S 6 2-2013 TAP CHl P H 6 N G C H O N S BENH SOT RET VA C A C BENH KY SINH TRpNG 3_
iTNG DUNG KY THUAT PCR DINH DANH NAM DU^PNG SINH SAN 6 PHU Ntr TUOI 18 -49 DA CO CHONG TAI
HUYEN TAM NONG TINH PHU THO 2011 - 2012
Cao Bd L9i', Nguyin Thai Dung^ Tran Thi Quynh Lien^
Tr^n Thj Kieu Anh^ vh CS T6m tat:
532 phu nii trong do tuoi sinh de 18 - 49 da co chong tai huyen Tam Nong - tinh Phu Tho da dugc kham sdn khoa va lay mau xet nghiem dich dm dao tim ky sinh trung. Kit qud co 135 mau nhiem nam Candida spp bdng phuong phap soi tuai, nhuom Gram, va nuoi cay trong moi trudng Saboraud. Sau do 93 mau duac xet nghiem hdng ky thuat PCR de dinh loai nam, kit qud: 69/93 (74,2%) mau cho kit qud bdng (giai) PCR, 24 mdu cho kit qud (-). Ky thuat PCR v&i gen mdi ITSl 5' TCC GTA GGT GAA CCT GCG G 3'vd ITS4: 5' TCC ICC GCT TAT TGA TAT GC 3' la phu hop. Ty le mdu nam phat hien duac bdng PCR tai cdc xd Quang Hucld 19,2%. TiLi26.3%vdTho Vdn3I,8%.
Kich thir&c ciia sdn pham cat DNA bdng enzyme cat han che cho kit qud nhu sau:
Candida gabrata (C. Glabrata) 557,314 bp; Candida tropical (C. Tropical) 340 bp; Candida albicans (C Albicans) 297,238 bp; Candida krusei (C Krusei) 261, 249 bp; Candida parapsilosis (C Parapsilosis) 176,243 bp
Ty le, thanh phan cdc loai nam chung a ba xa Id: C. glabrata chiim 43,5%, C. tropical chiem 34,8%, C. albicans chiem 14,6%, C. krusei chiim 4,3%, C. parapsilosis chiim 2,8%.
TIT khoa; - Duong sinh san;
- Nam,
1. DAT VAN DE
Nhilm khuan ducmg sinh san la nguyen nhan gay nhieu benh canh lam sang va roi loan ciia CO quan sinh san. Tai Viet Nam van de nhiem khuan sinh san do nam Candida spp dang CO xu hirong gia tang. Nghien cuu ciia Vien Da lilu Trung uong tai 5 tinh dong bSng Bac Bo (1999), cho thiy ty le phu nu 15-49 tu6i mSc it nhat mot loai nhiem khuan duong sinh san la 70,56%, tren lam sang cac viem nhilm thucmg gap a the phoi hop, co 35,6% viem co tii cung va viem co tu cung phoi hcrp vai viem am dao can nguyen do Candida spp va Trichomonas vaginalis [1], [2], [3j.
Tinh Phii Thp noi chung va huyen Tam Nong noi rieng, cho den nay chua co cong trinh nghien ciiu nao that dky dii va quy mo de danh gia tinh trang nhiem nam duong sinh san, dac biet la nghien cmi sau \h ty le va thanh phan cac loai nam gay benh. Nhu:ng cac yeu to nguy CO (YTNC) nhilm nkm duong sinh san a phu nu thi xkt cao [1], [2], [3], [4], nhu: Thoi quen
' Vien S6t ret - K S T - C T T U
^TT Da liiu Ngh? An
^ E)ai hgc Y Vinh
^ so 2-2013 TAP CHl P H 6 N G C H 6 N G B|NH SgT RET VA CAC BJNH KY SINH TJiUNS
song va sinh hoat mat ve sinh, su dung nguon nu6c ban, tinh trang nao ph4 thai v4 sinh ho?it tinh due bua bai, anh huong ciia dat vong tranh thai...da lam cho ty 1? b$nh tang cao. Vi vay viec ling dung ky thuat hien dai PCR nghien ciiu ty le thanh phan lo4i nam gSy b?nh du&ng sinh san a phu nil trong do tuoi sinh de la can thiet, de co dinh huong cho c4c bi?n phap phong chong nhSm cai thien silc khoe phu nii noi rieng va cpng dong n6i chung, chiing toi thuc hien d8 tai nghien ciiu: U'ng dung ky thu^t PCR dinh danh nam Juing sinh sin &pbu nOttuii (IS -49) di c6 chong tai huy$n Tam Nong tmh Phu Thf 2011 • 2012.
Mvc tieu dl tii: Xac djnh ty le va thanh ph4n loai nSm gay benh du6ng sinh sto b phu nQ trong do tu6i sinh de (18-49) da co gia dinh tai huyen Tam Nong tinh Phii Thp bang ky thuat ITS 1-ITS4 PCR.
2. D 6 I Tl/PNG VA PHlTONG PHAP NGHIEN Ctltl 2.1 Doi tu-ong, dia dilm, thoi gian nghien ciiu 2.1.1 Doi tygng nghien ciiu:
Phu nil do tuoi 18 - 49 da co gia dinh, tu nguyen tham gia nghien ciiu.
2.1.2 Dia diem va thM gian nghien cihi
- Dia diem nghien ciiu huyen Tam Nong - tinh Phii Tho: Cac xa Te Le, Quang Hue, Thp Van.
- Thai gian nghien ciiu tit 5/2012 - 5/2013.
2.2 Phu-ong phap nghien cuu
2,2,1 Tach chiSt DNA he gen cua nam [5], [6], [7], [8].
Diing que cay gat lop te bao nam cho vao eppendorf 2 ml chiia 0,5 ml lysis buifer, nhung que cay vai lan trong dung dich de te bao nam men roi va trong dung dich.
Bo sung 25 nl 100 mg/ml iysozyme va 10 jil 1% RNase vao mSi mau, ii h6n hop dung dich te bao a 37°C trong thai gian 60 phiit
Them 10 nl 20 mg/ml proteinase K va 55 (tl 10% SDS (n5ng do cudi dat 1%) vao mfli mau, li hon hop dung dich te bao a 37"C trong thai gian 30 phiit
Them 500 nl TE vao moi mlu, u hSn hop dung dich tj bao a 65°C trong thoi gian 120 phiit Li tam hon hop dung dich le bao 13000 vong/phiil, 10 phiit. Thu 1000 nl pha tren, b6 sung 1000 nl Phenol: Chloroform:isoamylalcohol (25: 24:1), lac d^u hdn hap.
Li tam 13000 vong/phiil, 10 phiit. Thu 900 nl pha tren, b6 sung 900 nl Chloroform:
isoamylalcohol (24:1), iac deu h6n hop.
Li tam 13000 vong/phiit, 10 phiit. Thu 800 nl pha tren, b6 sung 1400 nl 96% ethanol, ISc deu hon hop, de yen a nhiet dp phong 20 phiil.
Li tam 13000 vong/phiit, 10 phiit. Thu can, b6 sung 1000 nl 70% ethanol, IJc dSu.
Li tam 13000 vong/phiit, 10 phiit Thu can, d6 kho onhiei dp phong trong thoi gian lOphilt Hoa tua vao lOOnlTE
S6 2-2013 TAP CHJ P H 6 N G C H 6 N G BJNH S6T RIT VA CAC BJNH K9 SINH TRUNG 5^
Di$n di kiSm tra 2 nl san phim tach chiit tren gel agarose 0,8%
2.2.2 Lim sjch DNA he gen ciia n^m sau tich chiit
Thuc Men lai til buoc chiit bing Phenol: Chloroform: isoamylalcohol (25: 24:1) trong quy trinh tach DNA he gen de loai bo hoin toin nhilng chit ban con dinh vao DNA h$ gen.
2.2.3 Phan iing PCR
Bang 1: M6i trudng phin ling PCR (trong lOOnl) STT
1.
2.
3.
4.
5.
6.
7.
Thinh phan phan ihig DNA khuon
20 nM ITSl primer 20 pM ITS4 primer 2,5 mM dNTP I Ox PCR buffer Taq PCR polymerase H2O
Nong d$ cuoi Kh6ng x^c djnh
0.2 nM 0.2 pM 0.1 mM
Ix 2.5U Din V= 100 pi Bang 2: Chu trinh nhiet cua phdn iing PCR STT
1.
2.
3, 4.
5.
Nhi?« d9 (°C) 94 94 56 72
ThM gian 5 phiit 30 giay 45 giay 60 giay Lap Iai 30 chu ky til buoc 2 den buoc 4 72 j 7 phiit 2.2.4 Phan ihig cat DNA bang enzyme cat ban che
Phan ling cat san pham phan iing PCR bang enzyme cat han che Msp I duac thuc hien trong t6ng thS la 50 ^1 gom cac thanh phan nhu sau:
Bang 3: Thanh phin ciia phan iing cJt PCR bang enzyme cat han che Msp I STT
I.
2.
3.
Thanh ph4n phan ling AfepIbufferlOx
Msp I San phim PCR
Thi tich (^l) 5 0.5 45 - U han hop phan ling cSt 6 37°C hong thai gian 120 phiit
- Dien di kilm ha san phim cat tr&i gel agarose 1,7 %, 50 bp DNA tnaricer (femienlas).
2.2.5 Nguyen Ii$u nghien ciiu:
93 dong nim da duac dong hoa tren moi truong Saboroud c6 chiia hloramfenicol, duac bao quan trong tu im 37''C.
S6 2-2013 TAP CHl PHONG C H 6 N G BENH S6T RET vA C'AC^E^H kY SlN>l Tfeur^g ^
Hinb 1 Man nam co kef qua ('<-) bangphuxmgphap nuoi cay trong moi tru&ng Saboraud
Nhan xet • Chiing toi nhan duac tong so 93 ong nuoi cay nam, ve dSc diem cac khuan lac nam nhu sau:
Te bao vi nam mpc Ihanh cac khuan lac, mpt ong nuoi cay c6 rat nhieu khuan lac, kich thuoc khuan lac nho tir K5 - 4 mm; trung binh 2,5 ± 1,5 mm. Mau sac: Trang min nhu bong tuyet, mpt so mau co mau nga vang hoac hoi hong, mpt so mau vira co khuan lac mau trSng min vira co cac khuan lac mau nga vang, nhu'ng mau nay co the nhiem hai loai nam.
Quan sat phan moi trucmg thach nuoi cay van giiJu nguyen mau vang nau, khong co van due, khong bi nut va, bam chat vao thanh ong nuoi cay,
2.4.2 Cac loai hoa chat
Nghien cuu sir dung cac loai hoa chat can thiet theo quy trinh ky thuat PCR 2.4.3 Cac moi sir d u n g trong ngbien ciru
Nghien ciiu sir dung cac loai hoa chat can thiet theo quy trinh ky thuat PCR
3. K E T QUA VA BAN LUAN
3.1. Tach cbiet DNA tir cac dong nam da dong boa 3.1.1 Tach tho ban dau
bp IVT I 2 3 4 5 6 8 II 12 13 14
16 17 18 19 20 21 22 23 5 6 7 M Hinb 2: Hinh anh PCR tach tho ban Su bdng mdi ITSl va ITS4
S 6 2-2013 TAP CHi PHONG C H 6 N G BENH SOT RET VA C A C BgNH KY SINH TRUNG T_
Tir ket qua thu dugfc chiing toi nhan thay, DNA he gen ciia cac dong te bao deu da tach chiSt duac, dugc the hien la cAc duong ch^y dIu co mpt bSng DNA ro net. Tuy nhien, theo quan sat vin thay cac vet ma dual cac bang DNA he gen, chiing to san pham tach chiet chua sach. Cac miu DNA nay dugc Iam sach bing each chiet Iai mpt Ian niia bang phenol va xu li vai proteinase K va RNase.
3,1,2 San phim PCR x i 1^ viM RNase, proteinase K va chiet lai v*! phenol 4 S 6 8 10 11 12 13
M 14 15 16 17 IS 19 20 21 22 23
HinhS: Hinh anh PCR sau xir ly vai RNase. proteinase K' va chiit lai vai phenol Sau khi xii li blng phenol, proteinase K va RNase chiing toi da thu dugc san pham DNA he gen sach. Tren dien di d6 dugc thi hien bang cac bang DNA co kich thuoc Ion han 10000 bp, khong thay vet ma. Cac san phim nay du tieu chuin dl lam khuon DNA khuech dai mot doan DNA bing ky thuat PCR.
8 80 2-2013 TAP CHI PHONG C H 6 N G BJNH S6T RET VA CAC BJNH KY SINH TRUNG
3. 2, PCR bing mii ITSl va ITS4
M 1 2 3 5 6 7 8 10 11 12 13
Hinh 4: PCR bSng mdi ITSI va ITS4
Tat ca cac miu deu thu dugc san pham PCR la mpt bang DNA ro net, chiing to m6i va chuong tinh chay PCR la phu hgp. Rieng truong hgp s6 7 san phim PCR rit it. co thi do s6 lugng DNA khuon dugc dua vao phan ling chua phii hgp.
3,3, San pham PCR cat bang enzyme cit han chi Msp I 1 V I 1 2 3 5 6 7 8 10
) 2-2013 T^PFdHr^HONG CH6NG B|NH S6T RET VA CAC BENH KY SINH TRUNG
14 15 16 17 18 19 20 21 23 TVS Q4 M
Hinh 5: Hinh Sdn pham PCR cat hang enzyme cdt han chi Msp I De kiem tra sir da dang cua cac dong te bao, chiing toi su dung enzyme cat han che M.sp I de cat cac san pham PCR. San pham PCR duac phan tach tren gel agarose 1,7%. Ket qua cho thay, a tat ca cac dong deu co hien tupng san pham PCR bi cat bai enzyme cSt han che Msp I. So bang DNA dupe tao ra tir cac phan iing cat thuong la 2 bang. So lupng bang DNA va kich thuac cua timg bang dupc thong ke trong bang sau:
Xa
Quang Hiic TILI Tho Van
Chung Gia tri p
Bang 4: Tong hop ket qua dinh loai nam bang ky thuat PCR So mau nam xet
nghiem PCR
52 19 22 93
Ket quii xet nghiem bang ky thuat PCR So mau (+)
So mau (+) 42 14 15 69
Ty le (%) 80,8 73,7 68,2 74,2 (1:3)<0,01;(2: 3) > 0,05
So mau (-) So mau (-)
10 05 07 22
T y l e ( % ) 19,2 26,3 31,8 25,8 ( 1 : 3 ) < 0 , 0 1 ; ( 2 : 3 ) > 0 , 0 5 Nhan xet: Ty le phat hien ndm (+) bing ky thuat PCR vai cap moi chung ITS1-1TS4 la 74,2%, CO 24 mau chua tim thay hinh anh PCR tren di thach. Co su khac biet ve ty le phat hien dupe PCR giiia cac xa Quang Hue va Thp Van (80,8% so voi 68.
Bang 5: S6 luong san phSm PCR ITS1-ITS4 truac va sau khi cat bang Msp I
TT 1 2 3 4 5
Kich thuoc ITSl- ITS4 (bpp)
871 524 535 510 520
Kich thuoc san pham cat (bpp)
557,314 340, 184 297,238 261,249 176,243
Ten loai Candida spp C. glabrata C tropical C albicans C krusei C. parapsilosis Ting
So lugng 30 24 10 3 02 69
T y l £ ( % ) 43,5 34.8 14,6 4,3 2,8 100
10 S62-2013 TAP CHl PHONG C H 6 N G BgNH SflTRgTVA CAC B|NH KYSINH TRUNG
Nhan xet: San phim PCR ITS1-ITS4 truac va sau khi cit bing Msp I ciia cac loai nim gay benh DSS 6 dii tugng nghien ciiu la: C. glabrata la 871 bp va 557,314 bpp; C. tropical la 524 bpp va 340,184 bpp; C. krusei la 510 bpp va 261,249 bpp; C. albicans 14 535 bpp va 297,238 bpp, C. parapsilosis la 520 bpp va 176,243 bpp.
Tir nhiJng thing ke trong Bing 3 \k BSng 4 chiing toi nhto thiy ring trong s6 nhilng mlu ma chiing toi thuc hien thi loai C. glabrata chiim uu thi viii 43,5%, tilp din la loai C.
tropical chiim 34,8% va C parapsilosis 2,8% v4 C. krusei it nhit chiim 4,3 %. Rieng Candida albicans la loai nim rit nguy hilm, nhung trong nghien ciiu ciia chiing toi 14 14,6%, ket qua nay kh4c vol cdc nghi£n ciiu tru6c dIu khing dinh Candida albicans chiem da so.
So lieu nay co sir khac bi?l so voi cac cong bo ve thong ke da dang cac loai nam men gay benh ciia mpt so cong trinh gay benh tren the gi6i. Da so cac cong trinh deu cong bo la C albicans chiem so lugng nhieu nliat. Su khac biet ve so lieu nay co the do ca mau qua nho hoSe vimg lay mau qua hep. Vi the can phai co nlumg nghien ciiu rpng hem, so lugng mau nhieu han thi mcri CO the dua ra dugc bite tranli long quan ve su da dang ciia loai nam gay benh.
Tuy nhien, vol so lieu nay chiing toi de xuat nen giai trinh tu san pham PCR it nhat moi loai phan tich mpi mau, dac biet nhirng loai kho phan biel nhau bang kich thuoc san phim PCR va san pham cat thi nen tang so mau neu kinh phi cho phep.
4, KET LUAN
Cac mau thu dupe san pham PCR la mpt bang (giai) DNA ro net, chimg to mii va chuang tinh chay PCR voi gen mii ITS] 5' TCC GTA GGT GAA CCT GCG G 3' va ITS4:
5' TCC TCC GCT TAT TGA TAT GC 3' la phii hgp.
Kich thuoc cua san pham cat C glabrata 557,314 bp; C. tropical 340 bp; C. albicans 297,23 8 bp; C kruse, 261, 249 bp; C, parapsilosis 176,243 bp.
Ty le phat hien va dinh loai nam bang ky thuat PCR voi gen moi la ITS 1-1TS4 la 74,2%
(69/93). Ty le mau nam phal hien dugc bang PCR tai cac xa Quang Hiic la 19,2%, Tl Le 26,3% va Thp Van 31,8%.
Ty le cac loai nim ky sinh tai duong sinh san chung 6 ba xa la; C. glabrata chiim 43,5%, C. tropical chiem 34,8%, C. albicans chiem 14,6%i. C. krusei chiim 4,3%, C. parapsilosis chiim 2,8%,.
S6 2-2013 TAP CHi PHONG C H 6 N G BENH S6T RET VA CAC BENH KY SINH TRUNG l j _
TAI U^U THAM KHAO
1 Bp y te (1996), Cdc nhiem khudn du&ng sinh due va HIV/AIDS, Nhiem khuan duong sinh due va HIV/AIDS - Modun 10 - Tai Heu huan luyen to^n dien ve sue khoe sinh san, NXB Y hpc, Ha Npi, Trang: 59.-65.
2 Bp Y te (1998), Cdc benh lay truyin theo du&ng tinh due, Cham soc sue khoe ba me tre em tai cac tuyen y te, NXB Y hpc. Ha Npi, Trang: 128.
3 Bp Y te (2003), Huang ddn chuan Quoc gia vi cdc dich vu cham soc sire khoe sinh sdn, NXB Y hpc, Ha Npi, Trang: 126-144.
4 Wei YP, Feng J, Luo ZC et al (2010), "Isolation and genotyping of vaginal non-albicans Candida spp. in women from two different ethnic groups in Lanzhou China", Int J Gynaecol Obste.t. Vol.ll0(3), pp.227-30.
5 Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S et al (2011), "MEGA5 molecular evolutionary genetics analysis using maximum likeli-hood, evolutionary distance, and maximum parsimony methods", Moi. Bio, Vol. 28, pp:2731-2739.
6 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, Vol 12 ( 8), pp. 745-753.
7 White .P.L., Bretagne .S., Klingspor . L. and at al (2006), ''Aspergillus PCR: one step closer to standardization", PubMed - indexed for Medlin, niversity Hospital of Wales, Health Park, Cardiff CF 144XN, London, Kingdom
8 White .P.L., Carlo Mengoli and et al (2011), "Evaluation of Aspergillus spp PCR Rrotocal for Testing Serum Speccimens", Juonal of Clinical Microbiology, American Society for Microbiology.
12 so 2-2013 TAP CHi PHONG C H 6 N G BENH SgT RET VA CAC BJNH KY SINH TRUNG
Abstract
APPLYING PCR TECHNIQUE TO IDENTIFY VAGINAL FUNGI IN MARRIED WOMEN AT THE AGE OF 18 -49 IN TAM NONG, PHU THO IN 2011-2012
Cao B4 Lgi', Nguyin Thii Dune', Tr4n Thi Quynh Lien ', Triin Thj Kifu Anh 'et al.
A total of 532 married women at the age of child bearing 18 - 49 in Tam Nong -Phu Tho were gynecologically examinated and the samples of their vaginal discharge were taken to identify parasites. The resuhs produced 93 Candida spp samples by PAP tests, Gram staining and cuhure in Saboraud Enviroment. Then all of them were tested by PCR [5], [6], [7], [8], to identify fitngus types. The resuhs were follows: 69/93 (74,2%) of the samples were positive, 24 samples were negative (-). PCR with gene primers ITSI 5' TCC GTA GGT GAA CCT GCG G 3' and ITS4: 5' TCC TCC GCT TAT TGA TAT GC 3' 'is appropriate. The rate of fungus samples detected by PCR in Quang Hue, Te Le, and Tho Van was 19,2%, 26,3%
and 31,8% respectively.
Dimensions of the product having DNA cut by restriction enzymes produced the following resuhs: Candida gabrata (C. Glabrata) 557,314 bp; Candida tropical (C, Tropical) 340 bp; Candida albicans (C. Albicans) 297,238 bp; Candida krusei (C, Krusei) 261, 249 bp;
Candida parapsilosis (C. Parapsilosis) 176,243 bp.
Rate and composition of fungal species in three communes was C. glabrata accoimting for 43.5%, C. tropical occupying 34.8%i, C. albicans holding 14.6%, C. krusei taking 4.3%, and C. parapsilosis possessing 2.8%.
Can bo phan bien:
PGS. TS Le Xuan Hiing
Ngaynhgn bai: 15/03/2013 Ngay giri phan bien: 22/03/2013 Ngay dang bai: 30/03/2013
