Tp ch Vi sinh vt hc lm sng, thng Hai 2006, p. 302-306 0095-1137/06 / $ 08,000 doi: 10.1128/JCM.44.2.302-306.2006 Copyright 2006, American Hi Vi sinh vt hc.

. Tt c cc quyn. Nhanh chng pht hin Mycobacterium tuberculosis kiu gen Bc Kinh Chng Real-Time PCR

Vol. 44, s 2

Doris Hillemann, 1 Rob Warren, 2 Tanja Kubica, 1 Sabine Ru sch-Gerdes, 1 v Stefan Niemann1 * Forschungszentrum Borstel, Trung tm Quc gia tham kho cho Mycobacteria, D 23.845 Borstel, c, 1 v Trung tm MRC a Sinh hc phn t v t bo, S Y Sin , i hc Stellenbosch,Tygerberg 7505, Nam Phi Nhn c 10 thng 8 2005/Returned sa i ngy 16 thng 9 2005/Accepted 07 Thng 11 2005 Chng Mycobacterium tuberculosis kiu gen Bc Kinh ln u tin c xc nh Trung Quc v cc nc lng ging v t u t s c { c bit do xut hin ton cu ca h v kt hp vi khng thuc. tip tc phn tch s ly lan v cc c im c bit ca cc chng kiu gen Bc Kin , p ng php chnh xc, nhanh chng v nhy cm khc phc n c im ca cc p ng p p c in n IS6110 fingerprinting DNA hoc spoligotyping cho vic xc nh cc chng kiu gen ny l cn thit. Da trn cc trnh t nucleotide ca M. tuberculosis SAWC0780 v H37Rv, mi v fluorogenic 5 nuclease ( aqMan) t m d real-time PCR xt nghim c th i vi Bc Kinh v khng-Bc Kinh ging, tng ng, c thit k. Cc gii hn pht hin cho cc xt nghim PCR thi gian thc khong 5 v 10 bn sao ca DNA nhim sc th, tng ng. Trong hn hp ca Bc Kinh v Bc Kinh khng c DNA, mt th nghim multiplex c t pht hin (i) mt bn sao ca DNA Bc Kinh trong khong 1.000 bn sao khng Bc Kinh DNA v (ii) mt bn khng Bc Kinh DNA trong khong 2.000 bn Bc Kinh DNA. Trong mt phn tch m ca mt b su tp ca 103 chng a ng c phn lp c vo nm 2001, tt c Bc Kinh 62 v tt c 41 chng khng Bc Kin c xc nh chnh xc. Trong kt lun, xt nghim thi gian thc cho php pht hin nhanh chng v c th ca cc chng Bc Kinh v khng c Bc Kinh. Nhng li th chnh ca th nghim ny so vi cc p ng p p c c s dng cho vic xc nh cc chng Bc Kin l n gin v nhy cm ca n v thc t l khuc i v pht hin xy ra trong vng mt ng phn ng.

Trong nhng nm gn y, iu gen Bc Kinh Mycobacterium tu-berculosis t u t s c { c bit v xut hin ton cu ca mnh (2). Cc chng c m t ln u tin Trung Quc v cc nc lng ging trong nm 1995 (12), v sau oc currence ging kiu gen Bc Kin c ghi nhn trong mt s b phn ca th gii, c bit l cc nc chu m cn Lin X c, C u u, C u P i, v Hoa Kz (1, 2, 12). Kiu gen Bc Kin gy ra s bng pht ca bn lao a ng t uc (MDR) (8, 9), v mt s ngi trong vestigations c ra mt hip hi vi khng thuc (2). Ngoi ra, s khc bit trong hnh

nh lm sng ca nhim trng kiu gen Bc Kin v p ng min dch n gi ln mt kch thch tho lun v cc c im c bit ca cc chng kiu gen Bc Kinh (6, 7, 10). Cc p ng p p c n xc c o vic xc nh cc chng kiu gen Bc Kinh l mt iu kin tin quyt cho cc nghin cu phn tch s ly lan v cc c im ca gia n iu gen Bc Kin . Cc p ng p p c n c s dng c o n nay l IS6110 DNA ngn tay in (13) v spoligotyping (3), v gn y n ng a mt s ng thun da trn nhng p ng p p ny c xut bn (4). C hai k thut, tuy n in, lin quan n cc bc cng vic trong phng th nghim, v c bit l p ng p p du vn tay IS6110 i i mt mc cao ca cc th tc tiu chun a v bn t ng o m bo kt qu tng ng. Hn na, nhy ca c ai p ng p p xc nh s hin din ca tiu qun th ca Bc Kinh hay khng Bc Kinh chng, v d n , trong trng hp nhim phi hp, hoc phn bit Bc Kinh v Bc Kinh chng khng trc tip trong ti liu lm sng l thp. Warren v ng nghip (14) gn y m t mt sensi tnh rt PCR da trn p ng p p d dng n thc hin. H p dng thnh cng hai PCR nhm mc tiu xa somal nhim sc th l mt mn qu c th trong chng Bc Kin xc nh mt s lng ln cc bnh nhim trng hn hp vi cc chng Bc Kinh v khng-Bc Kinh trong cc mu m, mt pht hin vi implica chc quan trng cho s hiu bit v dch t hc bnh lao. S kt hp ca mt cng ngh PCR rt nhy cm vi subse-quent phn tch cc sn phm PCR ca agarose gel phoresis-electro, tuy nhin, vn b cn tr bi tim n vn lem nhim c th lm phc tp vic gii thch kt qu. Do , c ng ti p t trin mt multiplex real-time PCR kho nghim c o p p xc nh c th ca Bc Kinh v khng-Bc Kinh DNA trong mt phn ng mt ng. Xt nghim real-time PCR sau c n gi bng cch phn tch mt b su tp cc c trng ca cc dng MDR t c, m phn loi n Bc Kinh v khng-Bc Kinh chng c thc hin vi "tiu chun vng" p ng p p IS6110 DNA fingerprinting v SPO-ligotyping (5). LI PH PH P

Chng phn tch. Mt tp hp ca 103 chng MDR t u c t cc bnh nhn sinh sng c trong nm 2001 (5) c s dng n gi nng lc ca Bc Kinh xt nghim PCR thi gian thc c thnh lp trong iu tra ny ca s phn bit i x ca cc chng kiu gen ca Bc Kinh v khng c Bc Kinh. Kiu gen c tn . cho php mt phn loi chnh xc vo hai loi Bc Kinh v khng-Bc Kinh, tt c cc dng c phn tch bng IS6110 AD v spoligotyping n m t trc y (3, 11). DNA nhim sc th c c lp bng cch s dng mt th tc tiu chun n m t ni c (11). Kt qu xc nh kiu gien c m t chi tit trong iu tra trc y ca chng ti (5) BNG 1. Trnh t ca cc u d mi v aqMan c s dng pht hin ca Bc Kinh v khng-B eijing chng loi

Mi v t m d Tn trnh t mc tiu chng Non-Bc Kinh chng Forward mi Fluorogenic t m d o ngc mi Bc Kinh chng a. Mi v t m d c ng khng-Bc Kin c da trn khng gia nhp GenBank. BX842581, nucleotide 7896 n 7990. Cc on c to ra bi nhng on mi l 95 bp di. b. Mi v t m d c ng Bc Kin c da trn GenBank nhp khng. AF390039, nucleotide 820 n 948. Cc on c to ra bi nhng on mi 129 bp di. c. MGB-NFQ, rng buc rnh nh, nonfluorescent kh. d. YAK-DB, Yakima vng DABCYL (nonfluorescent kh ti).

- Thit k mi PCR thi gian thc v b t m d. Mi v t m d c tng hp bi ABI (Applied Biosystems, Weiterstadt, c) v TibMol-Biol (Berlin, c), tng ng Xt nghim PCR thi gian thc. Th nghim PCR thi gian thc c chy vi cc Rotor-Gene 2000 (Corbett nghin cu, Mortlake, Australia) v ABI TaqMan Mix Universal PCR Thc s di iu kin phn ng ca 95 trong 10 p t v 40 n 50 2 bc chu kz bao gm ca 92 C 15 giy v 60 C trong 1 pht. Nng mi trong cc hn hp phn ng ca 15 l l 500 nM cho mi mi xui v ngc v 200 nm cho mi u d. Tng cng c 1,5 l pha long 1:100 DNA nhim sc th c s dng n l mt mu PCR. KT QU V THO LU N Da trn cc trnh t nucleotide ca s nhp enBan BX842581 v AF390039, sn l t v nuclease fluorogenic 5 '( aqMan) t m d real-time PCR xt nghim c th i vi Bc Kinh v khng-Bc Kinh ging, tng ng, c thit k (Bng 1). (Mi v t m d c tng hp ca ABI v TibMolBiol, tng ng.) Phn tch vi BLASTN qu-strated gi thuyt rng cc on D A c to ra vi prim-ers nBjF / nBjR (non-Bc Kinh) v BjF / BjR (Bc Kinh) iDEN-tical vi mt mnh ca M. tuberculosis H37Rv hoc M. tuberculosis SAWC0780 (Bc Kin ), tng ng. Pht hin cc mnh v khc bit PCR da trn t m d aqMan c th c nhn vi cc thuc nhum huznh quang khc nhau. Probe BjTM lin kt vi on 129-bp c to ra bi cc nBjF mi / nBjR (Bng 1 v Hnh 1) c dn nhn vi mt FAM 5

(6-carboxyfluorescein) nhn v mt Tamra 3 (6-carboxytet-ramethylrhodamine) kh. gc li, nBjTM lin kt vi on 95-bp to ra bi mi nBjF / nBjR vi mt nhn 5 vng Yakima v mt 3 DABCYL [4 - acid (4-dimethyl-aminophenylazo) benzoic] kh (Bng 1 v Hnh 1.). Hiu sut ca PCR thi gian thc v c hiu ca b primer-t m d ban u c th nghim bng cc p n tc 10 c chn ngu nhin Bc Kinh v 10 chng khng Bc Kinh, phn loi theo IS6110 ADN v spoligotyping. Vi nhit mt nealing 60 C v mt s lng ti a 50 c u z PCR, c hai xt nghim thi gian thc mang li ch pht huzn quang i c phn tch ADN t cc chng kiu gen Bc Kinh hay khng Bc Kinh chng, tng ng (Hnh 1). Khng c phn ng cho ca Bc Kinh xt nghim PCR thi gian thc cho khng Bc Kinh DNA hoc, ngc li, xt nghim PCR khng Bc Kinh thi gian thc cho Bc Kin D A c quan st. Danh tnh ca cc sn phm PCR vi cc on gen tng ng c xc nhn DNA sequencing cng (d liu khng hin th). c tnh gii hn pht hin ca c hai xt nghim PCR, pha long lot cc nhim sc th D A ( n by bc ln) ca cc chng Bc Kinh v khng-Bc Kinh PCR khuc i. S bn sao gen tuyt i c c tnh da trn spec-trop otometric o nng DNA (UV ab-sorption 260 nm) v gi nh rng mt gen ca M. tuberculosis c trng lng phn t ca 4 fg. ng cong tiu chun cho mi chui mc tiu vi s lng quy nh ca D A t u c cho mi th nghim (bng cch s dng mt lot dung dch 1:10, bt u vi 107 bn), lun cung cp cc tng quan ng (gi tr r2 lun lun l 0,99, d liu ng c hin th). nhy ca c hai PCR l rt cao, k t khi cc gii hn pht hin ca PCR c th c tnh 5 v 10 bn sao ca DNA nhim sc th cho PCR khng-Bc Kinh v Bc Kinh PCR, tng ng. V my PCR thi gian thc th cng mt lc o lng thuc nhum vi nhiu bc sng pht x ring bit, mt multiplex PCR p dng c PCR trong mt phn ng c thnh lp. kim tra xem multiplex PCR lm vic prop-sn l t ng cc , v t m d c th nghim bng cch phn tch 10 mu DNA cha hoc Bc Kinh hay khng Bc Kinh DNA v 10 mu vi cc hn hp khng Bc Kinh v Bc Kinh DNA (t l 1 1 n 1: 100). Trong tt c cc trng hp, cc tn hiu r rng trong cc n tng ng huznh quang pht hin t u c khi Bc Kinh hay khng Bc Kin D A c mt, trong khi khi hn hp ca c hai loi AD c phn tch, tn hiu c ai n t u c (d liu khng hin th). V vy, khng c phn ng cho ca h thng Bc Kin PCR t m d ng Bc Kinh DNA hoc h thng PCR khng Bc Kin t m d Bc Kin D A c quan st. Gii hn pht hin ca PCR multiplex cho Bc Kinh v Bc Kin ng c D A sau c xc nh bng cch phn tch cc mu DNA c cha mt s tin c nh ca Bc Kinh DNA (106 cnh st cu) trn vi DNA froma lot dung dch 1:10 DNA khng Bc Kinh (bt u vi 106 bn) v ngc li. eo , gii hn pht hin ca kho nghim multiplex real-time PCR c tnh ton mt bn (i) ca Bc Kinh DNA trong khong 1.000 bn sao khng Bc Kinh DNA v (ii) mt bn sao ca khng-Bc Kinh DNA pht hin trong khong 2.000 bn Bc Kinh DNA.

Hnh. 1. Schematic ni a ha ca mi v t m d v t qu PCR 10 c chn ngu nhin Bc Kinh v Bc Kinh khng c chng thnh lp Bc Kinh v khng-Bc Kinh xt nghim PCR thi gian thc. Kh nng ca kho nghim multiplex PCR xc nh nhanh chng lm sng Bc Kinh v Bc Kinh chng ng c kim tra bng phn tch m ca mt b su tp ca 103 chng MDR phn lp ti c nm 2001 m phn tng chng vo Bei-jing v khng c Bc Kin t c hin da trn spoligotyping v DNA fingerprinting IS6110 d liu (5). C cu dn s da trn IS6110 D A fingerprinting c hin th trong hnh. 2. Da trn ging ht nhau IS6110 mu du vn tay DNA, 47 trong s 103 mu phn lp c nhm li trong 10 cm khc nhau bao gm t 2 14 phn lp. Kho nghim multiplex thi gian thc xc nh chnh xc 62 phn lp khi Bc Kinh Geno-type v 41 phn lp l khng-Bc Kinh kiu gen. Khng c tn hiu dng tn gi c quan st i vi Bc Kinh v Bc Kinh khng c ging, al-mc d PCR c chng min l c nhy cao. Nhn chung, mt mc lc 100% gia PCR thi gian thc v Geno-g d liu da trn spoligotyping v D A IS6110 ng n tay in t u c. vy, c hiu v nhy ca xt nghim PCR multiplex pht hin cc chng Bc Kinh v Bc Kin ng c c tnh ton c 100% trong cuc iu tra ny. Cc xt nghim PCR thi gian thc c thit lp cho php pht hin nhanh chng v nhy cm ca cc chng Bc Kinh v khng-Bc Kinh trong mt phn ng mt ng m khng c bt kz cc bc tip theo. Plicity sim ca th nghim ny cho php phn tch cc mu trn cc my PCR thi gian thc. Nhng li th chnh ca th nghim ny so vi cc p ng p p c c s dng xc nh cc chng Bc Kin l n gin v nhy cm ca n v thc t am-plification v pht hin xy ra trong vng mt ng phn ng. gc li, "tiu chun vng" IS6110 p ng p p D A du vn tay i i phi c mt vi bc lm vic, mt nn vn a cng n p t trin ca vi khun v phn tch 19 M. tuberculosis Bc Kinh Cc chng khi phn loi ti u l cn thit (4). Ngoi ra, IS6110 fingerprinting DNA phi c thc hin bng cch p dng mt p ng p p tiu c un a v cng lin quan n phn tch vi mt c ng trn phn mm phc tp c o p p bn t ng ca cc mu a n mnh hn ch chiu di (11). Cc k thut PCR da trn m t c o n nay, spoligotyping (3) v p ng p p m t bi Warren et al. (14), cho php pht hin d dng n n iu ca Bc Kinh chng, tuy nhin, trong c ai trng hp lng ther phn tch sn phm PCR l cn thit. S dng p ng p p t ai, cc bc cng vic b sung c th gy ra nguy c cao v nhim, c bit l khi pht hin mt lng nh cc mu lm sng DNAin l mong mun. guy c tim nng ny on ton c t trn c bng xt nghim thi gian thc c thnh lp, trong c mt li th n na n c o p p n lng s tin tng i ca Bc Kinh v khngBc Kinh DNA trong mt mu nht n . nhy v c hiu cao kt hp vi s n gin ca p ng p p lm c o Bc Kinh v Bc Kinh khng c xt nghim PCR thi gian thc mt cng c rt hu ch m s to iu kin thun li cho cc nghin cu c ng minh im-cc kha cnh khc nhau ca cc kiu gen M. Bei-jing bn lao trong trong tng lai. Hnh. 2. IS6110 DNA mu du vn tay cc chng MDR 103 phn tch. Cc di mu IS6110 c sp xp tng t trong mt dendrogram. V tr ca mi bng tn IS6110 c chun a m hnh di ca tt c cc chng ln nhau so snh. Quy m m t tng t IS6110 m hnh tnh ton vi h s Dice v p ng p p n m unweig ted cp s dng p ng p p lin t trung bnh.

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