88 TAP CHI PHONG CHONG BENH SOT RET VA CAC BENH KY SINH TRUNG SO 4 - 2011
UNG DUNG KY THUAT PHAN U N G CHUOI POLYMERASE (PCR) PHAT HIEN NAM CANDIDA SPP. GAY BENH 6 NGl/Ofl
Do Ngoc Anh', Nguyln KhSc Lire', Nguyen Duy BSc^
Nguyln Thj Huyen', Dirong Thuy Luih' ' Bo mon Ky sinh triing, Hoc vien Quan y,
^ Bo mon Giai phau. Hoc vien Quan y ' Sinh vien DH39, Hoc vien Quan y
Tom tdt
Chgn 9 mdu ndm men duang tinh tic kit qud nudi cdy dich dm dao di tdch DNA vd thuc hiin phdn img PCR.
Kit qud cho thdy 6/9 mdu nhdn dugc dogn gen dgc hieu vd dd chgn 01 mdu ndm dipha thdnh cdc dung dich cd ndng do khdc nhau di kiim tra do nhay cua phdn img PCR. Kit qud kiim tra do nhay cho thdy, vdi cap mdi FTSl vd 1TS4 phdn ting PCR cd khd ndng pfidt hiin ndm Candida spp. a ngudng > 0.5x10^ CFU/ml.
Tit khoa: Candida spp., do nhay, PCR
1. DAT VAN DE
Candida spp. la nam men gay benh phd bien d ngudi, chung cd the gay benh d bat kl co quan vd td chiic nao cua ca the nhung phd bien nhat la da va niem mac. Benh do nam Candida spp. cd the gap d ngudi binh thudng nhung thudmg gap ban d nhung ngudi cd nguy CO cao nhu benh nhan ung thu, benh nhan nhiem HIV/AIDS, benh nhan sii dung thuoc corticoid hoac thudc lic che mien dich keo dai. CJ ngudi, nam Candida spp. cd the gay ra nhumg hau qua nang ne nhu viem ndo, mang ndo, nhiem khuan huyet... Vi vay, viec chan doan sdm nguyen nhan gay benh la mgt yeu cau bet siic can thiet trong thuc hanh lam sang.
Tren thuc te, cd the chan dodn de dang nhiem nam Candida spp. trong cdc benh pham la dich tiet d da va niem mac nhd ky thuat soi tuoi hodc nudi cay. Tuy nhien, ddi vdi cdc benh pham la mdu, dich ndo tuy, md.. .cac bien phdp nay gap rat nhieu khd khdn. Phai mat hang chuc ngdy mdi xac dinh dugc sir cd mat cua nam Candida spp. trong cdc benh phdm nay.
Ky thuat PCR vdi uu dilm la do nhay, do ddc hieu cao, cd thi xdc dinh chfnh xdc loai ndm gdy benh. Them vao do thdi gian thuc hien ngdn, xdc dinh dugc tinh trang khang thuoc va cd the thuc hien thuan Igi d hau bet cac loai benh pham. Do vay, PCR hien la phuang phap dang dugc nhilu nha lam sang lua chgn de chan doan benh ndi chung vd chan dodn benh do nam Candida spp. ndi rieng. Hien nay, d Viet Nam chua nhilu ngudi quan tam din viec iing dung ky thuat PCR trong chdn doan benh do nam Candida spp. Xuat phat tir nhiing ly do tren chung tdi tiln hanh de tai "tfng dung ky thuat PCR phdt hiin ndm Candida sp gdy benh d nguoi" nhdm muc tieu:
Bir&c dau nghien cim vd thdm do ngudng phdt Men ndm Candida sp ciia ky thugt PCR dk tii dd tilp tuc nghien ciiu iing dung ky thuat PCR ti-ong lam sang chdn dodn benh do ndm Carulida spp.
2. DOI TirONG, VAT LIEU VA PHU'ONG PHAP NGHIEN CUtJ 2.1. Doi tu-gng nghien cun
9 mau nam men Candida spp. dugc phan lap tu ngudi nhiem nam Candida spp. dudng sinh
' ^ m., r
due den khdm, dieu tri tai BV 103 va Trung tam Md phdi Hgc vien Quan y. Chgn mdu nam cd PCR duomg tfnh pha thanh cac dung dich cd nong do O.SxlO'' CFU/ml, 0.5x10^ CFU/ml, 0.5x10^
CFU/ml, 0.5x10^ CFU/ml, 0.5x10^ CFU/ml, 0.5x10" CFU/ml dl thir do nhay.
2.2. Vat lieu nghien curu
Dung cii: may PCR (GeneAmp PCR system 9700 AB Applied Biosystem, My), may ly tdm lanh Mikro 22R (Hettech, Diic), may soi va chup gel Dolphin Doc (Wealtec, My), bd diendi...
- Sinh phdm hoa chdt: Mdi trudng Sabouraud, dung dich NaCl 0.9%, bd ta't tdch ADN ting sd (QIAGEN, My), cac hda chat can thiet khdc.
Cap mdi cho phdn ung PCR: Cap mdi ITSl (forward, 5'-TCC GTA GGT GAA CCT GCG G-3') vd ITS4 (reverse, 5'-TCC TCC GCT TAT TGA TAT GC-3') da dugc chgn (theo SH Mirhendi vd cs, 2001)
2.3. Phuong phap nghien ciin
2.3.1. Lay benh pham va nuoi cay tang sinh
Nhiimg benh nhan nir nghi ngd nhiem ndm dugc ldy benh pham va cay benh pham tren moi trudng Sabouraud. Sau 3 ngay quan sat khuan lac va soi kfnh hien vi kiem tra. Nhiing mau duomg tinh vdi ndm men dugc tach DNA vd thuc hien phan ung PCR.
2.3.2. Tach DNA va nhan doan gen dac hieu
Cdc mau nam men dugc tdch DNA theo qui trtnh cua hang QUIAGEN (My) va thuc hien phan iing PCR. Thanh phdn phan iing PCR gdm: Master mix, DNA tdng so, mdi va nude cdt kbit ion vua du 25^1. Thdi gian chay phan iing PCR nhu sau: 1 chu ky 95°C trong 5 phut;
35 chu ky moi chu ky gdm 95°C trong 30 gidy, 55°C trong 45 giay, ITC trong 1 phut; 1 chu ky 72°C trong 7 phut. San phdm phan umg PCR dugc bao quan d 4 "C.
2.3.3. Phuong phap kiem tra san pham PCR
San pham PCR dugc kiim tra bdng each dien di tren thach Agarose 1,2%. Nhirng mau duong tfnh Id nhung mau cd band dac hieu da dugc thdng bao {SH Mirhendi vd cs, 2001).
2.3.4. Kiem tra do nhay
Cdc mau ndm cd kit qua PCR duomg tfnh dugc chgn ra dl pha thanh cac dung dich cd ndng do khdc nhau. Sau dd cac dung dich nay dugc dem tach DNA va chay PCR trong nhirng dieu kien nhu nhau.
90 TAP CHI PHONG CHONG BENH SOT RET VA CAC BENH KY SINH TRUNG SO 4 - 2011
2.4. Dia diem va thdi gian nghien cun 2.4.1. Dia diem nghien cun:
Labo Trung tam nghien ciiu Y Dugc hgc Quan su - Hgc vien Quan y.
Phong xet nghiem Ky sinh triing va vi nam - Benh vien Quan y 103 va Labo ndm Bg mdn Ky sinh triing - Hgc vien Quan y.
2.4.2. Thoi gian nghien cun: tir 2/2011 - 7/2011 3. KET QUA VA BAN LUAN.
3.1. Ket qua xac dinh kha nang nhan gen dac hieu cua cap moi ITSl, ITS4 va lua chon mau nam Candida spp.
Do khdng cd cac chung Candida spp. chuan nen chung tdi tien hdnh nghien cuu tren nhiing mau nam tir nhijmg benh nhan dugc xac dinh nhiem ndm qua ket qua nudi cay. Nhirng mau cd ket qua cay nam duomg tfnh vdi nam men tren mdi trudng Sabouraud tiep tuc dugc xac dinh mgt each chac chan la 1 trong 6 loai nam Candida bang cap mdi ITSl va ITS4.
Ket qua hinh 1 cho thay, 6/9 mau xuat hien band ddc hieu. Trong do 2 mau cd band ddc hieu kfch thudc khoang 550bp va 4 mau cd kfch thudc band khoang 870bp.
Hinh 1: Mau nam cd ket qua PCR (+)
Theo SH Mirhendi vd cs (2001), cap moi ITSl va ITS4 cd kha ndng khuleh dai thanh cdng doan gen ITS cua 6 loai nam Candida la C. albicans, C. glabrata, C. tropicalis, C. krusei. C. guilliermondii. C. parapsilosis cho doan gen dac hieu cd kfch thudc dao dgng tir 510-870bp tuy loai nam Candida spp. Cu the C. albicans, C. glabrata, C. tropicalis.
C. krusei, C. guilliermondii vd C. parapsilosis cho doan gen cd kfch thudc tuomg iing la 535bp, 871bp, 524bp, 510bp, 608bp va 520bp. Ngoai ra ciing theo SH Mirhendi vd cs (2006), cap mdi nay ciing cd the phat hien nhd khueeh dai dac hieu doan gen cua cdc nam khdc gay benh d ngudi nhu A. fumigatus. Cr. neoformans va F solani vdi kfch thudc tuomg iing Id 597bp, 556bp vd 526bp.
Tren thuc tl, cap mdi ndy cung da dugc nhieu tdc gid diing dl phat hien cac nam Carulida sp, C. neoformans vd A. fumigatus gay benh d cac vi tri khac nhau cua ca the nhu mau, dich nao tuy, dich phi quan (P.V.C.Yong vd cs, 2008; Catherine Dunyach va cs, 2008).
Trong nghien ciiu nay, chung tdi thdy 4 mau ed band kfch thudc khoang 870bp phu hgp vdi nam C. glabrata, 2 mau cd kfch thudc khodng 550bp phu hgp vdi ndm C. albicans. Tuy nhien, de xdc dinh chfnh xac loai can tien hanh giai trinh tu hoac sir dung them cac ky thudt khac (sir dung enzyme gidi ban, PCR ldng...). Do muc dfch xac dinh do nhay cua phan ung PCR nen chung tdi khdng di xac dinh loai nam ma lira chgn 01 chung ndm Candida spp. ed PCR duomg tfnh de tiep tuc thir do nhay. Tir ket qua nghien ciiu chung tdi da lira chgn mdu sd 5 (Hinh 1) cho muc dfch nay.
3.2. Ket qua xac dinh do nhay cua phan ung PCR
Mau nam Candida spp. cho ket qua PCR duomg tfnh dugc pha loang thanh cac dung dich cd ndng do khac nhau. Xir ly bang nhiet cac dung dich nay rdi dem tach DNA trong ciing mot dieu kien nhu nhau. 3 |il san pham tach cd DNA dugc lay de lam khuan cho phdn iing PCR. Ket qua Hinh 2. cho thay phan iing PCR duomg tfnh vdi ede mau ed mat do nam
> 0.5x10^ CFU/ml.
Mau Mat do te bao
(CFU/ml) Kit qua PCR
1 Nude
eat
2 0.5x10-
3 0.5x10^
4 0.5x10"*
5
0.5x10' +
6
0.5x10^
+
7
0.5x10^
+ 7 6 5 4 3 2 1 M
Hinh 2.- San phdm PCR theo mat do te bao
(Gieng M- marker DNA lOObp, Gieng 1: nude cat, Gilng 2: nong do ndm 0.5x10^ CFU/ml, Gilng 3- 0.5x10^ CFU/ml, Gieng 4: 0.5xlO'*CFU/mi, Gilng 5: 0.5x10'CFU/ml,
Gilng 6: 0.5x10*CFU/ml, Gilng 7: 0.5x1 O^CFU/ml)
92 TAP CHI PHONG CHONG BENH SOT RET VA CAC BENH KY SINH TRUNG SO 4 - 2011
v l do nhay trong viec phat hien ndm Candida spp., Ni Made A. Tarini vd cs (2010) ling dung phan iing mutiplex-PCR vdi 2 cap mdi cd the phdt hien 5 loai nam Candida spp.
a nguang > 2.6 xlO" CFU/ml. Cung theo tac gia nay, multiplex-PCR cd do nhay cao, do chfnh xdc cao va thdi gian nhanh ban phuang phap nudi cay. Ben canh dd, tac gia cdn cho rdng, phuang phap nudi cdy dan thudn khd cd thi xac dinh duge loai trong khi do cac phuang phap sinh hge phan tir tbi ngugc lai. Cung vl vdn dl nay, K. MAKIMURA vd cs (1994) nghien cuu so sanh kha ndng phat hien ndm Candida spp. cua 2 phuang phdp PCR va nudi cdy vdi benh phdm la mau (chugt) cho thdy, phuang phap PCR ed do nhay, do dac hieu cao va thdi gian phat hien ngdn hom phuang phap nudi cay. Mdt nhdm tac gid khac la A.J.M. VAN DEVENTER vd cs (1995) cho rdng phuang phap PCR cd tbi phdt hien nam Candida albicans trong mau d nguang 10 den 15 te bao nam trong moi 100 pi mdu toan phan.
Nhu vay, do nhay cua phuang phap PCR phat hien nam Candida spp. trong nghien cuu eua ehdng tdi edn thdp hem so vdi eae nghien ciiu khac. Do nhay trong nghien ciiu nay thap cd thi do nhung ly do nhu hieu qua tach DNA nam chua cao, qua trinh tach mau cdn chua nhilu tap chdt lie chi phan iing PCR... Tren thuc te, do nhay eua phan iing PCR cd the phu thudc vao loai nam Candida spp. Do do, can tiep tuc nghien ciiu xac dinh do nhay vdi timg loai ndm Candida spp. va tim hieu cac yeu td anh hudng de tdng do nhay phat hien cua ky thuat PCR vdi cap moi ITSl, ITS4.
4. KET LUAN
- Cap mdi ITS 1 va ITS4 da nhan duge chfnh xac doan gen dac hieu.
Budc ddu da xac djnh dugc do nhay eua phan iing PCR vdi cap mdi ITS 1, ITS4 la >
0.5x10'CFU/ml.
Kien nghi
- Tilp tuc nghien ciiu de tang do nhay eua phuomg phap va nghien ciiu tren timg loai benh pham lam sang eu the.
I Tilp tuc nghien ciiu do dac hieu cua phuomg phap vdi cac can nguyen gay benh thudng gap d ngudi khac.
- Nghien cuu phat hien cac nam khac trong pham vi kha nang cua cap moi ITS 1 va ITS4.
TAI LIEU THAM KHAO
1. Le Bach Quang va cs (2008). Ky sinh triing vd cdn triing y hge. Giao trinh giang day dai hoc - Hoc viSn Quan y. Nha xuit ban QuSn d6i nhan dan, tr: 382-386.
2. Le Bach Quang va cs (2010). Ky thugt ky sinh trung. Giao trinh giang day dai hoc - Hpc vien Quan y.
Nha xuat ban Quan doi nhan dan, tr: 310-319.
3. Tr4n Xuan Mai va cs (2004). Vi ndm y hgc. Tniong dai hoc Y dugc Tp.HCM. Sach tham khao, tr: 107-120.
4. Phan Anh Tuan va cs (2007). Cdc lodi Candida spp. gdy viem dm dao tdi phdt vd do nhay vdi thudc khdng ndm. Tap chf phong ch6ng benh sot ret va cac benh Ky sinh trung, s6 1/2007, tr: 70-75.
5. Tran Phu Manh Sieu, Ho Quang Thing (2010). Tmh Mnh nhiim vi ndm Candida spp. trin ede binh nhdn nhgp vien tgi binh viin binh nhiit dai Tp.HCM 2009. Tap chf Y dugc Tp.HCM nam 2010.
6. P. Lewis White et al (2003). Detection of seven Candida species using the
Light-Cycler system. Journal of Medical Microbiology (2003), 52, 229-238.
7. L. Klingspor and S.Jalal (2006). Molecular detection and identification of Candida and Aspergillus spp. from clinical samples using real-time PCR. Clinical Microbiology and Infection Diseases, CMI, 12, 745- 753, Volume 12 Number 8, August 2006.
8. Hossein Mirhendi et a! (2006). A One-Enzyme PCR-RFLP Assay for Identification of Six Medical Important Candida Species. Jpn. J. Med. Myol. Vol. 47,225-229, 2006.
9. Rosa de Llanos Frutos et al (2004). Identification of species of the genus Candida by analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers. Antonie van Leeuwenhoek 85: 175-185,2004.
10. SH Mirhendi et al (2001). A PCR-RFLP Method to Identification of the Important Opportunistic Fungi: Candida Species, Cryptococcus neoformans, Aspergillus famigatus and Fusarium solani. Iranian J. Publ.
Health, Vol. 30, Nos. 3-4, PP. 103-106,2001.
11. P.V.C.Yong et al (2008). Molecular Identification of Candida orthopsilosis Isolated from Blood Culture. Mycopathologia (2008) 165:81-87. DOI10.l007/sll046-007-9086-8.
12. A.J.M. VAN DEVENTER et al (1995). Improved Detection of Candida albicans by PCR in Blood of Neutropenic Mice with Systemic Candidiasis. JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1995, p.625- 628. Vol.33, No.3.
13. Ni Made A. Tarini et al (2010). Development of multiplex-PCR assay for rapid detection of Candida spp. Med J Indones, Vol. 19, No. 2, May 2010.
94 TAP CHI PHONG CHONG BENH SOT RET VA CAC BENH KY SINH T R O N G SO 4-2011
DETECTION AND IDENTIFICATION OF CANDIDA SPP. ISOLATED FROM VAGINAL HUMAN BY POLYMERASE CHAIN REACTION
Do Ngoc Anh, Nguyen Khac Luc, Nguyen Duy Bac Nguyen Thi Huyen, Duong Thuy Linh
Department of parasitology of Military medical University,
Abstract
Nine Carulida spp. samples were collected from the cultured human vaginal mucus to extract DNA and perform PCR. The result of PCR showed that six of nine samples were positive with specific band, one of which was selected to make solution with different concentrations for checking the sensitivity of PCR method. We found that with the paired primers ITSl and ITS4 the assay was able to detect Candida spp. cells at dilution of 0.5x10^
Keyword: Candida spp., sensitivity, PCR
Can b6 phan bien
PGS.TS. Nguyen Van De Truong Dai hoc Y Ha Noi