1 VriUI^O £.\i lO
U'NG DyNG KY THUAT REALTIME-PCR D E PHAT HIEN VA PHAN BI^T CAC LOAI Ky SINH T R O N G S O T RET PLASMODIUM SPP. GAY BENH O NGLfOI TAI
M Q T S O T I N H K H U v y c M I ^ N T R U N G - T A Y NGUYENNgo Thj Huvng, Tri#u Nguydn Trung, Tran Thj Dung. Trin Binh Trong, Nguyen Thf Minh Trinh, Le Al Quoc, Le I " I H^nh Di$u
Vi§n Sot ret-Ky sinh tnJng-Con tnJng Quy Nhan
T 6 M TAT
Ky thu^ Realtime-PCR, sir dung mau chte EVAGreen duoc su dung dl phat hien va phan biet mpt so loai KSTSR thupc gi6ng Hfljmorfium gay b?nh d ngircti. Ket qua phan tich cho thiy RealUme-PCR 14 mot kj fliu|t dang tin cay trong phat h i ^ KSTSR va co ttie phan biet din?c cac lo4i KSTSR dya vao nhiSt d$ c h ^ (Tm). Trong nghien ciiu nay 74 mfiu P. falciparum, 39 mlu P.vivax va 5 mau P. malariae dugc phan tich , chiing toi da xac djnh duoc Tm tmng binh cua cic lo^ KSTSR k6 tren lan lirgt IS 75,24 ± 0,46;
78,55 ± 0,51 vi 74,16 ± 0,08. Ket qua nghiSn cmi cung cho thSy dp nh^y, do dac h i ^ v5 dp chinh xac ciia Realtime-PCR so voi Nwled PCR ciia lung loii KSTSR la r§t cao, d3y la 2 ky tirnat c6 the chpn lua dong chin doiin lam sAng b ^ i sk ret Realtime-PCR nhiai mi diem nhu: c6 the quan sit ket qua trvc t i ^ tren may khi dang hoat dong, tti&i gian lien hanh nhanh (tu luc bit dSu den khi ra let qi» cuoi cung chi mat khoang 3 gi&), khong phai dien di sau PCR n&i tranh dupe cac y6u to ngo?i nhiem anh hudng tm k6t qua.
C6 ttie dinh lupng dupe nong dp DNA nen c6 the dixpc sii dung di tiieo d6i hi^u qui va dq) ling ciia KST doi vdi thu6c sot rft trong qui trinh dieu tij de cd the dua ra phic dd ttiich hop.
Tir khoa: Realtime-PCR, Nested PCR, Plasmodium falciparum. Plasmodmm vivax. Plasmodium malanae, nhiet dp chay.
M&eAu
B§nh sot ret vIn la mgt v^n d^ si>c kh6e du-g-c quan tam cua loan cku. Theo i/6c tfnh md-i dSy nhat cda T6 chire y t l the gid-i, nam 2010 co khoSng 219 tri#u bgnh nhan mic sot r6t. trong d6 co khoang 660.000 ngiroi ti> vong.
Vige vAc djnh chinh xdc cac loai ky smh trOng s6t ret (KSTSR) c6 vai tro rit quan trpng trong vi?c chpn li/a bien phap dieu trj thich hg'p; dgc bi^t quan trgng \i d i l n dodn phan bi$t P. falciparum vdi cic loii ky sinh trCing s6t r6t khac, do loai ky sinh trting nSy co kha nang gSy ti> vong khoang 95%. Hien nay, co nhieu phuwng phSp chan dodn KSTSR nhu- phirong phap soi kinh hiin vi (Hanscheid, 2003). phirong phap miln djch huynh quang (Zhou e( a/.. 1998), test chan doin nhanh (Muray e( al., 2003)... Tuy nhifin cac phifcmg phip tr§n fliu c6 m$t s6 hgn che nhlt djnh. nhlt la khi m§t dp KSTSR thap du'a! ngu^ng phat hign (Payne, 1988; Tham e( a/., 1999).
Trong nh&ng nam gan day, ky thuSt PCR da du^c i>ng dung rong rai trong chin dodn KSTSR, trong dd Nested PCR Id mOt ky thudt aS dd nhgy cao vi phan bi$l duvc chinh xdc tCrng loai KSTSR (SntMJnou e( a/., 1993). Tuy nhien da s6 cac phirong phdp PCR da du'g-c cfing bo deu di,fa vao di?n di tr6n gel ho§c dira vdo ky thudt lai vdi cdc mau do. din din kit qud kto dai thfiri gian, quy trinh khong phii hgrp cho viec si> dgng Irong nghien ctfu idm sdng. Ky thuat Realtime-PCR ra ddi da Wiac phgc dirt^c nhifng nhm?c dilm cOa Nested PCR. Real Time PCR Id ky thugt PCR ma kit qua khuich dai DNA dfch du-grc hiin thi dong thdi vd'i phdn Cpng khuich dgi vd Realtime PCR cho phdp phan bigt chlnh xdc lodi KST thfoig qua phdn tich dirfmg cong chay (Meltng curve analysis), then gian tiln hdnh thi nghigm nhanh, cd t h i djnh lirgng dirge KST trong mau b$nh nhdn sau dieu tn. phi/cmg phdp nay khdng phai dign di san pham PCR nen logi tn> dif^c yeu
^ ngogi nhiem sdn phim khuech dgi.
Tr@n the gidri. Realtime-PCR dirge ung dgng rong rai d l phdt hien tdc nhan g3y bgnh nhu- vi khuin, virus, nam; trong do cd nhilu nghidn ciju i>ng dgng realtime-PCR d l chin dodn k^ sinh trung sot r^t (Mangold ef af., 2005; Shokoples ef aL.
2009). dVigtNam, vi§c Ong dgng Realtime-PCR trong chan doan KSTSR vln cdn hgn che (L§ Du-c Ddo etal. 2001).
Do dd, trong nghi&n CLPU nay chiing tdi li-ng dung k^ thugt Realtime-PCR de phdt hien vd phdn bi&t cac loai Plasmodium spp. gdy bdnh s i t ret d ngiroi tgi mgt so tinh thudc khu vufc mien Trung - Tay nguyen.
N G U Y £ N L I ^ U V A PHlfaNG P H A P
D i i tvQ'ng nghien ciiu Id ky sinh tmng s6t ret giing Plasmodium spp. bao gim P. falciparum {P.f), P. vivax (P.v), P.
malariae (P.m) d\rac thu thdp tai thi/c dia thudc vCing luu hdnh sot ret cCia cac tinh Khdnh H6a, Ninh Thu|in va Gia Lai tir nam 2010-2012.
Phvong phap thu th|ip miu tai thij>c dja: Cac b$nh nhdn sdt ret duuc lly mau trdn ddu ngdn tay. Moi b^nh nhdn dirge lly 1 lam mdu, gigt dac d l phat hi^n KSTSR bdng phu-ong phap soi kinh hiin vi vd mgt gipt l§n giay thdm Whatman (khodng 5-7|il) de phdn tich bang Nested PCR vd Realtime PCR.
Cac ky thuat su> dung trong nghien ciru
Ky thuit nhudm giem sa va soi kinh hien vl d l phdt hien va xac d[nh loai KSTSR; mdt dO KST du-gc dim ti/cmg dii theo quy udc"+","++" vd "£+++".
Ky thuit Nested PCR dui?c ddng de thIm dinh lodi cdc miu KSTSR dd du-gc xac dinh bang phirong phdp soi kinh hiln VI. Cdc mdu mdu cd KSTSR tren giiy thIm duwc tach chiet DNA ting so bdng bd smh phim QlAamp DNA Mini kit (QIAGEN Inc. USA). Si> dgng cdc cgp mil die hi§u. P5-P6; FF-FR, VF-VR; MF-MR; OF-OR (Snounou ef ai.. 1993) de ten hanh phdn i>ng PCR. Cac lodi Plasmodium da d\rac thim djnh bdng Nested PCR se duwc si> dgng lam chuin d l nghifin ciru bing ky thuat Realtime-PCR.
HOI NGHI KHOA HQC CONG NGHE SINH HOC TOAN QL
Kv thuit ReaKime PCR 6^ p h ^ hi?n va phan biet dc loai KSTSR d^ra vio nhi?t dO chdy (Tm). SCr dyng c$p mii (PL18F- 5' -TAA CgA ACg AgA TCT TAA- 3' va PL18R; 5' -gTT CCt CTA AgA AgC TTT- 3') OwfC Ihi^t ki trSn viing gen 18S rlbosome SJc hi^u cho 4 loSi Plasmodium \i P.I. P.v. P.m vi P.ovala (P.o) (Mangold et al.. 200S) d i phat h«n va timg loSi Plasmodmm dinjc phan bl#t dva vao nhiSt dO chay (Tm). ChM mau chan hujnh quang air<rc si> dgng la EvaGreen (hang Bio-Rad). Qua trinh l(huech dai dinjc Ihvc hien Hen may Realtime-PCR {Rotor Gene Q) cua hang QIAGEN theo chu trinh nhiat nhir sau: gs'c 10 phut, ti«p theo la 40 chu ky * 96°C 10 giay. SO'C 5 giay va 72 C 20 giay. Ti^p ngay sau d6 la chircmg trinh phan tich nhiet d« chjy ft 95"C 2 phut. 68 C 2 phut, sau db m6i buOc se tang 0.2'c;giay cho din iihi dst din 90°C.
Xi> li s6 llfir. Vi0c phan tich duftng cong chay d i xac dinh Tm (nhi#t do chay) dec hi#u cho tOng loai KST dut?c thi.ic hiSn bang phin mim Rotor-Gene Q Series Software. Tm trang blnh cila tOng loai Plasmodmm Aioc tinh toan bang phin mim STATA10.
K ^ QUA VA T H A O LUAN
K i t qua tham djnh ky sinh triing sot r6t (KSTSR) bing ky thu|t Nested PCR
Ky thuat Nested PCR difoc sir dung de thim dinh 191 lam mau da diroc xac djnh bang ky thuat nhu§m giSm sa (BSng 1) cho thiy c6 74 miu nhiim doii vdri P.f, 39 m'lu nhiim den vd'i P.v, 5 mau nhiim don vdi P.m: c6 29 miu nhiem phii h(?p 2 loai {P.f + P.v ho§c P.f +P.my, trong s i 45 miu {-) c6 1 miu dif?c xac dinh nhiirri P. vivax. Cac loai Plasmodium da dirvc thim dinh bing Nested PCR nay se diriyc si> dyng lam chuin d i nghl§n cOu bang ky thuat Realtlme-PCR.
B^ng 1. Kit qud thim djnh KSTSR bing ky U,u3t Nested PCR
Lodi KSTSR
P. falcipmrum P vivax P. malariae
(-)
T i n g s^KSt quA SOI giAm sa
90 51 5 46 191
K ^ qu£i Nested PCR P.f
66 6
74 P.v
3 35
1 39
P.m
5
5 Pf*P.v
17 10
27 Pf + Rm
2
2
(-)
44 44
K i t qua phdt hifn va dinh lu>9^g cdc lodi Plasmodium bang ky thuat Realtime-PCR
Trong k j thu§t Realtime PCR, si/ cd m$t cua cdc lodi Plasmodium (P.f, P.v. P.m. P.o) trong m l u vgt du^c xdc djnh bdng 1 cdp mii duy nhlt va 3u<?c quan sdt thong qua bilu d l cflnh li/^ng. Cac mau duancj tinh^(c6 KST) thi du'&ng bilu diin Id nhiing du-firng cong (Bilu d l 1) vd nlu mlu dm tlnh (khdng cd KST) thi du'dng bieu dien se nam ngang dU'd'i diF&ng nln (base line). Trong bilu d l djnh lifp-ng, quan frpng nhlt Id xac djnh du^c chu kjr ngu'dng ciSa tCrng mlu KSR. Chu ky ngu'&ng (Ct) Id no! md tin hi#u hu^nh quang cao han tin hi^u nln mOt cdch thiy rd. Ct phy thuOc vdo s l luyng DNA dich ban diu, s l luvng DNA ban ddu cdng nhilu thi Ct cdng s6m (Phgm Hung Vdn, 2009).
Phdn tich t i t cd 147 mlu KST ducng tlnh da duyc tham djnh bang Nested PCR, kit qud cho thiy cd 146/147 mlu KST iluvc quan sdt thay du'frng cong dinh luQ'ng. Cd nghTa hlu nhu' cdc mlu duong tlnh d i u phii hop vdi ky thudt Nested PCR chi khdc nhau d ddy Id moi mlu KST co 1 Ct khdc nhau tuy thupc vdo lu^ng KST ban diu cd trong mlu vgt Kit qud cung cho thiy Ct cGa cdc mlu KST trong nghidn ci>u ndy dao dgng tnsng khodng ti> chu ky 19 den chu k^ 36.
So sdnh v l si/ lidn quan giOa mdt dO KST duyc xdc djnh tucng doi bdng phuang phdp soi kinh hiln vi vdi chu ngudng Ct cda KST chiing tdi nh$n thiy nong dp KST trong mlu cdng cao thi Ci cdng sdm, nong dp KST cang thap thi Ct cang mu$n (Bdng 2).
Bilu d61.8l4u a6 phit hiin vi djnh lu^ng cic mau KSTSR,
= OC CONG NGHE SINH HOC TOAN QUOC 2013
^ * ^ 2- Bang p h i n tich s v H«n quan gKra m^t d$ KSTSR va chu ky ngirong (Ct)
- ^'""^ftrong 5-50 KSTIji iwau) jgTtaimhfagmBijtioildrtitifowi)
* (liwng ftfong SO^QQ KSTIji m
Km qud nay c6 I h l dwyc i>ng dyng de theo doi KSTSR trmw qud trinh dieu trj, ddc bi#t trong theo doi su- dap dng thulc sot ret trong cdc nghidn cCru ve KSTSR khang thulc.
K^ qud xac d{nh nhi^t d$ chay (Tm) cda P. fa/ciparum, P. wvax va P. malariae
IZ n ^ P. faldpanjm dvac phdn tich nhi^t 6d chay (Tm). kit qud cho Ihly Tm dao d^ng Kr 73,74''C din 75,70°C. Nhidt ao chay trung binh cua P. faldpanjm Id 75,34 ±0,46 (Bilu d l 2A).
Qua phdn tich dinh chdy cua 39 mau P. vivax cho thiy nhidt dd chay cua P. vivax dao ddng ti> 77,54"C den 79.34°C.
Nhi^ dd chay tmng binh cOa P. vivax Id 78,55°C ± 0,51 (Bilu do 2B). So sdnh nhi^t dg chay cua cdc mlu P. falcipanjm vi P. vtvax d cdc tinh khac nhau cho thiy khdng khac bidt nhieu.
5 mau P. malanae dinyc phdn tfch, nhi$t dg chdy ci3a P. malariae ghi nh^n duvc lan lugt Id 74,10; 74,10; 74,26; 74,24;
74,10 (Bi&j dd 3.4). Tm trung binh cda P. malariae Id 74,16 ± 0,08. Nhin vao biiu do 3 ta thiy dinh chay cua P. malariae tdch ra khdi dinh chdy cua P. fyldpamm vd t^p trung lai tgi nhiet dp 74''C, hjy nhidn lug'ng mdu P. malariae qud it ndn can phdn tich thSm.
TrcMig nghien ci>u ndy, Tm cOa P. ovale chira xdc djnh dugrc do chiing toi khdng dieu fra dirge mau ndo bj nhiim P ovale.
Bi4u dd 2. Bieu do dinh chdy cua P. falciparum (A) vd P. vi\
So sanh vdi kit qud nghidn cdu cua Mangold vd ding tdc gid (2005), Tm trung binh cua KSTSR trdn cac mlu mdu bdnh nhan nhir sau: P. falciparum 75,9 ±0,4; cua P. vivax Id 79,6 ±0,5 vd cua P. malariae Id 74,8 ±0,4. Cdc kit qud ndy cung khdng khdc bi^l Idm so vdi kit qud nghidn cdu cua chung tdi. Su* sai khac ndy cd Ihl do thiet bj Realtime-PCR dirgrc sd dung trong 2 ngh'i§n cdu Id khac nhau; Mangold sd dung may LightCycler (Roche Molecular Systems, Indianapolis, Ind.), trong nghidn cdu nay id mdy Rotor Gene Q (QiAGEN-USA); mau chdn cung khdc nhau. Mangold sd dgng SYBR Green, cdn ctiung tdi sd dung EvaGreen ggi la mau HRM (high resolution melting dye) co kha ndng phdt h u ^ h quang rit cao vd hem nda lgi hodn todn khdng dc c h l PCR ndn dd phan gidi cua cdc bilu do dinh chdy rat cao (Phgm Hung Vdn, 2009); thdm vao do cd the do cac miu KSTSR d Vi^t Nam cd mdt vai sai khac nao dd v l nucleotide so vdi mlu KSTSR trong nghidn cdu cua Mangold.
Nhl/ vdy, kit qud nghidn cdu da cho thiy cac lodi Plasmodium cd the du^c phdn biet dya vdo nhidt dd chdy.
HOI NGHI KHOA HOC CONG N G H f SINH H g c TOAN (
Biiu d i 3. Biiu d i dinh chiy cua P. falciparum, P. vivaxva P. Ualarfae
C6 2g mSu nhiem phii h m 2 loai KST (P./ * P.v hojc P.f *P.m) du.(»c phat hi«n bing Nested PCR. Kit qui phan tich bing Realtlme-PCR cho thay co 22/29 miu cho kit qua dilng. t h i hiin c6 2 dinh chay tucmg i>ng v61 Tm cua tCmg loai (biiu d i 4) va 7 miu cin 1^1 chi phat hi«n nhiim 1 ioai KST i>ng v*i loai duyc phat hiSn bang phuong phap sol kinh hiin «i Nested PCR phat hiSn duoc nhiim phii hffp trong cao tnfiing tvp nay do k j thujt nay gim 2 lin PCR, si lu-ffng copy tao ra ti, DNA dich dug'C khuich dai Idn hon rit nhiiu nen phat hi§n du^yc KST cf mi^c dO rat thap. VI vay Nested PCR Have xem la chuin vang trong phat hi#n nhiem phii hq'p (Shokoples el al, 2009). Bay la nhii(ic diiin cOa Realtlme-PCR so vdi Nested PCR.
Kit qua nay cOng tirong ty vfti nghiin cilu cua Shokoples va ding tac gia (2009). chl phat hien di«?c 13/16 miu nhlin phii hyp bing Realtime-PCR, c6 3 tnremg hijp khong phat hlSn duyc nhiem phii hgrp trong dd cd 1 tiuimg hyp chl phat hian nhiim 1 loai KST, 2 trvyng hyp con lai khSng quan sat thiy tren biiu d i khuech dai.
"A
M
&D3
01
" "^^ \
70 75
•c 35 90
Bidu dd 4. Bfiu 6h dinh chiy cua mau nhlftm phoi hgp P.bclparvm va P.vivax
Ket qud xdc d|nh dp nhay, dd d|c hi$u, dd chinh xdc gid tii d v dodn du'omg tinh va gia tri di^ dodn dm tinh cua Realtime-PCR
BO nhgy, dO dgc hi§u, dd chinh xac, gid fri d^r dodn duwng tinh vd gid tri dy dodn dm tinh cua Realtime-PCR dug'C tlnh todn dya vdo s i li$u theo bdng 3. Kit qud cho thiy dd nhgy, d$ ddc hieu, dd chinh xdc, gid trj dy dodn duang tlnh vS gid tri dy dodn dm tlnh cda Realtime-PCR so vdi Nested PCR rat cao. Vdi P. falciparum cac gid trj lln luvt Id 98,65%;
100%; 99.15%. 100% vd 97,78% lu'ang ty vdi ket qua nghidn cdu cda Mangold (94,1%; 100%; 98,7%, 100% vd 98,4%).
Dll vdi P. vivax cdc gid trj lln l u ^ Id 100%; 100%; 100%, 100% vd 100% cao han so vdi kit qud nghidn cdu cda Mangold (100%; 99,1%; 98,1%, 95,5% vd 98,3%). Doi vdi P. malariaeM cdc gid trj deu la 100%.
Bang 3. Bang so sdnh kit qud xdc djnh KSTSR bang k^ thu^ Realtime-PCR vd Nested PCR Roallime-PCR
P falcipanim P malano P irtvax T6ng
Chin aoAn b3ng k9 thu^l Nested PCR (sA m i u mdu) P fatciperum
73 0 0 1 74
P. malana 0 5 0 0 5
P vivax 0 0 39 0 39
Am tfnh 0 0 0 44 44
Cdc ket qud nghien cdu cho thiy Realtime-PCR la mdt ky thudt ddng tin c$y trong phdt hidn vd djnh lifyng cdc lodi KSTSR. C6 thi phdn bi§t cdc loai KSTSR dya vao nhi?t dp chdy.
Dd xac djnh di/grc nhi§t dO chdy trung binh ciJa lung loai KSTSR, cu the nhu' sau: P. falciparum Id 75,24 ± 0,46; CUE
HOC C O N G NGHE SINH HQC TOAN QUOC 2013
P- Wvax Id 78,55 ± 0.51 v d a i a P. m a t e n a e Id 7 4 . 1 6 + 0.08
^ p h d n Uch, s o s a n h d d n h ^ y , d d d $ c 1B$U v d d d c h i n h x d c . gid trj d y d o d n d u c m g h n h v a g i d tri d t f A ^ ^ " ^ " i **rtfi cOa R e a l t i m e 4 > c R s o v d i N e s t e d P C R c h o t h i y day Id 2 k y thuat o 6 t h e c h p n l y a trong c h i n d o d n lam s a n g b e n h sot r ^
^ 2 " f ' ' ^ . R e a W m e - P C R c d n h i l u i n i d i e m h o n n h i r c o t h e q u a n sdt k i t q u d l i v e t i e p tren m d y khi (feng hogt d d n g , P ^ a i a n t i l n h d n h n h a n h ( t d l i i c b a t d a u d i n kW r a k i t q u a c u l i c u n g c h i m a t k h o a n g 3 g i d ) . k h d n g p h a i d i d n d i s a u r ^ " e n t r d n h d u v c c d c y l u t l n g o g i n h i i m d n h h u d n g I d i ket q u d . c 6 t h e dinh lirenng d i r ^ n i n g d d DMA n § n g i i i p i ^ e o doi hidu l y e t h u l c t r o n g q u d t r i n h iTieu trj d l d i r a ra cdc p h d c d o p h u h o p .
T A I U g U T H A M K H A O
Le D d c D ^ , r i ^ i y g n VSn T u l n . BOi Quang Phuc (2001). NghȤn c d u c a cSu KSTSR tsB tw tinh Ldm O i n g . Binh Phirdc, Ddklak b i n g Ky thudt PCR. KJ y l u c6ng trinh nE^itfin c d u khoa hgc 199&-2000. NXB Y hoc 2001. Tr.:195-200.
Pham Hijng V5n (2009). PCR vd Realtifne-PCR c d c v i n d l c a b i i v d c a c d p dung thucmg ggp. NXB Yhoc.
C ^ MH. BL Bredenkamp. CH Wffiams. EJ Rossouw. VJ Kely. 1 Klemschmicfi, A Maitineau. and GF Henry (2002). FieW and Wxxatory comparative evahiatkan irf ten rapid malaria diagnostk; tests. Trms. R Soc Trop. Med. Hyg. 258-265
MangoW KA. Manson RU. Evelyn SC Koay, Ufxteey Stephens. M a y A n n Rebner, Rk^iard B Thomson, Jr. Lance K Peterson, Karen L Kaul (2005). Realtime-PCR for d ^ i c t k m and identiiKatmn o* PtasrixxJium spp. Jouma/ of Clinical Microbialogy: 243^^2440.
Murray CK. D BeB. RA Gasser. and C Wongsrichanalal (2003). R^)kJ diagnostic tesfing for malaria. Trop. Med. Int Health: 876-883.
Payne D (1988). Use and limitatnns of light microscopy (or (Jiagnos(r,g malaria at the primary health care level. Bta. W.H.O. 6 6 : 6 2 1 -
Shokoples SE, Momar ^4dao. Knga Kowalewske-Grochovvska, Stephv«e K Yanow (2009). Muttiplexed Realtime-PCR assay for disaiminatkm of P.asf7KX*iom species with improved sensitivity fe nMxed infectkxre. JcKirna^
Snourxxj G. Vkiyakosol S. Zhu XP (1993). High sensitivity detectkm of human malaria parasites by the use of nested polymerase chain reactkKi. Moi. Biochem. ParesHol. 6 1 . 315-320.
Tham JM. SH Lee. T M Tan, RC Ting, and UA Kara (1999). Detector and species determination of malaria parasites by PCR:
compaiison with microscopy and with ParaSigtti-F and ICT malaria Pf tests m a cUmcal enviionmenL Journal o/ Clinic^ Microbiology.
37:1269-1273.
Zhou M, Q Liu, C Wongsrichanalal, W SuwonkenJ. K Panart, S Prajakwong. A Pensiri, M Kimura, H Uatsuoka. MU Ferreira. S Isomura.
and F Kawamoto (1998). High prevaierK^ of Ptesmodum mabriae and Plasmodftim ovate ki malaria patients along the That-Myanmar txxder, as revealed t>y acrklhie orange staining and PCR-based ciagnoses. Trop. Med Int Health 3.304-312.
USING THE REALTIME-PCR ASSAY FOR DETECTION AND IDENTIFICATION OF HUMAN MALARIA PLASMODIUM SPP. FROM SOME PROVINCES IN THE CENTRAL WEST-HIGHLAND
N g o T h i H u o n g , T r i e u N g u y e n T r u n g , T r a n T h i D u n g , T r a n B i n h T r o n g , N g u y e n T h i M i n h T r i n h , L e A i Q u o c , L e T h i H a n h D i e u
Institute of Maiariology, Parasitology and Entomology Quynhon
S U M M A R Y
The Realtime-PCR using EvaGreen as an intercalator dye was performed to detect and identify human malaria Plasmodium spp. The result showed that Realtime-PCR is a reliable mediod for parasite detection and be able to differenciatc Plasmodium species based on melting temperanire (Tm). In diis study, the specimens inchiding 74 of P.faldpanim, 39 of P.vivax and 5 of P.malariae, »*ich were confirmed by Nested PCR, were analyzed, and ftie average melting tOT^waftires of each species was identified as follow: 75^4 ± 0,46; 78,55 ± 0,51 and 74,16 ± 0,08, respectively. The result also revealed that the sensitivity, specificity and accuracy of realtime PCR are higher compared to Nested PCR, Eiiber Realtime PCR or Nested PCR is alternative method for clinical malaria diagnosis.
However realtime-PCR reveals more advantagous such as: the result can be observed on the machine during running assay, short time requirement (ie.about 3 hours to complete); no need for electn^horesis. Hie parasites can be quantified, which allow to monitor the eflectiveness and the reqxinse of antimalaria d m ^ to parasites during treatment in order to give ^proriate therapies.
Keywords: Realtime-PCR, Nested PCR, Piasmodhmfalciparvm, Plasmodium vivax. Plasmodium malariae, melting ienq)ctanire.
Author for conesspondence: Mobile: 0983440069, Email: ngothihuong@gmail com
