Typing 殖 さ せ,コ 水mlに by 清 を 取 り除 い た も の を 用 い 株 か ら のDNA抽 準 的 で あ るlysostaphin 出 法 と し て9) )標 (SIGMA, て 溶 菌 し,0% sodium (SDS)とproteinase K(最 ら び に00μg
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1 Key words: arbitrarily-primed polymerase chain reaction (AP-PCR), methicillin-resistant StaNtylococcus aureus (MRSA)
2 Typing 殖 さ せ,コ 水mlに by 清 を 取 り除 い た も の を 用 い 株 か ら のDNA抽 準 的 で あ るlysostaphin 出 法 と し て9) )標 (SIGMA, て 溶 菌 し,0% sodium (SDS)とproteinase K(最 ら び に00μg/mi L4402) dodecyl K)処 てDNAを Purification 6030)を 用 い た 簡 便 なDNA抽 (BIO 5U/μl よ びtemplate れbufferをPCR DNAを な わ ちpH8.5に 加 え,そ M3 し て お い てMgC2を.5mmol/l 変 化 さ せ た も の と,MgCl,を (TTATG TAAAAC CATG),お 変 化 さ AAC よ びFerricら (CCCATGTG denaturation GTGGG)の 72 5分 72 2分 に て40サ typing法 よ るtypingの イ ク ル増 gelを 用 い40mAで Fig.2にMg濃 PCR泳 Gene法 度 とpHの に よ りDNAを 8.5, PCR泳 5分 gel 結 果 と比 較 検 パ タ ー ン を 示 す.標 準 的 にphenol/chloroform法 動 り識 別 能 件 と し て 以 下 の 実 験 で はpH MgCl23:5mmol/lを 用 い た. 応 の 温 度 と 回 数 の 選 択 に よ るAP- 動 パ タ ー ン の 差 異 をFig.3に (denaturation), annealing),72 5分 40 5分 92 (low-stringency (denaturation), 分 イ ク ル 反 応 さ せ た2段 泳 動 パ タ ー ン と,後 示 す. (extension)を2サ 反 応 さ せ た 後,92 分 sion)を40サ 績 度 とpH 動 パ タ ー ン は 大 き く異 分(high-stringencyannealing), 抽 出 法 に よ るAP-PCR泳 抽 差 異 に よ る AP- 動 パ タ ー ン の 差 異 を 示 す.Mg濃 の 差 異 に よ りAP-PCR泳 AP-PCR反 染 色 し た. 結 果 は 既 にMRSAの に よ るtypingの 成 平 成7年5月20日 の 実 験 に お い てInsta の た め以 下 の 高 いbuffer条 気 泳 動 は polya- 討 し た. Fig.にDNAの で もphenol/chloroform法 と 同 様 の 結 果 が 得 ら れ た(Fig.).こ イ クル 増 幅 を加 え た bromideで Matrix を比 較 した 出 し た. 60 と し て 確 立 し て い るpulsed-field electrophoresis法 Gene法 Purification Gene法)と な る こ と が 明 ら か に な っ た(Fig.2):よ も の の 結 果 と 比 較 し た.電 gradient 下Insta イ ク ル増 幅 を省 い た 際 の 反 応 結 果 に つ い て,2サ 0/20% 初 に イクル増 幅 し annealing こ で 最 初 の2サ と こ ろInsta 40 5分,お に て2サ 92 分, 75分 泳 動 し た 後,ethidium を 用 い た 方 法(以 3 温 度 設 定 と し て,最 よ びelongation 幅 し た5).こ TGAC- が デ ザ イ ン し たKZ TACGCGT た 後,denaturation を 用 い た も の とInstaGene AGT), AGCTA 92 5分,annealing よ びelongation crylamide sequencing GACGGCC (GGA 種 類 を 用 い た5).PCRの 分,お 3.5 し て お い てpHを に reverse れぞ (Invitrogen, せ た も の を 比 較 し た.はM3 Taq, 用 い 次 の よ う な 条 件 で 行 っ た.す 3.5mmol/lに mmol/lに 2.5 (TaKaRa Optimizer KA )を 732- mixture各 Taq-polymerase A),お ら に In- RAD, 出 法 を併 せ て行 い 比 較 し た.AP-PCRはd-NTP R00 phe- 抽 出 し ethanol Matrix 507 was used). and 5 are same strains. 2 and 6, 3 and 7, 4 and 8 are also same strains severally. to 4: DNA was extracted by the phenol chloroform method 5 to 8: DNA was extracted by the Insta Gene method. All lanes show same patterns. SDSな 理 の 後, タイ ピン グ Fig. Comparison of amplified products by APPCR according to the methods of DNA extraction (MRSA with coagulase type IV, M3 reverse sulphate で 沈 澱 さ せ る と い う 方 法 を 用 い た.さ stagene よ るMRSAの mg/ 終 濃 度 は0.5% proteinase nolとchloroformに mm, ロ ニ ー を 白 金 耳 に て 掻 き 取 り生 理 食 塩 浮 遊 さ せ,上 た.MRSA菌 mlに of MRSA 階PCRに イクル (extenよる 半 の40サ イ ク ル の み 行 っ た 泳 動 パ タ ー ン と を 比 較 し た.2段 階AP-PCRの 方が
3 北条 聡子 他 508 Fig. 2 Comparison of amplified products by APPCR according to the buffer conditions (MRSA with coagulase type II, M3 reverse was used). Lane : ph 8.5 MgCl2.5mmol/l, 2: ph 8.5 MgCl2 2.0mmol/, 3: ph 8.5 MgCl2 2.5mmol/l, 4: ph 8.5 MgCl2 3.5mmol/l, 5: ph 9.0 MgCl2 3.5mmol/l, 6: ph 9.5 MgCl2 3.5mmol/l, 7: ph 0.0MgCl2 3.5mmol/l. Lane 4 shows the most clear pattern. Fig. 4 Comparison PCR according of amplified, 2 in each gel: strains 3, 4 in each a): M3 reverse sequencing Comparison PCR between AP-PCR of amplified products the two step AP-PCR (MI3 reverse was to 4: one step AP-PCR, step AP-PCR, by AP- with coagulase gel: strains,, d): b): IV, with coagulase KZ, M3 reverse II, c): M3 & KZ, e) M3 sequencing & KZ, f): M3 reverse & M3 sequencing a) Fig. 3 products to the s. b) c) by AP- and one step used). 5 to 8: two, 2, 5, and 6: strains coagulase IV, 3, 4, 7, and 8: strains coagulase II, 5 to 8 are better with with than to 4. 験 に お い て は2段 階AP-PCRを Fig.4にの 選 択 し た. 種 類 に よ るAP-PCR泳 パ タ ー ン の 差 異 を 示 す.M3の とKZの2本 と,M3と M3 を使 っ た もの は泳 動 パ タ ー ン識 別 能 が 不 良 で,か typingに み の も の と, M3 を 使 った も の reverse2本 動 つ増 幅 され な い株 が 目立 つ た め に は 適 さ な い と考 え ら れ た.KZの み の も の は本 来 同 じで あ る べ き菌 株 間 にお い て もパ タ ー ン が 異 な っ て お り,同 じ くtypingに 考 え ら れ た-M3 reverseとkzの2本 reverseの は適 さ な い と み の も の と, M3 を使 っ た も の は泳 動 パ タ ー ン 識 別 能 が 適 切 で あ っ た. よ るtypingの gelelectrophoresis法 結 果 とpulsed-field のtypingの 結 果 とは今 回 検 討 し た 菌 株 に お い て は 一 致 し た(デ 考 バ ン ドが 鮮 明 か つminor よ り正 確 なtypingが bandも 消 失 し て お り, 可 能 と 考 え ら れ た.以 下 の 実 近 年 printing法 ー タ 省 略). 察 よ るgenomic は 各 種 細 菌 のtypingに 感 染 症学 雑 誌 DNA finger- 広 く用 い られ 第69巻 第5号
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5 epidemiological marker for study of nosocomial infections caused by methicillinresistant Staphylococcus aureus. J. Clin. Microbiol., 29: , 99. 5) Fang, F. C., McClelland, M., Guiney, D. G., Jackson, M. M., Hartstein, A. I., Morthiand, V. H., Davis, C. E., McPherson, D. C. & Welsh, J.: Value of molecular epidemiologic analysis in a nosocomial methicillin-resistant Staphylococcus aureus outbreak. J. A. M. A., 270: , ) van Belkum, A., Bax, R., Peerbooms, P., Goessens, W. H. F., van Leeuwen, N. & Quint, W. G. V.: Comparison of phage typing and DNA ngerprinting by polymerase chain reaction fi for discrimination of methicillin-resistant Staphylococcus aureus strains. J. Clin. Microbiol., 3: , ) Welsh, J. & McClelland, M.: Fingerprinting genomes using PCR with arbitrary s. Nucleic. Acids. Res., 8: , ) Williams, J. G. K., Kubelic, A. R., Livak, K. J., Rafalski, J. A. & Tingey, S. V.: DNA polymorphisms amplified by arbitrary s are useful as genetic markers. Nucleic. Acids. Res., 8: , ) Jordens, J. Z. & Hall, L. M. C.: Characterisation of methicillin-resistant Staphylococcus oureus isolates by restriction endonuclease digestion of chromosomal DNA. J. Med. Microbiol., 27: 7-23, ) Boom, R., Sol, C. J. A., Salimans, M. M. M., Jansen, C. L., Wertheim-van Dillen, P. M. E. & van der Noordaa, J.: Rapid and simple method for purification of nucleic acids. J. Clin. Microbiol., 28: , 990. DNA Fingerprinting by Arbitrarily Primed Polymerase Chain Reaction (AP-PCR) for Methicillin-resistant Staphylococcus aureus Satoko HOJO), Jiro FUJITA), Kiyoshi NEGAYAMA3), Takayuki OHNISHI), Guang XU), Yasufumi YAMAJI), Hiroki OKADA2) & Jiro TAKAHARA) Recently, nosocomial outbreaks of MRSA have become an important social problem in Japan. To examine the routes of transmission of MRSA, the establishment of accurate MRSA typing system is essential. However, more recently, because MRSA strains with type II coagulase have been increasing, it is difficult to discriminate MRSA strains by the coagulase typing method. Under this background, our study was designed to evaluate the clinical significance of DNA fingerprinting by arbitrarily primed polymerase chain reaction (AP-PCR). Several MRSA strains isolated from patients in our department were used in this study. To optimize the condition of AP-PCR, the differences of amplified products by AP-PCR were evaluated according to the following conditions: extracting methods of DNA from MRSA strains, buffer conditions, the temperature of AP-PCR, cycles of AP-PCR, and several s. As a result, the optimal conditions of AP-PCR were as follows: extracted DNA using the InstaGene kit, amplified DNA by two-step AP-PCR using a M3 reverse with a buffer condition of 3.5 mm of magnesium chloride, and a ph of 8.5. The results of AP-PCR correlated well with the results of pulsed-field gel electrophoresis. In conclusion, DNA fingerprinting by AP-PCR seems to be useful in examining the nosocomial MRSA outbreak.
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