Quan tif ica tion of Polym era se Cha in Reaction Products Using Solid Pha se Hybr id iza tion-enzym e Color im etr ic D etection

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1 14 2 V o l114,n o Ch inese Jou rnal of B iochem istry and M o lecu lar B io logy A p ṙ (, ),,,, 125 g, :,,, Quan tif ica tion of Polym era se Cha in Reaction Products Using Solid Pha se Hybr id iza tion-enzym e Color im etr ic D etection W ang Feng2Shu i W ang Yu Chen Hong2Song Cong Xu (Institu te of H ep atology, P eop le s H osp ital, B eij ing M ed ical U niversity, B eij ing ) Abstract A sim p le, rap id po lym erase chain reaction (PCR ) based on DNA quan tification techn ique w as estab lished Fo r detecting specific viral nucleic acids, the p rim er labeled w ith b io tin w as u sed to am p lify viral gene fragm en ṫ T hen the 1st PCR p roduct w as com p lem en tly hyb ridized w ith the specific p robe covalen tly coup led on to m icrop late w ellṡ F inally, Strep ta2 vidin2pod w as u sed in co lo rim etric detection W ith th is m ethod, the sen sitivity of the detec2 tion system cou ld be 1 5 cop ies of hepatitis B genom e and hepatitis C genom e respectively Sim p le, rap id w ith h igh specificity, sen sitivity and sem iquan tification, the m ethod described can be of general app lication fo r detection of any fo reign pathogen in b lood, o ther body flu ids and tissue sam p les, u sed bo th in clin ical diagno sis and estim ate of the efficacy of an tiviral therap y Key words: N ucleic acid quan tification, Po lym erase chain reaction, So lid phase hyb ridization, Enzym e linked co lo rim etry,,, 180 (H IV ) 10, H IV CD ( ) : , :

2 [ 1, ],,,,,,,,,,,,, [ 2 3,, ], , N uck (Covalink N uck) 30, 5 2GA T GA GGCA TA GCA GCA GGA T GAA GA G GAA 23, 401n t 430n t 5 2CT CA TA CTAA CGCCA T GGCTA GA CGCT T TC23, 5 71 n t 100 n t, 5 (EDC) [ 4, 2N H ] : 10 mm o lgl 12, ph 710, m in, 5 m in,, 75 Λ1, 25 Λ1 25 mm o lgl EDC,, 50 5 h 50 1 (2 SSC2011% SD S) 50 5, 1 m in, 112 CH , 10 8 g m l [ 5 ] ( ) phbv 29 CH , PCR 10 6 gm l [ 6 ] Λ1 310 Λ1 KgSD S (50 mm o lgl T ris ghc I ph 810, 200 mm o lgl N ac I, 10 mm o lgl ED TA, 2% SD S, 1 m ggm l K) 60 1 h, g 2,, (D EPC) 2 : 10 Λ1, 100 (Superscrip tg IBCO 2BRL ), 5 2TA TCA GGCA GTA CCA CA A GG23 ( HCV 279 n t 298 n t ) m in, cdna 5 2CT GT GA GGAA CTA CT GTCT T 23 ( HCV 45 n t263 n t), 254 bp s; s; s, Λ1 2% PCR, 5 Λ1 PCR 193

3 , 145 bp : 50 Λ1 10 Λ1dH 2O, 95 7 m in 5 2CAAA CGGGCAA CA TA CCT T G23, 455 n t 474 n t, 5 5 2CA GA GTCTA GA CTCGT GGT G23, 242 n t 261 n ṫ s, s, ṡ 35, 233 bp Λ1, m in 2 m in, ( 50%, 5 SSC, 1 FPG, 25 mm o lgl KH 2PO 4, 012% SD S, 5%, 200 Λggm l DNA ) 100 Λ1, m in , 1 m in 115, 3% m in,, 100 Λ1 2 Λggm l (V ecto r, SA 25004, ) m in, 2 (100 mm o lgl T ris2hc I, 200 mm o lgl N ac I, 013% Tw een220) 4, 1 m in 15 m in, 100 Λ1 1 m o lgl, A 492nm T E, ngg100 Λ1g, 10 Λ1,, A 492nm A 492nm F ig ng, A 492nm,, 200 ngg, 2 Λ1 PCR, Λggm l (F ig 2) A 492nm, 2 Λggm l F ig 1 Capability of the various o ligonucleo tide p robe used fo r binding amount hybrided w ith the PCR p roducts 194

4 F ig 2 T he co lo rim etric results are affected by the various concentrations of enzym e2avidin conjugate 212 -, DNA 2 pg T E 10 3 (F ig 3) A B C, PCR , T ab le 1 F ig 4a 27 HCV 01034, 01015, Cu t off 3, F ig 3 A garo se gel electropho resis pho tograph of the HCV RNA RT 2PCR p roducts A, B, and C are the 1st PCR p roducts of th ree tim es perfo rm ance using the sam e serum 102fo ld dilution a, b, and c are N ested2pcr p roducts of the above2m entioned samp les 1-7: the serum 102fo ld dilution from 10 0 to 10-6 M :M o lecular w eigh t m arker, 100 bp laddeṙ N : negative contro l 195

5 Table 1 Sensitivity of the nucleic acid so lid phase hybridization enzym e co lo rim etric detection (A 492nm ) Samp les N C A B C n g102fo ld dilution of serum, 10-4, 5 F ig 3 a b c PCR, 10-5, 1, 2 PCR 2, , (F ig 5) , 01483, F ig 4b F ig 4 (a ) The DNA quantification by so lid phase hybridiza2 tionenzym e co lo rim etric assay A, B, and C: the detected samp les of the m icrow elles co r2 responding to the A, B, and C samp les of F ig 3 the co rre2 sponding op tical densities are show ed in T able 1 (b) The DNA quantificative analysis pho tograph of the standardize HBV serum samp le by so lid phase hybridiza2 tion2enzym e co lo rim etric assay 1-8: co rresponding to the 1-8 samp les of F ig 5 F ig 5 A garo se gel electropho resis pho tograph of the standardize HBV serum samp le 1st PCR p roduct M : M o lecular w eigh t m arker, 100 bp laddeṙ 1-8: the standardize serum 102fo ld dilution from 10 0 to ,, DNA PCR, PCR, PCR, 196

6 ,,, PCR 100 2,, PCR,,,, (1),, 35,, (> 10 5 ), , (2), 0-2, ,,,,,, 2,,, References 1 M ello rs J W, R inaldo C R J r, Gup ta P,W h ite RM, Todd J A, Kingsley L A P rogno sis in H IV 21 infection p redicted by the quantity of virus in p lasm a S cience, 1996, 272g M antero G, Zonaro A, A lbertini A, Berto lo P, P rim i D DNA enzym e imm unoassay: general m ethod fo r detecting p rod2 ucts of po lym erase chain reaction C lin Chem, 1991, 37g Keller G H, H uang D P,M anak M M A sensitive noniso top ic hybridization assay fo r H IV 21 DNA A nal B iochem, 1989, 177g Rasm ussen S R, L arsen M R, Rasm ussen S E Covalent immobilization of DNA onto po lystyrene m icrow ell: the mo lecules are only bound at the 5 end A nal B iochem, 1991, 198g O kamo to H, T suda F, A kahane Y, Sugai Y, Yo sh iba M,M o riyam a K, T anaka T,M iyakaw a Y,M ayum im H epatitis B virus w ith m utations in the co re p romo ter fo r an e antigen2negative pheno type in carriers w ith antibody to e antigen J V irol, 1994, 68 (12) g O kamo to H, O kada S, Sugiyam a Y, T anaka T, Sugai Y, A kahane Y,M ach ida A,M ish iro S, Yo sh izaw a H,M iyakaw a Y D etection of hepatitis C virus RNA by a two2stage po lym erase chain reaction w ith two paris of p rim ers deduced from the 5 2noncoding region J p n J E xp M ed, 1990, 60 (4) g