The Korean Journal of Microbiology, Vol. 43, No. 3, September 2007, p. 179-185 Copyright 2007, The Microbiological Society of Korea Nested PCR DNA Enzyme-Linked Immunosorbent Assay w Ralstonia solanacearum š Á y 1 * v w œw polymerase chain reaction (PCR)» DNA enzyme-linked immunosorbent assay (DNA ELISA)» w m ü ³ Ralstonia solanacearum wš w. m l R. solanacearum DNA w» w ƒ sƒw» DNA w Guanidin isothiocyanate Chelex-100 resin w m ü w w w R. solanacearum š w PCR w w z. R. solanacearum p w» w flic p primer w. p ùkü set primer RsolfliC (forward; 5-GAACGCCAACGGTGCGAACT-3 and reverse; 5-GGCGG CCTTCAGGGAGGTC-3, designed by J. Schönfeld et al.) RS_247 (forward; 5-GGCGGTCTGTCGGCRG-3 and reverse; 5-CGGTCGCGTTGGCAAC-3, designed by this study) w nested PCR ww š w. Nested PCR primer biotin t w š nested PCR ü p w probe w PCR DNA-EIA y w w. Primary PCR nested PCR» y w, nested PCR 10 2 ùkü š DNA-EIA 10 2 ~10 3 g y. Key words ý DNA enzyme immunoassay, Ralstonia solanacearum Ralstonia solanacearum»z sww 450 t w ³ (9). p š w y ƒ ƒ j vw öeš (13), w û w š (26). 1998 7 ü t ù û t š. š m m l t, ƒ š (1). R. solanacearum r w d x w z r ù ƒ t w (27, 28). w m wš 5 ƒ w 30 cm dm tmd w w» ƒ w (3). š y w»ù» m l R. solanacearum y w w (21). R. solanacearum w» w PCR» *To whom correspondence should be addressed. Tel: 82-2-940-7187, Fax: 82-2-919-0345 E-mail: hbcho@skuniv.ac.kr rrna sw w (2, 7, 18, 25, 29), PCR-ribotyping RFLP ww R. solanacearum ww» w (17). ù, R. solanacearum 16S rrna ƒ 98%» R. picketti ³ ùkû (23). z 16S rrna wì endoglucanase, hrpb (19)» w x w R. solanacearum strain 1 w» w (IS)ü p sw w š (15). Schfeld (21) flagellin (flic) target w Southern blot hybridization m ü w R. solanacearum w. flic œm š ³ p y ù m DNA z PCR û z š. m l w DNA z z wš, primer š w PCR p w wr DNA enzyme-linked immunosorbent assay (DNA ELISA) w m ü w R. solanacearum z w» wš w. 179
180 Young-Jin Ko and Hong-Bum Cho Kor. J. Microbiol ³ w Ralstonia solanacearum strain Escherichia coli, Pseudomonas aeruginosa, Burkholderia glumae. Xanthomonas xonopodis x w ³ Table 1 w. R. solanacearum ³ CPG (casamino acid 1 g/l, peptone 10 g/l, glucose 5 g/l) 1.8% w, 0.05% 2,3,5- triphenyltetrazolium chloride (TTC)ƒ sw TZC š s q 28 C o š,»k ³ Luria 9 broth -3¾ 34 C o. m 1g 1 10 w R. solanacearum w š ³ Haemacytometer (Marienfeld, Germany) w w. z R. solanacearum m l DNA w x w š ³ ƒ m w. DNA m l R. solanacearum DNA w. 1 ml w cell pellet š phosphate buffer saline (PBS, 0.01 M phosphate beffer, 0.0027 M potassium chloride, 0.137 M sodium chloride; ph 7.4) 3z w 200 µl PBS xk g. m 500 µl v w. DNAzol (MRC, USA) 1 ml ƒw š, z DNAzol w w. DNAzol z DNA pellet 200 µl chelex-100 resin 5% ƒw 10 ò wš 5µl w PCR DNA Table 1. Strains used in this study x w. Primer, probe PCR x primer probe Table 2 ùkü. Primer probe Primer3 v (Whitehead Institude, MT center for Genome research) mw. RsolfliC forward reverse primer flic ü 400 bp PCR š RS_247 forward reverse nested PCR mw 247 bp. Probe nested PCR. PCR AmplitronII mw w. Primary PCR 10 PCR buffer 10 mm datp, dgtp, dctp, dutp 0.5 µl 10 pmol primer set 1 U Taq wz 5µl x DNA w.» 94 o C 5 z, 94 o C 1, 61 o C 1, 72 o C 1 40 cycles z 72 o C 5. Primary PCR nested PCR x DNA nested PCR w, 94 o C 30, 55 o C 40, 72 o C 30 30 cycles. PCR 2.5% agarose» y w. DNA enzyme immunoassay DNA Enzyme-linked immunosorbent assay (DNA ELISA) nested PCR probe w. DEIA ww» w plate w» w, 1- Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC, Sigma E-7750, USA) probe bovine serum albumin (BSA, Sigma, USA) š jš 96-well plate Species Strain Biovar Host plant Geographic origin Source of reference a R. solanacearum DSM9544 1 Tomato United States KCCM R. solanacearum KACC10698 4 Tomato Korea KACC R. solanacearum KACC10699 2 Potato Korea KACC R. solanacearum KACC10702 4 Potato Korea KACC R. solanacearum KACC10706 4 Potato Korea KACC R. solanacearum KACC10708 4 Tomato Korea KACC R. solanacearum KACC10711 3 Pepper Korea KACC R. solanacearum KACC10722 2 Potato Korea KACC R. solanacearum KACC10815 ND b Potato Egypt KACC R. solanacearum KACC10816 3 Potato Fidji KACC E. coli KCCM11234 KCCM P. aeruginosa KCCM11328 KCCM B. glumae Seoul National Univ. X. xonopodis Seoul National Univ. a KCCM, Korean Culture Center of Microorganisms; KACC, Korean Agricultural Culture Collection b ND, not determined
Vol. 43, No. 3 Ralstonia solanacearum DNA enzyme-linked immunosorbent assay 181 Table 2. Characteristics of primers and probe used to detect R. solanacearum Primer or probe a Sequence (5' 3') Length Modification 5' end RsolfliC b -F GAA CGC CAA CGG TGC GAA CT 20 RsolfliC b -R GGC GGC CTT CAG GGA GGT C 19 RS_247-F GGC GGT CTG TCG GCR c G 16 RS_247-R CGG TCG CGT TGG CAA C 16 Biotin RS_200-F CAA CTA CAA CGG CAA CAA GC 20 RS_200-R TCA GGG AGG TCA GAT CGG TA 20 RS_250-R CCG TAC TGG AAG GTC GTC G 19 RS_122-F TTC GAC GAC CTT CCA GTA 18 RS_122-R CGG TCG CGT TGG CAA C 16 RS_fliC-P GTC ACC AAC GTC GAC ATG TC 20 Amine a F, forward; R,Greverse; P,Gprobe b primer RsolfliC from Schönfeld et al. c R, A or G (F96 well plate, Nunc) 0.2 pmol/µl 100 µl w 18 g. z Tween20 s w PBS (PBST, 0.1% Tween in PBS) 3z wš 0.5% BSAƒ sw PBS blocking w ww. Nested PCR 95 C 10 jš o 96-well plate ƒ 60 C w o w. z PBST wš streptavidin-peroxidase (1:2000, Sigma S-5512, USA) 100 µl ƒw 37 C w k z wš o 3,3',5,5'-tetramethyl-benzidine (TMB, Sigma T0440, USA) 100 µl ƒw streptavidin-peroxidase 15 g. 2 M H 2 SO 4 50 µl z jš ELISA plate reader (Molecular Device, USA) 450 nm Ÿ d w. dutp Uracil nucleosidic glycosylase w» w x dutp-uracil-n-glycosidase (UNG) w, PCR w dutpƒ ƒ dntp yw w dutpƒ ƒ w. 10 U UNG ƒwš» 37 C 30 ww o UNG w w. In vivo pot assay š j R. solanacearum y w» w in vivo pot assay ww. 10 cm sp f m(w z ) 180 g š š q w 25~35 C w o w. 4»ƒ üš R. solanacearum xk 30 ml (10 CFU/ml) w 7 w. z y wš l R. solanacearum w, ³ genomic DNA z w PCR DNA EIA x DNA w. DNA m l R. solanacearum genomic DNA w» w SDS w, STET ò, resin ƒ w ò DNAzol w ƒƒ w w x ww. w l ƒ mw genomic DNA wš 16S rdna flic sw» PCR w. DNAzol w DNA w w PCR s z ƒ w, w z w j, d w g e j ƒ» ù š rw. z x w chelex-100 resin š ò m ü w PCR w w z (Fig. 1). PCR mw R. solanacearum DSM9544 KACC 9 R. solanacearum RsolfliC primer w w s 400 bp PCR y w. ³ B. glumae, X. xonopodis, E. coli P. aeruginosa s. z primary PCR x w nested PCR ww 247 bp PCR y w (Fig. 2), x R. solanacearum flic ƒ œm w y w.,
182 Young-Jin Ko and Hong-Bum Cho Kor. J. Microbiol primary PCR ƒ û š, nested PCR primary PCR w 2. Fig. 1. Results of agarose gel electrophoresis of amplified genomic DNA of R. solanacearum extracted by each method. Lane 1, genomic DNA extracted by SDS lysis method; 2, STET method; 3, boiling with chelex X-100 resin method; 4, DNAzol method; 5, DNAzol treatment and boiling with chelex X-100 resin; L, ladder; N, negative control. DNA enzyme immunoassay Nested PCR primer biotin t š, probe amine t. 5' amine t probe EDAC MES mw BSA w z 96-well plate gq. DNA ELISA biotin t primer s nested PCR 96-well plate gq probe w. Probe nested PCR streptavidin-peroxidase w š z streptavidin-peroxidase» TMB ƒw z w d w (Table 3). wš d w ƒ ƒ, w ù 96-well plate gqw probe š wù ww. Probe 5~10 pmol/microwell, 60 C ƒ š o ùkü. Fig. 2. Nested PCR products of R. solanacearum strains studied. Lanes 1 to 10, reference strains DSM9544, KACC 10698, 10699, 10702, 10706, 10708, 10711, 10722, 10815, 10816, respectively. L, ladder; N, negative control. x ww 4 cell/g ¾ 400 bp» y w 3 cell/g ¾ 247 bp y w (Fig. 3). m 400 bp 5, 247 bp 3 cell/g ¾ y w (Fig. 4). x m w In vivo pot assay t l R. solanacearum wš genomic DNA w PCR DNA EIA mw w š w. 4» R. solanacearum wš z w, z 2 ü t l R. solanacearum x w PCR y w (Fig. 5). š x primary PCR mw swš w flic R. solanacearum GMI1000 696 1095 e w x R. solanacearum flic 400 bp PCR y w R. Fig. 3. Threshold of detection for R. solanacearum in culture broth by PCR. Results of agarose gel electrophoresis of amplified DNA from tenfold serial dilutions of cell concentration. Lanes 1 to 10, cell concentration of R. solanacearum diluted 8 to -1, respectively. (A) Amplification of 400 bp PCR fragment by primary PCR; (B) Amplification of 247 bp PCR fragment by nested PCR. L, ladder; N, negative control.
의 DNA enzyme-linked immunosorbent assay Vol. 43, No. 3 Ralstonia solanacearum 183 Threshold of detection for R. solanacearum in soil by PCR. Results of agarose gel electrophoresis of amplified DNA from tenfold serial dilutions of cell concentration. Lanes 1 to 10, cell concentration of R. solanacearum diluted to, respectively. (A) Amplification of 400 bp PCR fragment by primary PCR; (B) Amplification of 247 bp PCR fragment by nested PCR. L, ladder; N, negative control. Fig. 4. 8-1 Symptom of R. solanacearum infection in pepper plants and results of nested PCR. (A) Agroinoculated wild-type pepper plant; (B) Agroinoculated pepper plant infected by R. solanacearum (C) Amplification of 247 bp PCR fragment by nested PCR from wild-type and R. solanacearum infected plants in vivo assay; 1, (A); 2, (B); L, ladder; N, negative control. Fig. 5. 과 유사한 토양내의 이종 세균에서는 검출되지 않 았다 유전자 내의 위치는 3종류의 biovar를 포함하는 6종류 의 아종 - DSM9544와 K60 (biovar 1), 1609와 1737과 267 (biovar 2), GMI1000 (biovar 3) -의 유전자 서열을 비교하였을 때, flic의 가장 중앙에 위치하고 있었으며 종간의 유전적 다양성도 가장 심하였다. flic 유전자는 편모 단백질의 비기능적 위치를 담고 있기 때문에 병원성을 좌우하는 부분도 아니며 유전자의 수평적 이동도 발견되지 않는다(30). 즉 flic 유 전자는 종내와 아종간의 연관성이 깊기 때문에 R. solanacearum 의 검출을 위한 유전자 지표로서 매우 유효함을 확인할 수 있었 다. 토양으로부터 직접 추출한 DNA로부터 분자생물학적 기법을 수행하는 데 있어서는 humic acid와 같은 반응 저해물질로 인한 solanacearum. flic R. solanacearum 어려움이 많다. 이들이 포함되어진 PCR 혼합 용액 내에서는 Thermus aquticus의 DNA 중합효소, Amplitaq Gold 뿐만 아니라 다른 다양한 종류의 내열성 DNA 중합효소 모두가 활성에 저해 를 받는 것으로 알려져 있으며(16), 본 연구의 수행과정에서도 R. solanacearum의 액체 배양액과 토양 시료에서 PCR 반응을 저해하는 또 다른 물질이 존재한다는 것도 알 수 있었다. 이에 본 연구에서는 일반적으로 사용되는 DNA 회수 방법을 개선하여 토양으로부터 빠르고 단순하면서도 PCR 저해물질을 효과적으로 제거할 수 있는 방법을 고안하였다. Chelex-100 resin을 넣고 끓 이는 방법은 기존의 guanidine thiocyanate 분해법이나 킬레이트 물질을 첨가하여 독성을 갖거나 인체에 유해한 시약을 사용하는 방법들과 비교하였을 때 알코올 침전 반응을 거치지 않고도 PCR에 바로 사용할 수 있는 순수한 DNA를 얻을 수 있었다. Absorbance values of DNA ELISA from amplified DNA according to tenfold serial dilutions of R. solanacearum Cell density of R. solanacearum PCR product of R. solanacearum from Culture broth 1.889 1.732 1.004 0.719 0.368 0.033 0.004 Soil 1.684 0.965 0.522 0.211 0.027 0.006 0.001 Table 3. 4 3 2 1 0-1 -2 0.002 0.001-3
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