Y HQC VlgT NAM THAHG 6 - SO 1/2013 2. Ket qua dieu trj va tac dung phu:
Benh nhan sau khi du'dc chan doan xac djnh du'dc dieu trj thudc khang vi rut va thay rSng:
Benh nhan cai thien dp bao hda oxy nhanh sau tmng binh 4.93 ± 6.13 ngay benh nhan da cai du'dc oxy mac dii trUdc dd benh nhan ludn trong tinh trang phu thuoc oxy.Tinh trang sdt du'dc khSc phuc sau 2.3 ± 1.5 ngay. Tai lu'dng vi riit giam dang ke ngay sau tuan dau tien va da sd benh nhan chi sau 2 tuan dieu tn benh nhan da cd cai thien ro ret tren lam sang va ket qua xet nghiem vi riit am tinh. Ket qua ciia chiing tdi cung phii hdp vdi 1 sd nghien ciru khac tren the gidi [2].
Ve theo doi cac tac dung phy tren lam sang thi khd khan vi da so benh nhan ciia chiing tdi lira tuoi du'di 3 thang nen chii yeu thdng qua hdi ngUdi cham sdc va tham kham lam sang, du'a vao cac xet nghiem sinh hda huyet hpc de phat hien kip thdi cac trieu chirng bat ldi. Tuy nhien nhu'ng tac dung phu dUdc theo ddi thi chu yeu thoang qua va khdng cd hau qua nang ne. Ket luan ciia chiing tdi cung phii hdp vdi nghien cu'u ciia cac tac gia tren the gidi [ 2 ] .
V. KET LUAN
Ganciclovir an toan va cd hieu qua tren 9 0 % benh nhan dUdc do lu'dng bang Real Time PCR CMV trong mau am tfnh d cudi ddt dieu tri.
T A I UEU T H A M KHAO
1. Khuc Van Lgp ( 2 0 0 9 ) , NghiSri cud dac dim lam sang, can lam sang cac thi' binh do CMV is tri em tai Benh vien Nhi Tmng ddng, Luan van cao hoc, TrUdng Oai hoc Y Ha Npi.
2. Avila - Aguero ML, Paris MM, Alfaro I , etal.
( 2 0 0 3 ) , "Ganciclovir therapy in cytomegalovirus (CMV) infection in immunocompetent pediatric patients", Int J Infect Dis, 7, pp. 278-278.
3. Demmler G.L ( 2 0 0 4 ) , Cytomegalovims Infections (Vol. 17), Infectious Diseases of children.
4 Eddleston M, Peacock S and WarrellMaria DA Juniper M ( 1 9 9 7 ) , "Severe Cytomegalovirus Infection in Immunocompetent Patients", Clinial Infectious Diseases 2A, pp. 52-56.
J. Ho M ( 2 0 0 8 ) , "The History of cytomegalovirus and Its diseases", Medical Microbiology and Immunology 197, pp. 65-73.
6. Karen KB ( 2 0 0 6 ) , "Antiviral drugs for cytomegalovirus diseases". Antiviral Research, 71, pp. 154-163.
7. Karine Kadaya, Werner Wunderll, Christelle Deffernez, et al. ( 2 0 0 3 ) , "Monitonng of Cytomegalovirus Infection in Solid-Organ Transplant Recipients by an Ultrasensitive Plasms PCR Assay", Journal of clinical micnobloioy, 41(8), pp.3757-3764.
THIET KE KY THUAT PCR PHAT HIEN VI KHUAN LISTERIA MONOCYTOGENES TRONG THin; PHAM
Vu Xuan NghiaS P h a m Cu'c Minh^, Nguyen Hung Long^
TOM ih
Trong thdi gian gan day, an toan thuc pham la van de du'dc nghanh y te quan tam vi so lu'dng ngUcfi mac va qui mo cac vu djch, Mpt trong nhUng nguyen nhan gay ngo dpc thuc pham do vi smh vat la VI khuan Listeria monocytogenes. Chan doan nhanh va chinh xac mam benh gay ngd dpc thUc pham se giiip ich cho cong tac dieu tr; va phong chong mat an toan thi/c pham, Trong nghien ciTu nay, ky thuat PCR da dUdc thiet ke, irng dung de phat hien vi khuan Listena monocytogenes trong
^ Hoc vien Quan y
^ Cue An toan Thu'c pham - Bg Y ti' Phan bien khoa hgc: GS.TS. Hoang Van Lu'dng
thyc pham. Ket qua cho thay, ky thuat PCR co kha nang phat hten dUpc mau nhiem vi khuan Li^eri3^
monocytogenes cd ndng dp lO^cfu/mL. So vdi ky thuat nuoi cay, ky thuat PCR co dd nhay la 100%, dp dac hieu la 75%. Vdi Uu diem ciia phu'dng phap PCR la thdi gian tien hanh nhanh, dp chfrih xac cao, dp dac hieu cao nen ky thuat nay cd the irng dung trong thUc tien.
Td khoa: PCR, vi khuan Listeria monocytogenes, ngd doc thUc pham.
Y HQC VIET NAM THANG 6 - SO 1/2013 SUMMARY
DESIGN PCR TO DETECT LISTERIA MOA/OCVTOGENESBACTERIA I N FOOD In recent years, food safety issues are health care intention because of the number of infected people and outbreak scale. One of the causes of food poisoning by microorganism Is Listena monocytogenes bacteria. Quick and accurate diagnosis of pathogens causing food poisoning will help the treatment and prevention of food insecurity, In this study, PCR techniques have been designed, applied to detect Listena monocytogenes in foods. Results showed that PCR was able to detect sample contamination by Listena monocytogenes at concentration of lO^cfu/mL Compared with culture techniques, PCR has a sensitivity of 100%, a specificity of 75%. With the advantages of PCR are short time to conduct, high accuracy, high specificity, this technique can be applied in practice.
Keywords: PCR, Listeria monocytogenes, food poisoning.
I.
OAT VANof
Nguyen nhan thi/c pham bj nhiem vi smh vat gay ngd ddc thdc pham dang la van de nd'i com hien nay. VI khuan Listeria monocytogenes la mpt trong nhij'ng mam benh gay ngd ddc thu'c pham quan trong, dac biet la cac thUc an che bien diing ngay (Gallagher et al, 2003). Benh Listeriosis cd the de lai nhO'ng hau qua nghiem trpng nhu" gay nhiem khuan huyet, viem mang nao, say thai, thai chet lu'u va de non d phu nu' mang thai, v.v. dac biet d nhdm nhti'ng ngu'di cd he mien djch suy giam. 6 cac nUdc phat trien, / . monocytogenes la mdt chi tieu dddc kiem soat nghiem ngat trong quan ly chat lu'dng thUc pham. Tuy nhien tai Viet Nam, van de phat hien va kiem soat su" cd mat ciia i. monocytogenes trong thy'c pham chu'a thyc s y dypc cac nha quan ly va ngydi tieu diing quan tam [1], [ 2 ] .
Phat hien nhanh va sdm sy cd mat ciia L.
monocytogenes se cho phep cdng tac dieu trj va d y phdng dat hieu qua cao [3], [4]. Nghien ciTu nay da thiet ke cap moi dac hieu vdi vi khuan L monocytogenes, diing trong ky thuat khuech dai gen (PCR) de phat hien vi khuan. Gia tri ciia PCR dUdc danh gia qua so sanh vdi phydng phap nudl cay.
II. o 6 l TUONG, VAT LIEU VA PHUONG PHAP NGHIEN COU
2 . 1 . Ooi tu'dng nghien cu'u
Nhdm nghien ciru: vi khuan Listeria monocytogenes PJCC 35152
Nhdm chuTig 1: Listeria ivanovi/AJCC B/\A-139 Nhdm chutig 2: Escherichia coii BL21 (Vien Cong nghe Sinh hpc, Vien Khoa hpc va Cong nghe Viet Nam).
Cac vi khuan la chiing chuan quoc te, chiing chuan tai cac phdng thi nghiem, dUdc tang sinh va bao quan theo tieu chuan quoc gia tai phdng xet nghiem khoa Vi sinh vat - Vien 103 -Hoc vien Quan y.
Mau thu'c pham: 100 mau thyc pham la cac san pham cd nguon goc tir thit nhU xuc xfch, pa- te dUdc thu thap tai mdt sd ciia hang thyc pham va cac hd gia dinh trong thdi gian tir 3/2012 den 6/2012.
2.2. M o i cua phan uTng k h u e c h dai g e n Mdi ciia phan iTng PCR dac hieu vdi Listeria monocytogenes dddc thiet ke bang phan mem CLC Genomics Workbench 4.0.3 (CLC bio, Oan Mach) dya tren dCi' lieu cd sd ciia Trung tam thdng tin cdng nghe sinh hpc quoc gia Hoa Ky - NCBI (National Center for Biotechnology Information).
2.3. Tach c h i e t ADN
Tach ADN td'ng sd td mau nghien ciiu Qiagen ADN mini kit theo quy trinh chuan (Qiagen, Dire) va cat giu' d -80°C den khi sir dung.
2.4. Phan Ctng k h u e c h d a i g e n PCR Phan irng PCR: 95°C trong 2 phiit va 40 vdng ciia cac giai doan: duoi xoan d 94'=C trong 30 giay, bam moi 55°C trong 1 phiit, keo dai 72'^C trong 1 phut. Cuoi ciing la giai doan keo dai 72°C trong 10 phiit. San pham PCR dUdc chay tren agarose gel 1,5% d dien ap lOOV va chup tren he thdng may doc gel.
2.5. T h i e t b j , hoa c h a t
2.5.1. Thiet br. may luan nhiet t y dpng GeneAmp PCR system 9700 AB (Applied Blosystems, Hoa Ky), may ly tam lanh Mikro 22R (Hettich, £)u'c}, may soi va chup gel Dolphin Doc (V\/altec, Hoa Ky), bd dien di.
2.5.2. Hoa chat: Cac hda chat chinh ddric sir dung la: dung dich dem HB (8x SSC; 0,5%
SDS), nUdc khir ion, thach Agarose, SYBR-green de nhudm gel, thang DMA chuan do hang Sigma cung cap.
2.5.3. Moi tru'dng nuoi cay va xac djnh vi khuan: Hddc pepton 0 , 1 % ; mdi trUdng thach Oxford; mdi trydng thach Tryptone soya cd 0,6%
cao men (TSA-YE); canh thang Tryptone soya cd 0,6% cao men (TSB-YE); thach mau ciru; mdi trydng Clurk-Lubs; canh thang dydng Manitol, 55
Y HQC VI$T NAM THANG 6 - S6' 1/2013
Xylose, Rhamnose 5%; bp nhupm Gram. I^au thiTc pham se du'dc tang sinh tren moi tru'dng
chpn loc Difco Fraser Broth trong 20 gid trifdc khi tien hanh phan irng PCR.
III. KET QuA NGHIEN ClTU
3.1. OodSc hieu cua moi phat hi^n Listeria monocytogenes in sillico
Trinh ty cac cap moi nhu" sau
Lis_A: TGA GCA TTG TAT COT GCA GAG Lis_B: TGA TCG ACG GAA ACC TTG G
S s q u a n c s B p r o d u c i n g slgnincant a l l g n m s n i s . SeleCL Pi itiiiit: Selectod 0
,ri
38 S 30 2 3 0 2
3 8 2 3 S 2 33 2 38 2
.r:
30 2 36 2 JO 2 JO 2 3 8 2 3 8 2 3 0 2 30 2
cowei 100«, lOOOi
100^.
1 O 0 ' . l o o f . 100°,*
lOOfg F
0 075 0 075 0 0 / 5
0 075 0 075 0 075 0 075
d e r l 0 0 % 0 0 ^ 00°l.
0 0 ^ o o « . 0 0 % 0 0 %
Acct»irot1
aFjHSTELL H M S E ^ U ESiflfljaj
" " • w . , !
" - ' " » T 1 n.L;iiti nmiixi
Hinh 1:Q6 dac hieu ciia moi Lis_A in sillico tren ngan hang gen NCBI
Ket qua hinh 1 cho thay moi Lis_A cd kha nang phat hien vi khuan Listeria monocytogenesy&\
dp dac hieu 100%, khdng cd dydng tfnh gia tren in siiiico.
S e q u e n c e s p r o d u c i n g s i g n l l i c a n t a l i g n m e n t s
100% 0 002 100% 0 002 1 0 0 % 0 002 100% 0 002 100% 0 002 100% 0 002 100% 0 002 100% 0 002 100% 0 002 100%
100% - t - . ' : ; . . ; • 100% H.i.:^Vi I
Hinh 2: Dp dac hieu ciia mdi Lis_B in siiiico tren ngan hang gen NCBI
Ket qua hinh 2 cho thay moi Lis_B cd kha nang phat hien vi khuan Listeria monocytogenesvd\
do dac hieu 100%, khdng cd dydng ti'nh gia tren in sillico.
3.2. Do dac hieu cua m'oi tren mau chii'ng du'dng va doi chu'ng
San pham PCR dddc chay dpc ket qua tren gel Agarose 1,5%
Y HQC VI$T NAM THANG 6 - SO 1/2013 Trong d d , cac vi khuan nghien ciTu bao gom:
C l : L. monocytogenes, nong dp: 10^ CFU/mL C2: L. monocytogenes, nong dp: 10^ CFU/mL C3; L. monocytogenes, nong dp: 10^ CFU/mL C4: L. monocytogenes, nong dp: 10'' CFU/mL C5: L. monocytogenes, nong dd; 10'' CFU/mL C6: L. monocytogenes, nong dp: 10^ CFU/mL C7.1: Listeria IvanoviiATCCBAA-139, nong dp: 10^ CFU/mL C7.2: Escherichia coli BL21, ndng dp: 10^ CFU/mL
Ket qua hinh 4 cho thay tren in vitro, cap moi Lis_A va Lis_B cd kha nang phat hien dac hieu vi khuan Listeria monocytogenes, khong cd phan u'ng cheo vdi vi khuan Listeria Ivanovii vh Escherichia coii Ky thuat PCR cd the phat hien dypc mau vi khuan cd nong dp 10^ CFU/mL.
3.3. U'ng dung t r e n m a u benh p h a m so sanh phu'dng phap PCR vdi nuoi cay Oe so sanh, danh gia gia trj ciia cac phUdng phap phat hien tryc khuan L. monocytogenes cung nhy kha nang sir dung cac phUdng phap de thay the ho trd lan nhau, cac mau thyc pham nghien ciTu da dUdc diing de phat hien vi khuan L. monocytogenes dor)g thdi bdi ca hai phydng phap nuoi cay va PCR. Ket qua cac ciia hai phUdng phap dydc so sanh lan nhau ve dp nhay, do dac hieu, gia trj chan doan du'dng tinh, gia tri chan doan am tinh va he sd phu chan doan Kappa.
Bang 1: Ket qua thir nghiem PCR tren thyc pham so vdi nuoi cay
PCR Td'ng
Ket qua (+) H Kappa = 0,84; Se =
Nuoi cay (+)
9 0 9
(-)
3 88 91 100%; Sp = 97%; PPV = 75%; NPV = 100«/Tonq 12 88 100 Se: Sensitivity; Sp: Specificity;
PPV: Positive Predict Value; NPV: Negative Predict Value.
So sanh ket qua phat hien vi khuan L. monocytogenes cda phUdng phap PCR va nuoi cay cho thay he sd phii hdp chan doan (Kappa) ciia hai phUdng phap nay la 0,84 nen cd the diing thay the lan nhau. So vdi phu'dng phap nuoi cay, phudng phap PCR cd dp nhay 100%; dp dac hieu 97%; gia tri chan doan dydng tfnh 7 5 % ; gia tri chan doan am tfnh 100%.
IV. BAN LUAN
Nhiem khuan Listeria thydng lien quan den nhu'ng thyc pham tu'di song va qua trinh che bien thiTc an, chii yeu la bao quan lanh trong vai ngay. Trong thdi gian lyu tru' lanh, tryc khuan Listeria se sinh trydng va nhan len. Trong thdi gian day, ty le mac benh va ty le vong tren the gidi do Listeriosis tang dan hang nam [ 4 ] . Tai Viet Nam da xuat hien nhu'ng ca benh nhiem Listeria gay benh canh lam sang phii'e tap, khd khan trong chan doan va tien lUdng. Neu benh nhan dydc chan doan sdm va dieu tri sdm se dem lai liieu qua tdt. Neu chan doan mupn, vi khuan cd the gay nhiem khuan huyet, viem mang nao, viem nao, v.v. ty le tir vong cao. Tran Thj Hong Chau va cs (2010) da phat hien dydc 03 ca nhiem Listeria monocytogenes gay viem mang nao tai benh vien Lam sang nhiet ddi thanh pho Ho Chi Minh [ 3 ] . Benh vien Lam sang Quoc gia
nam 2010 cung phat hien dddc mdt ca benh nhiem Listeria monocytogenes (Bs. Nguyen Trung Cap va cs, 2010).
Mdt trong nhu'ng phu'dng phap chan doan mdi dUdc dUa vao trong vai thap nien trd lai day la phudng phap khuech dai gen. U'u diem cua phUdng phap nay la cd do nhay va dp dac hieu cao. 6 nUdc ta PCR da dUdc sir dyng rdng rai de chan doan cac benh nhiem triing nhU viem gan virus, HIV, lao... Trong nghien ciru nay, ky thuat PCR dydc thiet ke, irng dung va so sanh vdi phydng phap kinh dien la nuoi cay, phan lap, xac dinh tinh chat smh hda hpc, xac dinh ty le nhilm L. monocytogenes trong thdc pham. Trong 100 mau thyc pham nghien ciru, ky thuat nuoi cay phat hien dUdc 9 mau nhiem L. monocytogenes, ky thuat PCR cung phat hien dUdc vi khuan trong 9 mau nay va cdn phat hien them dydc 3 mau khac nhiem vi khuan L. monocytogenes. Nhu vay
Y HOC VI$T NAM THANG 6 - S6 1/2013 phydng phap PCR cd dp nhay cao hdn phydng
phap nudi cay. Kha nang phat hien L.
monocytogenes ciia ky thuat PCR trong nghien cu'u nay la 10^ CFU/mL, he sd phu hdp chan doan giii'a PCR va nudl cay la 0,84 nen cd the diing mdt trong hai phydng phap de ho trd hoac thay the lan nhau.
Ngydng gay benh cua L. monocytogenes rat thap chi khoang 10^ CFU/g ddi vdi ngUdi trydng thanh va 10 CFU/g ddi vdi tre em va phu ntT mang thai (Harrigan, F.Wilkie, 1998). Chfnh vi vay viec phat trien ky thuat chan doan va phat hien VI khuan d ndng dp thap va giai doan sdm ciia benh cd vai trd het sii'c quan trpng dieu trj va d y phdng. Dac die'm ciia PCR la cd the nhan hang ty lan mpt doan gene dac hieu ciia vi sinh vat cd trong benh pham ma khdng can den qua trinh nudi cay [ 5 ] . Hdn nu'a, phydng phap nay cd the thyc hien nhanh chdng trong vdng 24 gid tir khi thu thap benh pham, trong dd 20 gid dau se tang sinh mau, 4 gid sau dd danh cho phan irng PCR. Trong khi dd, phydng phap nudi cay truyen thong phai mat it nhat tif 5 - 7 ngay.
V. KET LUAN
Da thiet ke thanh cdng cap mdi cho phan
irng PCR phat hien vi khuan L. monocytogenes trong thyc pham. Ky thuat cd kha nang phat hien du'dc vl khuan trong thdi gian 4h sau khi da tang sinh vdi ngydng phat hien la lO^cfu/mL, co dp dac hieu tuyet doi.
TAI LIEU T H A M K H A O
1, Nguyen Chi Phu'dng (2005). An toan thu'c pham. nhilm khuan Listeria. Tap chf y hpc diT phdng so 6 nlm 2005, tr. 97 - 99.
^, Nguyen Minh Thi/c va cs (2008). BUdc dau khao sat sU nhiem Listeria monocytogenes tronq mpt so thyc pham tren thi trUdng Ha Noi bSng ky thuat PCR. Tap chf Khoa hoc va Cong nghe, tap 46, so 2, 2008, tr. 67-77.
^. Tran Thi Hong Chau et al ( 2 0 1 0 ) : Three Adult Cases of Listeria monocytogenes Meningitis in Vietnam. PLoS Medicine. Vol7 (2010) pp.1-6.
4. Angela Ingianni va cs (2007): Isolation and Identification of Listeria monocytogenes in Processed Meat by a Combined Cultural-molecular Method. American Journal of Infectious Diseases 3 (3): 159-164, 2007,
5. Levin R.E. Application of the polymerase chain reaction for detection of Listena monocytogenes in foods: a review of methodology. Food Biotechnol. 2003, 17, pp.99-116.
SO SANH HIEU QUA KIEM SOAT AP LlJC NOI SO BANG DUNG DjCH MUOI IAI TRirONG VA MANNITOL 0 BENH NHAN TAI BIEN MACH NAO CO
TANG AP LUIC NOI SO CAP TINH
Nguyin Anh Tuan*, Dong Van He**, Bang Quoc Tuan*
T O M TAT
Dat van der. Trong di'eu tn phii nao va tang ap lu'c noi so con nhieu quan diem chua thong nhat giu'a viec sir dung dung dich kinh dien la mannitol va muoi u'u trUOng. Nghien ciTu nham so sanh hieu qua kiem soat ap luc npi so ciia hai dung dich nay va so sanh cac tac dpng tren lam sang va can lam sang trong qua trinh dieu tn tang ap lUc ndi so bang hai dung dich tham thau. Phu'dng phap nghien cdu: tien ciTu can thiep ngau nhien c6 nhom chirng. Kit qua: Ca hai dung dich co tac dyng giam ap lUc noi sp c6 y nghla thong ke, tai thdi diem bat d'au truyen nhom benh nhan dung mannitol co ap lUc noi so trung binh la 38.9
•* Benh vien Bach Mai; ** Benh vien Viet Ddc Phan bien khoa hoc: PGS.TS. Nguyin Van Lieu 58 •
± 11,26 mmHg, tai thdi diem 120 phiit sau truyen la 23.75 ± 8.7 mmHg, tUOng tU 6 nhom diing NaCl irtJ trUOng la 37.4 ± 10.77 va sau truyen 120 phiit la 22.44 ± 4.16. Su khac biet ve giam ap IUc npi sod hai nhdm dieu tri khong cd y nghfa thong ke. Thay doi do mau 6 nhom diing NaCl Uu trUOng dUdc ghi nhan. Kit luan: Ca hai phac dd truyen mannitol va NaCl 3% deu CO hieu qua nhu nhau trong viec kiem soat ap lu'c noi sp trong giai doan dau. Viec lya chpn giu'a hai phac do tren phu thudc vao tinh trang huyet dpng, tim mach ciia benh nhan, tinh trang Na mau, Oo mau va tren tirng benh nhan cu the.
Tir khoa: ap lye npi sp, tai bien mach nao.
