JOURNAL OF 108 - CLINICAL MEDICINE AND PHARMACY Vol 10 - N°1 /2015
Xay dung quy trinh PCR da m6i phat hien mot s6 vi IchuSn thuoc ho Enterobacteriace gay benh 6* nguoi
Establishing a novel multiplex PCR to detect some Enterobacteriacae causing human diseases
Dao Thanh Quyen*, Le Hihi Song*, Nguyin Thi Kim PhiroTig*, "Benh viin Trung uang Qudn ddi W8 Bui Thanh Thuyet*, Trinh Van Son', Phan Quoc Hoan*, '"Benh vien Qudny 103 Nguyen Thi Mai L y " , Ngo Tat Trung*
Tom t ^ t
Muc tieu: Xdy dUng quy trinh PCR da mdi phdt hien mdt sd vi khuan thude bg Enterobacteriace gay benh d ngudi. E^i tugng vd phuang phdp: SUdung eae loai vi khuan thudc ho Enterobacteriace (Escherichia coll, Klebsiella pneumoniae, Salmonella sp, Proteus mirabilis, Enterobacteriaceae sp) da biet ndng do va duge pha loang ndng do tU 10^-^10°CFU/ml) lam ehuan dUong vd de danh gid ngudng phdt hien cua k?
thuat, PCR da mdi dugc thiet ke dua tren trinh tU tham ehieu tren ngan hdng gen. 0 d dac hieu cua PCR da mdi dugc xac djnh bdng gidi trinh t u vd thU nghidm tren cdc ADN tU vi khuan khdng thudc ho ndy, ADN virut, ndm, ngUdi. Ketqud: PCR da moi eho ketqud rd n d t t r e n hinh dnh dien d i ; gidi trinh t u g e n s o sdnh vdi ngdn hang gen cho thdy do tuong dong dat 94-99%; PCR da moi cd the phdt hien duge ddng thdi cdc tdc nhan vi khuan quan tam va ngudng phdt hien Id 10 CFU/ml, khdng ed hldn tUdng dUdng tinh gid vdi AON ngUdi vd cdc tdc nhdn vi khuan khac, ADN nam, virut. Ketludn: Quy trinh PCR da moi de phat hidn mdt sd vi khudn thudc bg Enterobarteriae dd duge xay dUng thdnh cdng vdi ngudng phdt hien la 10 CFU/ml va do dac hieu Id 94-99%.
TU khda: PCR da mdi, Escherichia coli, Klebsiella pneumoniae. Salmonella sp, Proteus sp, Enterobacteriaceae sp.
Summary
Objective: The aim of the study is to establish a multiplex PCR for detecting some pathogenic Enterobacteriace spieces. Subject and method: This technique is based on simultaneous amplification of a plural of target gene in a single optimized reaction tube with relevant negative and/or positive controls.
Detection limits was determined by piking assays in a range from 10" -=-10° CFU/ml. Result: Our data demonstrated that the selected target gene fragmnents are suitable for calling up Escherichia coli, Klebsiella pneumoniae. Salmonella sp, Proteus sp, Enterobacteriaceae sp from clinical samples. Specificity and sensitivity of endpoint PCR were 94-99%. Conclusion: A multiplex PCR for detecting mentioned pathogens was established with a detection threshold of 10 CFU/ml and 94-99% specificity.
Keyword: Multiplex PCR, Escherichia coli, Klebsiella pneumoniae. Salmonella sp, Proteus mirabilis, Enterobacteriaceae sp.
Plian bi?n khoa hpc: PGS.TS. LE VAN DON 124
TAP CHl Y DU-QC LAM SANG 108 Tgp 10-So 1/2015 1. Oat van de
Nhiem trung Id mdt benh ly thudng gap, nhat la d cac bdnh vidn. Trong do nhiem trijng dudng mau (Nhiem khuan huyet, NKH) gay bien chUng va nguy Cd tCr vong eao nhat. Vi khuan gay bdnh rat da dang, tuy nhien, theo eae nghien eUu d Viet Nam eung nhu trdn the gidi ehi ra ra rdng tde nhdn gdy bdnh chu yeu Id vi khuan thudc hp Enterobacteriaceae, nghien cUu eho thay ede vi khuan gdy NKH thudng gap nhat la Escherichia coll (22,1%), tiep theo la Klebsiella species (12,6%) vd Staphylococcus aureus (8,4%) [4].
Vide xae djnh can nguyen gay benh nhanh vd ehfnh xde Id eo sd eho vide ehl djnh dung khdng sinh dieu tri. Cho den nay cay khuan van duge coi la phUdng phdp ehuan trong phat hidn eae tac nhan truyen nhiem. Tuy nhien, vdi nhUng benh nhdn da sU dung khdng sinh tU trUde, vide cay mau sd rat khd ed ket qud duong tinh; ngodi ra eay mdu thudng ed thdi gian ehd ket qud keo ddi ndn neu dua vao ket qua cdy mau thi dieu tri thudng cham, de nguy hiem den tinh mang bdnh nhdn. Trong nhUng nam gan ddy nhd sU tien bd eua sinh hge phdn tU ma dien hinh Id ky thudt phdn Ung chuoi PCR da mdi cho phdp phat hidn nhanh axit nhan dae hieu cua nhieu loai vi sinh vat trong cdng mdt phdn Ung vdi thdi gian ehan doan ngdn khdng can nudi eay. Oe hidn thuc hda khd nang nay chung tdi tien hanh tdi Uu hda phdn Ung PCR da mdi phat hidn ddng thdi 04 tac nhdn gay bdnh dien hinh thudc ho Enterobacteriace la Escherichia coli, Klebsiella pneumoniae. Salmonella sp vd Proteus mirabilis nhdm xdy dung quy trinh chan dodn va danh gia do nhay, do dac hieu ky thudt eCia phuong phdp.
2. Doi ti/cmg va phUdng phap 2.1. Doi tuang
Nhdm ehUng duong: SU dung ede loai vi khuan thudc ho Enterobacteriace (Escherichia coli, Klebsiella pneumoniae, Salmonella sp, Proteus sp, Enterobacteriaceae sp) dd biet ndng do va dugc pha loang ndng do tU10''-^10° CFU/ml lam ehuan dUdng va de ddnh gid ngUdng phat hldn cua ky thudt.
Nhdm chUng am: Nhdm kiem tra do dde hieu cua ede bd mdi thiet ke: ADN dugc taeh ehiet tU cac loai vi sinh vat khac khdng thude hg Enterobacteriaceae lam ehUng am: Staphylococcus aureus; Streptococcus pneumoniae; Pseudomonas aeruginosa; Acinetobacter baumanil...;ADH dugc tdeh ehiet tU mau mau ngudi khde manh; ADN virut viem gan B; H5V, EBVva nam.
2.2. Thiet kemoi vd quy trinh PCR Cac cap mdi ddn dugc thiet ke bdt cap dac hieu vao cac khu vUc bao tdn eao cho mdi loai nhU cac gen ma hda cho protein ddc td dde trung eua loai.
Cae cap mdi khdng bdt cap cheo vdi nhau (Khi thuc hidn phdn Ung da mdi) va khdng bat cap vdi genomics ADN ngudi. Su khac bidt ve kich thude giUa cac doan san pham tao ra ndm trong khodng 80 - lOObp, nd dam bdo cho vide eae doan sdn pha'm cd the de dang dugc phdn biet khi didn di trdn gel agarose 1,5-2,5% [6]. Phdn Ung PCR da mdi dua trdn su khuech dai ddng thdi hai hay nhieu cap mdi trong cung mdt phdn Ung nen gidm ehi phi va thdi gian thuc hldn, ky thuat nay ddi hdi sU can bdng cua eae thanh phan phdn Ung nhU ndng do mudi, mdi, tag ADN polymerase, nhiet do ndng ehdy eCia mdi.
Do vdy, vide tien hanh thU nghiem dd' tdi Uu dieu kien phdn Ung het sUc phUc tap va ton thdi gian.
Chung tdi sU dung phan mem tin sinh hoc VectorNTI 10.0 de thiet ke mdi, md hinh cu the nhu Hinh 1.
Gen aldehyde dehydrogenase dac hieu eho Klebsiella pneumoniae cd ma ky hieu AB106869.1 duge sU dung de thiet ke bg mdi dac hieu cho PCR vdi kieh thUdc tren bang dien di la 324bp.
JOURNAL OF 108 - CLINICAL MEDICINE AND PHARMACY Vol10-N"l/201S
Hinh 1 . Mo hinh thiet ke moi ddn dac hieu
Theo nguyen ly co ban cua viee thiet ke mdi nhU da m d ta t r o n g phan phUdng phdp ehung tdi da thi^t ke duge cac cap mdi dae hieu ed trinh t U t u o n g Ung Bdng 1 sau:
Bdng 1 . Trinh t U cac cap mdi phat hien vi khuan gay benh
2|£
773
406
334
254
424
180
Ten tac nhan
E coli
Proteus mirabilis
K.pneumoniae
Salmonella sp
Enterobacteriace aesp Gen npi chuan
Ten moi
S-uidA-F S-uidA-R ureR-PM-F ureR-PM-R KP-F KP-R NTS-F NTS-R Ent-F Ent-R Pglobin F Pglobin R
Trinh tif
GTCGCGAGTGAAGATCCCTTTC
TCACAGTCACCAaAATCTCACGTTGA AGATGAGTGATCGCCTGAI1111IGGC CCGGGGGAGAATATCCTTGTCTT
CGCCGCAGCGGCCATAATTTTT AGCCTGTCGaGTTGACCCAGAAT
CGCACACGaGCAGGTTGTTGTT GTTGTAAAG(C/r)ACmCAGtT/OGGCT/G)GAGGAA
GCCTCAAGGGCACAACCTCCAA AGAAGAGCCAAGGACAGGTACG TGaAGTGAACACAGTTGTGTCAGA
gen dieh
S-uUf.
UreR
aldehyde dehydrogenase
flagellin gene
leSrlbosomal RNAgene
hemoglobin betaglobin chain (HBB) gem
TAP CHl Y DUOC LAM SANG 108 T|ip 10-So 1/2015 3. Ket quS
3.1, Kit qud toi Uu cho phdn dng PCR da moi phdt hiin hg vi khuan Enterobacteriace vd moi ndi chudn Chu trinh nhiet eho tdi uu phdn Ung PCR: 94°C, 2 phut; (94°C, 30 giay; 56°C, 30 giay; 72°C ,40 giay) x 38 chu ky; 72°C, 7 phut; 4°C, co
M50-mal:ei SObp
!-CknigMi 2- Ent-B.!ta,.\DS ngim 424 bp S-Ent-B-2ta Saimomiiasp
4-Enl-Beiii K ymiimomiic 5-Enl-Belii Proieiis mirabilis 180 bp 6-Ein-Beia Ecoii
Bo moi Eni-Betagom:
-Bg moi noi chuan bernglobiilin- ISO bp
-Bo moi diimgphiit hien lig vi l;hui.viEiirerobiicteh -424bp
Hinh 2. Ket qud PCR da mdi phat hien ho vi khuan Enterobacteriace va mdi noi chuan
^f/^l5nxef; hinh dnh dien di eho eae bang rd net, dae hieu tren ede mau ADN chuan dUdng.
3.2. Ket qud tdi Uu cho phdn dng PCR da moi phdt hiin mdt sovi khuan thudc hg Enterobacteriace Can cU vao nhiet do ndng chdy cua mdi trdn ly thuyet va khde phuc nhUng khd khan ma phdn Ung PCR da mdi gap phai, de ed dugc dieu kien tdi uu nhat eho phan Ung chung tdi khao sat nhiet do gdn mdi, ndng do mdi vd cdc yeu td khac. TU ket qud thi nghidm chung tdi da thiet lap duge mdt phan Ung PCR da mdi. Ket qud dugc trinh bay d hinh 3.
M50 maicer 50 bp 1-EPKSCinmgcmi 77:,bp 2-EPKSSalmoiidla sp 254 bp
S-EPKSKpneumoniae SS4bp l y , 4-EPKSProteus mirabilis 406bp jMbp 5-EPKSE.col! "3 bp -•^-H'P 6-Bd mdi tdng iwpEPKS tren
.4D\'cliudn ditovg mix
Bo moi EPKS gdm:E.coli. Proteus mirabilis. Kpneumoniae. Salmonella sp Hinh 3. Ket qud PCR da mdi phat hien mot so vi khuan thuoc ho Enterobacteriace Nhdn xit: Sa\j nhieu lan tdi Uu, ehung tdi thay d nhiet do 56°Ccho ketqud rd net nhat, 6 dieu kien tdi uu dugc, bd mdi chung tdi thiet ke ed the phat hien dugc eae tde nhdn don le hay ddng thdi ed 4 loai vi khuan tren deu duac phdt hien rd.
JOURNAL OF 108 - CLINICAL MEDICINE AND PHARMACY VoMO-N°1/2015 3.3. Ket qui gidi trinh tif gen cic mam binh
Pho sSc ky (chromatogram) chuoi giai trinh ti/ diTOc doi chieu tren ngan heing gen http://www.ncbi.nlm.nlh.nov/tools/primer-blast cho l<et qua nhif sau (hinh sau):
K. pneumoniae
GGTGACCACCGGGATCCTGCCGTGGAACrrCCCGTTCTTTCTTATCGCCCGCAAGCTGGCGCCGGaaGATa aGGAAATACCATTGTCATTAAGCCCAGCGAATTTACGCCCAATAATGCCATCGCCTTTGCCGAGATTGTCCATaG GTTGGGTTGCCGAAAGGGGTCTTTAACCTTGTGCTTGGCCGCGGAGAAACCGTTGGCCAGGAGaGGCCGGCAATC CGAAGGTGGCGATGGTCAGCATGACCGGCAGCGTGGCGGCGGGAGAAAAAATTATGGCCCGaGCGGCGA
Hinh 4. Hinh Snh tin hieu huynh quang cua doan gene dac triing cho vi Ichuan K. pneumoniae Nhdn xet: Ket quS xac dmh trinh VJ ADN khang dinh doan ADN ma chung toi nhan duqc la doan gen aldehyde dehydrogenase. Khi so sanh vdi cac trinh tir gen aldehyde dehydrogenase da cong bo tren ngan hSng gen the gidi tai d|a chi truy cap http://blast.ncbi.nlm.nih.nnv/ cho thay do tuong dong dat 99%.
Tuong tl/vdi K. pneumoniae b tren, giJi trinh tu gen cac loai vi khuan khac cho ket quS nhu sau: - Ket qui xac djnh trinh tu ADN doan gen I6S ribosomal RNA cCia ho Enterobacteriace chung, do tuong dong 96%; gi^i trinh tu doan gen S-uldfi. cua £co//, do tuong dong dat 97%; g\i\ trinh t J doan gen UreR cCa Proteus mirabilis cd do tuong d6ng dat 98%; doan gen flagellin ciJa Salmonella sp do tuong dong dat 94%.
TAP CHI Y DUO-C LAM SANG 108 Tgp 10-So 1/2015 3.4. Xdc dinh ngudng phdt hiin vd do ddc hiiu cua phuang phdp
3.4.1. Ngudng phdt hiin
De xac dinh ngudng phat hien cua phuang phdp ehung tdi tien hanh pha eae mam benh thanh cac ddi ndng dp khac nhau tU (10*CFU/ml -^ 10°CFU/ml). Sau do ehung tdi tien hanh taeh ehiet ADN tdng sd cua cac vl khuan va chay PCR.
jOg* lOe* lOe^ lOe^ lOc" CFV'ml
Enrerobacteiace— 424 bp Xpi chudn belaglobiilm— ISObp
EPKS'Salmonellosp 254hp
EPK.S'Prx>teii3mirabilis 406bp
EFtiS/ICpneiimcniae 334 bp EPKS'E coll "3 bp
Hinh 5. Ket qud xac dinh ngUdng phat hien cua PCR da moi tren cac mau chuan dUdng Nhdn xit: Qua hinh anh dien di d trdn cho thay: Ngudng phdt hien la 1-10 CFU/ml, tuy nhien de gidm thieu yeu td ngoai nhiem chung tdi lay ngudng phdt hidn cao hdn Id 10 CFU/ml.
3.4.2. Ddnh gid dd ddc hieu cCia PCR do moi
Thi nghidm sU dung cde mau ADN dugc tdeh ehiet tU cac lodi vi khuan gay benh khac nhU:
Staphylococcus aureus; Streptococcus pneumoniae; Pseudomonas aeruginosa; Acinetobacter baumanii, ADN ngudi, ADN nam, ADN virut khde. Ket qud the hien qua hinh anh dien di sau:
M50 1 2 y 4 1 6 7 8 9 10 II i : 14 15 16 17 Ent-Btia ^za bp Ent-BeI.Tl80bp
EPKS chii.in duimg
Ghichii M50-maker SObp.
2- Pseudomonas aei iiginosn 3-Staphylococcus ainniis 4- Snvplococi 5-Acweiobaciei baum e-Ndni Coduia alhicai
• -Nam .-Ispei-giUas sp S-Nam Fiisaniim sp 9-J0-AD.\'vinii viem gan B U-12.ADS vinii Epsrain - Ban 14-15. AD\ xinir Met pes simpler ISADX mail ngiiai khoe maiih 1'': Chudn dirffng
Hinh6. Ket qud kiem tra do dac hieu cua bo mdi tren ADN tac nhan gay benh khac Nhdn xit: Qua hinh dnh didn di cho thay khong cd ket qua dUdng tinh gid nao duge ghi nhan tren eae bdnh pham thU nghiem. Ket qud eho thay eae bd mdi chung tdi thiet ke ed do dae hieu eao, khdng nham lan vdi cac mam bdnh khde cung nhu ADN cua bd gen ngudi.
JOURNAL OF 108 - CLINICAL MEDICINE AND PHARMACY Vol10-N''l/2015
4. Ban luan
De thiet lap duge quy trinh PCR da mdi trude het chung tdi phdi tdi Uu cac dieu kien cua PCR n h u trinh tU mdi, ndng do mdi, Mg*^^ nhiet do, sd chu ky [1], 16] ... Ket qud eho thay khi thU nghidm PCR da mdi eae san pham dien di cho hinh anh dep, rd net, rat de nhan biet cd 4 lodi vi khuan quan tam {hinh 2, hinh 3). De xde djnh mdt each chinh xae sdn pham cd dung la loai vi khuan quan t a m , chung tdi da giai trinh t u gen va so sdnh vdi ngdn hang gen Qude te NCBI. Ket qud cho thay do tUdng d d n g rat cao (94- 98%). Nhu vdy, bd mdi va sdn pham PCR la dac hieu, khdng nham lan vdi cae mam benh khae eung nhU ADN eua bd gen ngudi (hinh 6). TU nhUng dieu kien phdn Ung PCR da duoc tdi uu, d gieng sd 6 hinh 3, vdi gid dinh mdt benh pham ed ed 4 tdc nhan vi khuan quan tam thi phuong phdp PCR da mdi cCia chung tdi eung ed the phat hien duge, ket qud rd rang, de phan biet cdc tae nhdn bdnh. Tuy nhien, day la tinh hudng gid dinh, vi t r o n g thUc te khdng ed khi nao mdt bdnh nhdn ed nhiem d d n g thdi 4 loai vi khuan ke trdn. Nhung nhU vdy chUng t d tinh uu viet eCia phUdng phap phdt hidn eCia chung t d i . ThUc te sau khi tien hdnh danh gia ngUdng phdt hidn va t i n h dac hieu eua phuong phap ehung tdi thay ngUdng phat hien Id 10 CFU/ml va khdng cd phdn Ung cheo vdi cdc mam benh khae. Ket qud nghidn eUu eua ehung tdi cung tUdng t u n h u nghidn eUu khae vdi do nhay ky thudt dao d d n g t r o n g khodng 10-50 CFU/ml [2], [5]. Nghidn cUu cua Hossein Fazzeli vd cs cung ed ket qud tUdng tU khi thay PCR ed do nhay 100%, do dde hieu 100%, vdi ngudng phat hidn lalO copies/1 phdn Ung [3],
5. Ket luan
Phuong phap PCR da mdi ehan dodn nhanh mdt sd lodi vi khuan thude hg Enterobacteriae: Escherichia
coli, Klebsiella pneumoniae. Salmonella sp, Proteus sp.
Enterobacteriaceae sp t h u d n g g a p gay bdnh d ngi/cn da d u g c t h i e t Idp t h a n h edng vdi n g u d n g phat hi&i la 10 CFU/ml va d d t u o n g d d n g la 94-99%, khdng co phan Ung cheo vdi ede m a m b d n h khae.
Tai lieu t h a m khdo
1. Chamberlain, J.S.,R.A. Gibbs,J.E. Ranier,P,M.
Nguyen,C.T. Caskey (1988) Deletion screening oftix Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res,16(23): p.
11141-56.
2. GosiewskI Tl,Jurkiewicz-Badacz D,Sroka A,Brzychczy-Wtoch M,B. M.(2014) A novel, nested, multiplex, real-time PCR for detection ofbaaeria and ft/ng/'/n Wood, BMC Microbiol.,14{l): p. 144.
3. Hossein Fazzeli, M.R.A., Bahram Nasr Esfahani, Farzin Khorvash,l Mohammad Reza Pourshafie,2 Sharareh Moghim, Hajieh Ghasemian Safaei, Jamshid Faghri, and Tahmine Narimani (2012}
Development of PCR-based method for detection ol Enterobacteriaceae in septicemia. J Res Med 5ei,17(7):p.671-675.
4. Park, D.W., B. C. Chun, et al. (2012) Epidemiological and clinical characteristics of community-acquired severe sepsis and septic shock: u prospective observational study in 12 university hospitals in Korea, J Korean Med Sei,27(ll):p. 1308-1314.
5. Pletz, M.W., N. Wellinghausen, et al. (2011) Will polymerase chain reaction (PCR)-based diagnostics improve outcome in septic patients?, A clinical view, Intensive Care Med 37(7): p. 1069-76.
u. Sambrook J., F.E.F., Maniatis T. (1989) l\Aolecular cloning Nil. A Laboratory Mannual London, UK:
Cold Spring Harbor Laboratory Press.
