat hien
L. monocytogenes da dirpc khang dinh tuong dong vdi phuong phap phan tich thong thudng. Viec khao sat budc dau mCrc do nhiem L. monocytogenes trong cac san pham thjt va sda trong thuc pham tren thj trudng Ha Npi dupe thpc hien tren 141 miu thpc pham, trong dd 92 mau sira va sua nguyen lieu, 49 miu san pham thit. Ket qua phan tich bang ky thuat PCR cho tha'y khong phat hien dupe L. monocytogenes trong cac mau sua nghien cuu trong khi do, 11/49 miu san pham thit nhiem L. monocytogenes & mut phat hien cua phuong phap. Ket qua phan tich dupe khang dinh khi xac dinh cac khuan lac cho miu duong tinh vdi L. monocytogenes trong ky thuat PCR tren moi trudng dac trUng Oxford, dinh ten chung bang ky thuat 16S rDNA. Ke't qua phan tich khang dinh tat ca 11 chung phan lap dupe tP cac miu nghi nhiem la L. monocytogenes. Day la cong bd dau tien ve nguy co thpc pham nhiem L. monocytogenes tren thi truPng Ha Npi. Can thiet cd khao sat rong hon cho cac thpc pham, dac biet cac thpc pham an lien ve mdc dp nhiem vi khuan nguy hiem nay.
PRIMARY INVESTIGATION OF LISTERIA MONOCYTOGENES INFECTION IN FOODS IN HANOI MARKET
USING POLYMERASE CHAIN REACTION Abstract:
Using PCR techniques developed with LM-F76, LM-R543 primers, the investigation of the
infection of L. monocytogenes was done with 141 food samples including 92 dairy- and 49 meat- based foods. The PCR analysis showed no contamination with L. monocytogenes was detected in dairy samples, in contrary, 11/49 meat samples was positive with L monocytogenes. This result was verified by identification of the strains isolated from positive sample on Oxford agar by the 16S rDNA technique. The result confirmed that all strains isolated from PCR positive samples were identified as L. monocytogenes. This primary analysis gave a first warning to the contamination risk of L. monocytogenes in food in Hanoi market. It demands a widely investigation for the dead bacterium infection risk in the marketed foods.
I . D A T V A N D E
Listeria monocytogens cd mat trong thirc pham la mpt trong 400 tac nhan gay benh tir thirc phim cho nguoi. Nhiem L.monocytogenes b nong dp 100 CFU co the gay benh listoriosis vdi mpt phd rat rpng cac trieu chung: viem mang nao, nhiem trung mau, say thai, va viem rupt d ngudi va dpng vat, dac biet nguy hiem vdi ngudi b i suy giam midn dich [1,2, 3]. Viec phat hien su cd mat cua L.monocytogenes theo phuong phap tieu chuin 52 TCN - TQTP 0002: 2003 gap nhieu khd khan do tinh phuc tap cua phuong phap, va dac biet kho thuc hien khi can phan tich sang lpc mpt sd lupng ldn m i u . Nhieu ky thuat hien dai g i n day da dupe phat trien va ap dung de phat hien nhanh L.monocytogenes vdi thao tie don gian hon, dp chinh xac cao va thdi gian phan tich n g l n hon, trong dd, phuong phap dua tren ky thuat PCR la dupe coi la thich hpp de phan tich sang lpc m i u trong phong thi nghiem [4, 5, 6,7].
Phuong phap phan tich L. monocytogenes vdi cap mdi L M - F 7 6 va LM-R543 dac hieu vdi gen dich listeriolysin O (hlyA) da dupe thiet ke va thu nghiem dac hieu vPi L.
monocytogenes [8] va dupe phat trien cho tirng loai san pham thuc p h i m [9, 10, 11], cho phep phan tich mpt lupng m i u ldn vdi dp chinh xac cao. Bai bao de cap ket qua su dung quy trinh phan tich dua tren ky thuat PCR ap dung cho phan tich mpt sd san pham thit, sua [10, 11] dd budc dau khao sat mdc dp nhidm loai vi khuan nguy hiem nay trong mpt sd san pham cd nguy co cao thupc nhom nay.
II. DOI TUONG VA PHUONG PHAP NGHIEN CUU 2.1. Mhu thuc pham
M i u thuc phim su dung trong nghien cuu bao gdm mpt sd sin pham cd ngudn gdc td thit, sua dupe lay tai cac nha may hoac mua tai cac cua hang thuc phim va sieu thi Uen thanh phd Ha Npi trong khoang thdi gian td 2006 -2008.
2.2. M d i trudng
MPi trudng LEB, Difco (Listeria Enrichment Broth) dupe sd dung cho tang sinh vi khuin.
Moi trudng Oxford, Sigma su dung de kiem tra phuong phap. Dung dich pha loang m i u la nude mudi sinh ly 0,9% NaQ.
2.3. Hda chat
Hda chit cho dien di tren gel agarose, thang A D N chuan do Sigma cung cap. Hda chat sd dung cho phan dng PCR do Amersham Phamacia Biotech cung cap. Cac hda chit khac cd dp tinh sach phan tich. Cap mdi LM-F76, LM-R543 [8] va cap mdi 16S/F rRNA
[12] do Invitrogen cung cip.
2.4. Phuong phap lay mau
M i u dupe l i y ngiu nhien theo me san xuit, theo 10 san p h i m tai co so sin xuat hoac mua tai cua hang, sieu thi. Thdi gian l i y m i u theo mua: mua dong (thang 12-thang 1) hoac mua he (thang 4, 5).
Miu sua su dung trong nghien cuu gdm sua nguy6n lieu, sua thanh triing, cd ddng
gdi san hoac ban rdi. M i u sua thi nghiem la cac m i u sua nguyen lieu hoac thanh p h i m dupe lay theo quy trinh lay m i u tai nha may hoac cac m i u sua thanh triing va tiet trung cd hoac khong cd bao gdi uen thi trudng. M i u san pham thit la san pham che bien, da qua xu ly nhiet hoac khong (xuc xich, nem chua), ban tren thi trudng. Cac m i u dupe l i y , bao
quan dung quy djnh va phan tich trong thoi han sir dung ciia nha san xua't, m l u khong bao goi duoc phan tich ngay sau khi lay m i u .
2.5. Phurong phap chuan bi mau
Bao bi m l u dupe k h u trung bang con 70°. Diing dung cu cat mau da dupe khir trung cat bao b i , cat va can trong dieu kien vo triing 25g mau cho mpt lan phan tich.
MSu da la'y dupe chda vao tiii chuyen dung chda 225ml moi truong L E B da tiet triing de tien hanh dong hoa mau tr6n thiet b i dap m l u Stomacher 400.
Phdn tich phdt hien L. monocytogenes bdng PCR
Viec phan tich phat hien su co mat cua L. monocytogenes trong cac mau thuc p h i m dupe thuc hien theo cac quy trinh tuong ung cho cac san pham thit va sua da dupe xay dung trong cac nghien cuu trudc [10, 11]. Cac budc thuc hien bao gdm:
- Tang sinh: 25g mau dupe nghien (hoac trpn) trong 225ml moi trudng LEB, dupe dem tang sinh trong thiet bi lac dn nhiet d 37°C tdc dp lac 200 vdng/phut trong thdi gian tuong ung 20-24h.
- Phan ung PCR va x u ly ket qua: Tien hanh thu nhan D N A td cac mau tang sinh theo phuong phap da trinh bay trong cac tai lieu trudc [10,11] lam khuon cho phan dng PCR. Phan ung PCR dupe thuc hien vdi the tich 25pl vdi chu trinh nhiet gdm 94°C trong 3 phut, (94°C 30 giay, 60°C 30 giay, 72°C 1 phut) x 35 chu ky, 72°C 5 phut va dn dinh san phdm d 10°C. Ket qua cua phan ung PCR dupe danh gia bang dien di san pham PCR trSn gel agarose 1,5%.
- Phuong phap dien di DNA: 5pi san pham ciia phan dng PCR dupe kiem tra bang
phuong phap dien di D N A tren gel agarose 1,5%. Dong dien su dung cho dien di la 100mA. Sau k h i dien di, D N A dupe nhupm trong dung dich ethydium bromide va soi dudi budc sdng 254nm.
- Danh gia ket qua: Cac m l u xua't hien cac bang D N A tuong ung vdi vi t r i 468bp trong cac phan dng PCR vdi cap mdi LM-F76, LM-R543 la cac m i u bi nhidm L.
monocytogenes.
Phdn tich khdng dinh ket qud
Cac mau tang sinh cho ket qua PCR duong tinh dupe quet tren moi trudng Oxoford.
D N A tu cac k h u i n lac den dupe dung lam khuon cho phan dng PCR vdi cap mdi 16S/F.
San pham PCR vdi cap mdi 16S/F dupe tinh sach vdi kit tinh sach D N A do Invitrogen cung cap. San p h i m PCR vdi doan gen 16S rDNA dupe gui xac dinh trinh tu va so sanh mdc tuong ddng vdi cac trinh tu 16S r D N A da cPng bd tren ngan hang gen.
III. KET QUA NGHIEN CUU VA BAN LUAN 3.1. Cac mau phan tich
V d i kha nang phat trien dd dang cua L.monocytogenes trong didu kien khac nghiet nhu moi trudng mudi, nhiet dp lanh, cac thuc p h i m gia nhiet khong day du va bao quan lanh dai ngay la cac thuc pham cd nguy co nhidm L.monocytogenes. Cac m i u thuc p h i m dupe lua chpn dd khao sat kha nang nhidm L.monocytogenes trong nghien cuu nay la cac thuc pham cd ngudn gdc sua va thit, bao gdm 49 san p h i m thit (xiic xich, nem chua), 116 m i u san pham sua (sua tuoi nguyen lieu, sua ban thanh p h i m , sua thu hdi trong nha may, sua thanh trung, tiet triing ban tren thi trudng) (bang 1). Cac m i u dupe phan tich ngay sau
l;hi mua, hoac bao quan nhu dieu kien quy dinh cua san pham. M o i phan tich m i u deu dupe thuc hien trong han sir dung cua m l u .
Bdng 1. Cdc mdu san phdm diing cho phdn deh
STT Loai mau Sd luong
mau T h d i gian lay mau
1 San phim thit hun khoi 17 4-5/2006
San pham thit hun khoi 9 12/2006 - 2/2007
3 San phim thit che bien 10 4-5/2006
4 San pham thit che bien 11 12/2006 - 2/2007
5 San pham nem chua 2 12/2006 - 2/2007
6 Sua tuoi nguyen lieu 35 3-4/2006
7 Sira tiii thanh triing, co duong 19 4-5/2006 8 Sua tiii thanh trung, khong duong 17 4-5/2006 ) Sua thanh triing, dong hop 1 lit 10 4-5/2006 10 Sira thanh trung, khong bao goi 11 4-5/2006
3.2. Ket qua phan tich L . monocytogenes bang PCR
Cac mau dupe tang sinh nhu trinh bay trong phan phuong phap. 3pl dung dich DNA
duoc sir dung lam khuon cho phan dng PCR vdi cap moi LM-F76, LM-R543 dac hieu cho /-. 'twnccywgenes .
Bdng 2. Cdc mdu duong dnh vdi LM-F76, LM-R543
rrr Loai mau Ky hieu mau Ngay lay mau
I San pham thit hun khoi M l - 2 12.04.06
2 San pham thit che bien M l - 6 18.04.06
San pham thit hun khoi M2-12 28.04.06
A San pham thit hun khoi M2-14 13.03.06
5 San pham thit che bien A l l 20.11.06
6 San pham thit che bien B l l 01.12.06
7 San pham thit che bien B13 15.12.06
o San pham thit hun khoi B23 30.01.07
San pham thit hun khoi A31 01.02.07
10 San pham thit hun khdi A32 01.02.07
11 M l u nem chua C10 01.02.07
Hinh 1: Ke't qud PCR cua mdu duong tinh vdi doan gen hlyA Ladder lkb
l.Mdu Ml-2 2. Mdu Ml-6 3. Mdu M2-12
4, Mdu M2-14 8. Mdu B 23 9. Mdu A31 10.Mdu A32 ll.MduC12 5. Mdu All
6. Mdu Bll 7. Mdu B13
Phan ung PCRvdi cac mau sua nguyen lidu va san pham cho ket qua am tinh trong khi do, 15/ 49 phan ung PCR cho ket qua duong tinh voi mau san phim thit (bang 2, hinh 1).
Do nguong phat hien cua phuong phap la 10 CFU/25g, m l san phim [10, 11], nhu vay, cac san pham sua phan tich hoac khong nhiem hoac nhiem L.monocytogene dudi muc phan tich, trong khi do, cac m i u thit khao sat co 11/49 m i u n h i i m L.monocytogenes a mdc phat hien cua phuong phip.
Theo cic canh bao tren the gidi, sua tuoi nguyen lieu va sua tuoi thanh triing cd nguy co n h i l m L. monocytogenes cao. Ket qua phan tich cho thay cac m i u sua phan tich ngay ca sua tuoi nguyen lieu deu khong phit hien nhidm L. monocytogenes. Dieu nay cho t h i y dieu kiSn thu nhan sira tuoi hien dang dupe dam bao. San lupng sua tuoi thip trong sua sin phim cung la mpt dac diem ghi nhan trong ket qua phan tich nay.
Ngupc lai, cac san p h i m thit da lua chpn de phan tich deu cd muc n h i i m L.monocytogenes kha cao, 11/49 m i u .
Mac du mdi chi la ket qua khao sat budc d i u kha nang n h i l m vi khuin nay trong thuc pham nhung ket qua phan tich cung cho thiy ro, cac san pham khong dupe gia nhiet d i y du va dupe bao quan lanh dai ngay la cic thuc p h i m thuc su cd nguy co n h i l m L.monocytogenes. Tuy vay, do sd lupng m i u dupe khao sat chua nhieu, cac sd lieu cong bd o day chi cd tac dung canh bao tinh trang mpt sd thuc p h i m tren thi trudng n h i l m loai vi khuin nguy hiem nay c i n phai dupe kiem soat.
3.2. K h a n g dinh ket qua phan tich
Phuong phap PCR su dung trong phan tich nay da dupe kiem nghiem vdi phuong phap tieu c h u i n va dupe cap giay xac nhan tinh tuong dong cua phuong phap tieu c h u i n [13].
Hinh 2. Cluing BK7 phdn lap tic mdu C12 phdn rich tren mdi trudng Oxford 48h (a) va nhudm Gram (b)
6 day, chung toi tien hanh khang dinh lai cac chung L.monocytogenes phan lap tir cac m l u tang sinh cho phan ung duong tinh vdi phan tich PCR b i n g kiem tra khuan lac va bing ky thuat 16S r D N A . Cac dich tang sinh duong tinh dupe quet len moi truong thach Oxford, nuoi d 37°C trong 48h. Tren moi truong thach Oxford, toan bp cac m i u tang sinh duong tinh tao thanh cac khuin lac mau den hoac xanh den dac trung (hinh 2a). Cac chiing phan lap dupe tren moi truong Oxford dupe nhupm Gram. Ket qua nhupm cho thiy day la vi khuin Gram duong, khong hinh thanh capsul, khong sinh bao tir (hinh 2b).
Bdng 4. So sanh trinh tit chiing BK7 thu tit mdu C12
Bdng 5. Dd titang ddng ciia trinh tit 16S rDNA ciia cdc cluing phdn lap tain Desaiptioi Total Query E M
tain Desaiptioi score score coverage nine iimt 993 im 0.0 100%
; 'jstenawKfa/m*• EGO,awtetw*sjwt82 85 » 0,0 100%
P";;M ww MMMJMS stran EGO, trap 'fs pun, agws 53 1991 ion 11.0 100%
USKIUmonocrtcgenesoaroal 16SrfWAgene,isolate 9? I 995 ion 0J1 100%
ListenamonociftogenBspartial 16SrRNAgene..scHste44 Ct; 995 ion 0,0 100%
I iistenawmqms»aKD.MA»genoresegment 33 1991 100'* u m
; •jsten;Towtfccer,esai.>b'23C,o)n[Hegenera cc; 5973 100% n \m
; Listenamonocrtogenes23SrKNAgsne.strasnATCC'.9115 : 989 mm n S9%
».-:••. liienaM%f(togentSl6SrksMalK^r«,oaitial<:}jsttt 9?9 939 INK u m
; mm raxflcgenes 165 ntoul M4 gene, paroal sum 98! m 0.0 99%
: ; Liiowtog:nes»'ilor!6Sjb3stiiJalKS p; 9;9 10W 0.0 99%
L"KMCttegenes C* (or US nbosomai Btt 0"'. 979 10W 0.0 99%
Chung/mSu D6 tirong ddng
L I/All 98%
L4/BU 99%
L6/B13 100%
BK 1/B23 99%
BK 4/A31 98%
BK 5 /A32 98%
BK.7/C12 100%
VN1/M1-2 99%
VN2/M1-6 100%
VN3/M2-12 98%
VN4/M2-14 100%
DNA thu dupe tir cac k h u i n lac tach tir moi truong Oxford cua cac dich tang sinh duong tinh dupe dung lam khuon cho phan ung nhan doan gen 16SrDNA v6i cap moi 16S/F. Sin p h i m PCR vPi doan moi nay dupe tinh sach, xac dinh trinh tu, so sanh v6i trinh tu /;/yA ciia L. monocytogenes tren ngan hang gen, cho thay gen hly A cua chung nghien ciru tuong dong voi cac trinh tu 16S r D N A ciia L. monocytogenes khac da dupe cong bo' (bang 3, 4). Ke't qua nay chiing minh cac khuan lac den tren moi truong Oxford thu dupe tir cac dich tang sinh duong tinh voi doan gen lily A. chinh la L. monocytogenes. V i the mdc dp nhilm L. monocytogenes trong cac san pham thit phan tich dupe xac nhan la 11/49 m i u phan tich.
I V . K E T L U A N
Ke't qua khao sat budc ddu cac mau san p h i m thit co tren thi truong cho tha'y su n h i i m L. monocytogenes d mure can phai duoc quan tam. Hien nay, L. monocytogenes chua duoc quan tam nhieu trong cac van d i an toan ve sinh thuc pham, nhung nguy co nhilm loai vi khuin nay trong cac thuc p h i m , dac biet la cac thuc phim an lien nhu trong phan tich nay la co that. Day la cong bd d i u tien ve muc n h i l m loai vi khuin nay trong thuc p h i m tren thi trudng Ha Noi. C i n thiet cd cac khao sat tren dien rpng hon ve mdc dp nhilm L.monocytogenes tren thuc p h i m hien nay de co cac giai phap c i n thiet bao ve sdc khoe cpng ddng.
Nghien ciat nay duoc thuc hien dudi su tdi tro cua de tdi AP05\Prj3\Nr01.
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