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1 The Korean Journal of Microbiology, Vol. 41, No. 4, December 2005, p Copyright 2005, The Microbiological Society of Korea Mycoplasma w PCR» Á½ w * Á Á Á xá yá w ü mycoplasma w» w polymerase chain reaction (PCR)» 2ƒ ty PCR kp sƒw. PCR» x mycoplasma p w ù, 2ƒ ty PCR kp mycoplasma w w. w, PCR» p mycoplasma w 4 t 7 w. PCR» 9 CFR Mycoplasma w Mycoplasma ³ Acholeplasma ³ w 1~100 colony forming units/ml¾ w. w PCR» ƒ sƒw» w, k yw q ü A. laidlawii œ w z, PCR» w w w w. PCR» ü mycoplasma wš w w q. Key words ý mycoplasma detection, PCR, veterinary live viral vaccines Mycoplasma Mollicutes w x ¾ w š (16). Hayflick (10) 20 Mycoplasma Acholeplasma s, 5~35% s 6 mycoplasma (M. arginini, M. fermentans, M. hyorhinis, M. orale, Acholeplasma laidlawii) š šw., x x w, x ü s w s y (4, 10, 19). Mycoplasma s ³ s x kƒ w, 0.2~2 µm s 0.22~0.45 µm vl m w» s mw (9). w, s ƒ š mycoplasma yk, ph y, š s z ƒ yƒ» š š (9, 13, 18). s, s, w wš. œ ³ w Áw³ ƒ š w œ ü ³ w w mycoplasma»z ƒ w (22). w, mycoplasmaƒ s s w k sxk y s w w z w k, w öe (1, 5, 8, 22). x ƒ t» s w ww ³ x w mycoplasma ( ) x wš (3). Mycoplasma y w» w,, p w w (2)., s seed virus ü mycoplasma Á yw w» w DNA fluorochrome, z, xÿ, yw, š DNA probe» š š (1, 11, 18, 20). ù w» w s mycoplasma w w ƒ (18). x ƒ wš w mycoplasma p rrna w PCR» š š, kpƒ ty (7, 8, 11, 13, 20). t w w Wong-Lee Lovett(26)ƒ w PCR» 2 PCR kp w w mycoplasma z w, mycoplasma q ü mycoplasma œ w z PCR» w Á w mycoplasma z w» w PCR» ƒ sƒw. *To whom correspondence should be addressed. Tel: , Fax:

2 270 Woo-Jin Jeon et al. Kor. J. Microbiol ³ Table 1 ùkù ³ 11 mycoplasma t ³, 1 mycoplasma, 7 M. synoviae (15) 9 CFR Mycoplasma 37 o C, 5% CO 2 w 10 ³w. ³ y w» w 10 w ƒ 100 µl 9 CFR Mycoplasma w 3 plates w 5 w z ³ x w colony forming unit (cfu) w s³e w. 8 ³ ƒƒ «w x w. Genomic DNA Genomic DNA Sasaki (17) w., template DNA mycoplasma 1ml 12,000 g 30 w d w z 25 µl lysis buffer [10 Table 1. List of bacterial and Mycoplasma species used in this study Species Source M. bovis ATCC AG M. flocculare ATCC M. gallisepticum ATCC M. gallisepticum ATCC M. gallisepticum ts-11 B M. hyorhinis ATCC M. hyopneumoniae ATCC M. orale ATCC M. synoviae WVU 1853 M. iowae ATCC M. gallinaceum ATCC A. laidlawii ATCC M. synoviae(7 isolates) Field isolates, NVRQS c Staphylococcus aureus Field isolate, NVRQS Escherichia coli Field isolate, NVRQS Pasteurella multocida Field isolate, NVRQS Salmonella pullorum Field isolate, NVRQS Salmonella gallinarum Field isolate, NVRQS Salmonella enteritidis Field isolate, NVRQS Rimerella anatipestifer Field isolate, NVRQS Ornithobacterium rhinotracheale Field isolate, NVRQS A ATCC, American Type Culture Collection. B ts-11, Mycoplasma gallisepticum (MG) live vaccine strain. C NVRQS, National Veterinary Research and Quarantine Service. mm Tris-HCl (ph 8.3), 100 mm KCl, 2.5 mm MgCl 2, 1% Tween 20, 1% Triton X-100, proteinase K (120 µg/ml)] ƒ w 60 o C, 60 g. 100 o C, 10 k 5 ew. 12,000 rpm 10 w z d template DNA w. ³ PBS w w, Qiamp Tissue Kit (Qiagen, Germany) w genomic DNA w. PCR Mycoplasma universal primers 2 q kp mycoplasma z w» w 8 mycoplasma t, 1 M. gallisepticum (ts-11), š 8 ³ w w. w, mycoplasma universal primers mycoplasma z mycoplasma t 4 M. synoviae 7 w ƒ w. Mycoplasma universal primers ƒ 16S rrna» w 464 bp PCR s š Wong-Lee Lovett (25) w w, w PCR ƒ (94, 60, 72 C) o cycle ramp time 1 w ù x ramp time w (Table 2). PCR 2µl 10 PCR buffer(mgcl 2 ƒ, 20 mm), 1 µl dntp(10 mm), ƒƒ 1µl universal primer A B(10pM/ µl), 0.1 µl Taq DNA polymerase(5u/µl), 2 µl sample DNA y ww z 20 µlƒ ³ ƒw DNA thermal cycler(geneamp PCR system 9600, Perkin- Elmer, USA) w Table 2 w. q kp PCR Mycoplasma detection kit(takara Bio inc., Japan) Mycoplasma PCR ELISA kit(roche, Germany) «w x w., x PCR» mycoplasma p w R Mycoplasma PCR ELISA kit lysis reagent PCR premix w. PCR s ethidium bromide (0.5 µg/ml) ƒ 1.5 % agarose gel» w z UV transilluminator p band w. PCR» p Universal primers, R T PCR kp w ƒƒ PCR p w» w M. orale w, ƒ 8 ³ w w PCR PCR w ƒƒ PCR Table 2. PCR conditions for detection of mycoplasma DNA by using universal primers Primers A B Nucleotide sequence(5' 3') GGC GAA TGG GTG AGT AAC ACG CGG ATA ACG CTT GCG ACC TAT G Reaction condition Denaturation Annealing Extension Cycles 94 o C, 1min 60 o, 1min 72 o, 5min 30

3 Mycoplasma 검출을 위한 PCR 기법 Vol. 41, No. 4 반응의 특이성을 조사하였다. 271 및 음성대조군에 대해 PCR을 실시한 결과, 음성대조 군을 제외한 모든 mycoplasma에 대하여 464 bp의 특이 band가 관찰되었다(Fig. 1). 반면에 시판 검출킷트인 R사의 킷트를 사용 하여 검출 유효성 조사를 실시한 결과 음성대조군, M. synoviae 및 M. flocculare를 제외한 모든 mycoplasma에 대하여 약 550 bp의 특이 band가 관찰되었다. 그리고 T사 킷트를 사용한 경우 A. laidlawii를 제외한 모든 mycoplasma를 검출할 수 있었으며, 증폭산물의 크기는 균종에 따라 M. orale는 423 bp, M. hyopneumoniae는 681 bp, M. hyorhinis는 448 bp, 양성대조군은 810 bp 산물이 관찰되었다. 또한, 제조사의 사용설명서에 언급되 지 않은 균주에 대한 검출능을 조사한바 M. gallisepticum과 백 신주인 ts-11는 약 850 bp, M. synoviae와 M. bovis는 약 510bp, M. flocculare는 약 590 bp 크기의 다양한 유전자 증폭산물을 확 인할 수 있었다(Fig. 1). flocculare PCR 기법의 민감도 조사 민감도를 조사하기 위하여 4주의 mycoplasma 표준주를 이용 하여 universal primers로 PCR을 실시하였다. 4개의 표준주를 10 일간 배양한 후, 9 CFR Mycoplasma 액체배지로 10진 희석한 다음, 계수를 위해 각 희석단계별 배양물 100 µl를 9 CFR Mycoplasma 한천배지에 접종하였으며, PCR 실시를 위해 각 희 석단계별 배양물 1 ml를 사용하였다. 동물용 생 바이러스 백신에 대한 PCR 기법의 민감도 조사 인공 배양한 mycoplasma를 이용한 PCR 민감도 결과를 근거 로 시판 백신에 대하여 적용 가능한 PCR 기법의 민감도를 조사 하기 위해 배양한 mycoplasma를 시판되는 동물용 생 바이러스 백신에 접종한 후 검출한계를 비교조사 하였다. 돼지 전염성 위 장염 및 로타바이러스 생 혼합 건조백신과 개 파보바이러스 생 건조백신을 각각 희석액으로 용해한 후 A. laidlawii 배양액(10 cfu/ml)을 첨가한 다음 10진희석하여 37 C에서 20시간 배양하고 증 식된 집락을 계수한 후 PCR 기법의 검출한계를 비교 조사하였다. 6.0 o 결 과 각각의 PCR 조건에 따른 포유동물 유래 mycoplasma 검출 9주의 mycoplasma에 대하여 universal primers와 시판 검출 킷 트(T사, R사)간의 검출 유효성을 비교조사 하였다. Universal primers의 경우, A. laidlawii, M. orale, M. gallisepticum, M. synoviae, M. bovis, M. hyopneumoniae, M. hyorhinis, M. Universal primers를 이용한 조류 유래 mycoplasma 검출 조류 유래의 mycoplasma에 대해 universal primers의 검출가능 성을 추가로 조사한 바, 4주의 mycoplasma 표준주 및 7주의 M. synoviae 분리주에서 모두 464 bp의 특이 band가 관찰되었다 (Fig. 2). 각각의 PCR 조건에 따른 mycoplasma 검출 특이도 Mycoplasma 속균이 아닌 S. aureus 등 일반세균 8주에 대하여 universal primers와 시판 검출 킷트인 T사 및 R사의 검출특이성 을 비교 조사한 결과, 사용한 모든 PCR 기법에서 양성 대조균 인 M. orale를 제외하고 공시한 일반세균들에 대해서는 DNA증 폭산물이 관찰되지 않아 각각의 PCR 검출기법의 특이성이 인정 되었다(Fig. 3). PCR 기법의 mycoplasma 검출 민감도 Universal primers를 이용한 PCR 기법으로 A. laidlawii 등 총 4주의 Mycoplasma속균의 PCR 검출한계를 조사한바, A. laidlawii와 M. bovis는 10 cfu/ml, M. orale는 10 cfu/ml, M. gallisepticum은 10 cfu/ml로 균주에 따라 각각의 민감도가 상 이한 것으로 확인되었으나 약 1~100 cfu/ml의 농도에서 mycoplasma 검출이 가능함을 확인하였다(Fig. 4) Detection of Mycoplasma spp. by PCR using universal primers(a), R company's kit(b), and T company's kit(c). Lane M, 100 bp DNA ladder (Bioneer, Korea). Lane 1, A. laidlawii; lane 2, M. orale; lane 3, M. gallisepticum; lane 4, M. gallisepticum (vaccine strain, ts-11); lane 5, M. synoviae; lane 6, M. bovis; lane 7, M. hyopneumoniae; lane 8, M. hyorhinis; lane 9, M. flocculare; lane 10, mock; lane 10*, positive control template of T company's kit. Fig. 1. Detection of avian Mycoplasma spp. by PCR using universal primers. Lane M, 100bp DNA ladder (Bioneer, Korea); lane 1, M. gallisepticum; lane 2, M. synoviae; lane 3, M. iowae; lane 4, M. gallinaceum; lane 5 to 11, M. synoviae isolates. Fig. 2.

4 272 Woo-Jin Jeon et al. Kor. J. Microbiol Specificity of PCR using universal primers (A), R company's kit (B), and T company's kit (C). Lane M, 100 bp DNA ladder; lane 1, M. orale; lane 2, Staphylococcus aureus; lane 3, Escherichia coli; lane 4, Pasteurella multocida; lane 5, Salmonella pullorum; lane 6, S. gallinarum; lane 7, S. enteritidis; lane 8, Rimerella anatipestifer; lane 9, Ornithobacterium rhinotracheale. Fig. 3. 동물용 생 바이러스 백신 내의 mycoplasma 오염검출 동물용 생 바이러스 백신인 돼지 전염성 위장염 및 로타 바이 러스 생 혼합 건조백신과 개 파보바이러스 생 건조백신에 대하 여 PCR 기법의 적용가능성 및 그 민감도를 확인하기 위하여, A. laidlawii의 배양액을 각각의 백신에 접종하여 10진 희석한 후 20시간 배양한 결과, 배양균의 최종농도는 10 ( )cfu/ml 로 확인되었고, PCR 기법의 검출한계는 돼지 전염성 위장염 및 로타 바이러스생 혼합 건조백신의 경우 10 ( )cfu/ml, 개 파보바이러스 생 건조백신의 경우 10 ( )cfu/ml로 조사되어, 동물용 생 백신내의 mycoplasma 오염 검출한계는 약 10~100 cfu/ml로 균 배양액에 대한 검출한계와 유사한 민감도가 확인되었다(Fig. 5) Sensitivity of the PCR for detection of mycoplasma spiked into the vaccine. A. laidlawii (10 cfu/ml) was artificially inoculated into swine transmissible gastroenteritis-rota virus combined vaccine (A) and canine parvovirus vaccine (B). Decimal dilutions of the mixture were incubated for 20 hr and then were amplified by the PCR. The numbers of organisms in the PCR assay were indicated on both upper and lower panels (in log cfu/ml). Lane M, 100 bp DNA ladder. Fig 고 찰 각종 세포배양 등에 오염된 mycoplasma를 검출하기 위한 많 은 PCR 기법 및 시판킷트가 개발되어 실용화되고 있다(8, 11, 12, 13, 17, 18, 20, 21, 26). 본 연구에서는 시판되는 2종의 mycoplasma 검출킷트와 mycoplasma 검출 특이성과 민감도가 높 다고 알려져 있는 Wong-Lee와 Lovett (26)의 universal primers 를 자체 제작하여 PCR 반응조건을 일부 변경한 후, 각종 mycoplasma 및 세균들에 대한 PCR 검출특이성을 비교 평가하 였다. 또한, 동물용 생 바이러스 백신의 안전성 및 품질향상을 위해 universal primers를 이용한 동물용 생 바이러스 백신에 대 한 PCR 검사법의 적용 가능성을 평가하였다. 기존의 연구자들에 의하면 universal primers는 16S rrna 유전자를 근거로 하여 세 포배양내 주된 오염균인 M. hyorhinis, M. arginini, M. pneumoniae, M. fermentans, M. orale, M. pirium, A. laidlawii, Spiroplasma mirum Fig. 4. Sensitivity of the PCR assay for the detection of A. laidlawii(a), M. orale(b), M. bovis(c), and M. gallisepticum(d) using universal primers. The numbers of organisms in a assay were indicated over the panals(in log cfu/ml). Lane M, 100 bp DNA ladder. 등을 특이적으로 검출하는 것으로 알려져 있 으며 다른 세균이나 효모 등과는 교차반응이 일어나지 않도록 설계되었다고 보고하고 있다(26). 본 시험에서는 기존 연구자들 이 보고한 균주 이외에 송아지에서 만성폐렴을 야기하는 M. bovis, 돼지에서 유행성 폐렴을 일으킨다고 알려진 M. hyopneumoniae, 돼지의 호흡기계에 흔하게 존재하며, 비병원성인 M. flocurlare, 닭에서 만성 호흡기성 질병을 야기하는 M. gallisepticum과 닭에서 호흡기 질병 및 관절염을 야기하는 M. synoviae을 대상으로 universal primers의 검출특이성을 조사한 바 사용균주 모두에서 특이증폭산물을 확인되어 광범위한 mycoplasma를 검출하기 위해 충분히 사용가능할 것으로 판단되 었다. 또한, 기존 연구보고에서 적용한 적이 없는 조류유래의 M.

5 Vol. 41, No. 4 Mycoplasma w PCR» 273 iowae M. gallinaceum ü M. synoviae 7 w PCR» w, x³ p s y w,, y, mycoplasma 1 y ƒ w. Mycoplasma» j» ƒ 16S~23S rrna spacer s g ƒƒ ³ w j» s w ƒƒ mycoplasma ³ PCR s j» w w T kp w 8 mycoplasma t w, A. laidlawii wš bp 850 bp w j» s y w. PCR s biotin-labeled capture probe hybridization g z mycoplasma y w R kp, M. synoviae M. flocculare w s y w. wr, x PCR» w mycoplasma p x ww» PCR 2 mycoplasmaƒ z (ELISA) ƒ ƒ w w, kp ü primers p w mycoplasma ³ w» kp z w w PCR s» w ƒ w w. Eldering (7) š w mycoplasma mw w B. subtilis 7 ³ w universal primers PCR w, S. epidermidis, B. cereus, B. subtilis, S. bovis, C. sporogenes, M. luteus, C. pseudodiphteriticum 430~464 bp s y, šwš. Enterococcus faecalis (ATCC 29212), Bacillus subtilis (ATCC 6633), Staphylococcus epidermis (ATCC 12228), Streptococcus gallolyticus (ATCC 9809), Clostridium difficile (ATCC 9689) ³ Pseudomonas aeruginosa (ATCC 27853), z ³ Candida albicans (ATCC 90028) ƒ œ w xw, E. faecalis, B. subtilis, S. epidermis, C. difficile 400~500 bp w j» PCR s y Eldering (7) w (data not shown). ù, xw ƒ» mycoplasma w p ƒ ³ w ³( ³) x w ³ w k ³ x PCR p ³ w w ƒ, ƒ PCR s w» ww w ³ w p w ƒ. s mycoplasma PCR» 10 cfu/ml w š 4 ù(18, 20, 23), DNA (17), nested PCR (20), hotstart Taq DNA polymerase Touch-down PCR (7), PCR (21, 24) mw (15) (6) w w 1~100 cfu/ml w š šwš. p Wirth (25) 16S rrna ƒ w primers w z nested PCR ww 1-2 genome copy w w 1fg Mycoplasma DNA w šw. wr, Sasaki(17) w lysis buffer w œ ƒ k M. hominis w PCR w ƒ 300 cfu/ml šw, x 4 mycoplasma w 2 A. laidlawii w 20 w z xw PCR w ƒ 10~100 cfu/ml w ùkü» w y w. ù, Kojima(14) M. gallisepticum š w M. gallisepticum ƒw z 7 w PCR w, 10 (1.6)cfu¾ w šw x 0.2 mycoplasma ƒ ùkû ù» t w yw ƒ. w mycoplasma ³ PCR» ƒ w ùkù t w cfu/ml ³ w,, ³ ³ ƒ ùkú. š 1 w PCR» ww wš yw mycoplasma w. mw ww universal primers w PCR» Ÿ w mycoplasma ùkü ü mycoplasma Á yw w 1 j» y ƒ w q. š x 1., ½Ÿx s Mycoplasmas. w wz 28, , ½ w,,, x,,, w s Mollicutes w. w g v wz 15, w txz ƒ t» Barile, M.F., H.E. Hopps., M.W. Grabowski., D.B. Riggs, and R.A. DelGiudice The identification and sources of mycoplasmas isolated from contaminated cell culture. Ann. NY. Acad. Sci. 225,

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Razin, S., D. Yogev, and Y. Naot Molecular biology and pathogenicity of mycoplasmas. Microbiol. Mol. Biol. Rev. 62, Sasaki, T., R. Harasawa., M. Shintani., H. Fujiwara., Y. Sasaki., A. Horino., T. Kenri., K. Asada., I. Kato, and F. Chino Application of PCR for detection of mycoplasma DNA and pestivirus RNA in human live viral vaccines. Biologicals. 24, Spaepen, M., A.F. Angulo., P. Marynen, and J.J. Cassiman Detection of bacterial and mycoplasma contamination in cell cultures by polymerase chain reaction. FEMS Microbiol. Lett. 99, Stipkovits, L., L. Bodon., J. Romvary, and L. Varge Direct isolation of mycoplasmas and acholeplasmas from sera and kidneys of calves. Acta. Microbiol. Acad. Sci. Hung. 22, Tang, J., M. Hu., S. Lee, and R. Roblin A polymerase chain reaction based method for detecting Mycoplasma/Acholeplasma contaminants in cell culture. J. Microbiol. Method. 39, Teyssou, R., F. Poutiers., C. Saillard., O. Grau., F. Laigret., J.M. Bove, and C. Bebear Detection of mollicute contamination in cell cultures by 16S rdna amplification. Mol. Cell. Probes. 7, Thornton, D.H A survey of mycoplasma detection in veterinary vaccines. Vaccine 4, Toji, L.H., T.C. Lenchitz., V.A. Kwiatkowski., J.A. Sarama, and R.A. Mulivor Validation of routine mycoplasma testing by PCR. In Vitro Cell. Dev. Biol. Anim. 34, Uphoff, C.C. and H.G. Drexler Comparative PCR analysis for detection of mycoplasma infections in continuous cell lines. In Vitro Cell. Dev. Biol. Anim. 38, Wirth, M., E. Berthold., M. Grashoff., H. PfUtzner., U. Schubert, and H. Hauser Detection of mycoplasma contaminations by the polymerase chain reaction. Cytotechnology 16, Wong-Lee, J.G. and M. Lovett Rapid and sensitive PCR method for identification of Mycoplasma species in tissue culture, p In Persing D.H., T.F. Smith, F.C. Tenover, and T.J. White (eds.), Diagnostic molecular microbiology principles and applications, American Society for Microbiology, Washington, D.C. (Received October 17, 2005/Accepted November 24, 2005) ABSTRACT : Application of a PCR Method for the Detection of Mycoplasma in Veterinary Live Viral Vaccines Woo-Jin Jeon, Byoung-Han Kim*, Byeong-Yeal Jung, Dong-Jun An, Chul-Hyun Yi, Hwan Jang, and Gab-Soo Chung (National Veterinary Research and Quarantine Service, Ministry of Agriculture and Forestry, Anyang , Korea) We evaluated the PCR assay and two commercialized PCR kits for the detection of mycoplasma in veterinary viral live vaccines. The PCR assay could specifically detect all the tested Mycoplasma spp. and Acholeplasma spp., whereas two commercialized PCR kits did not. Also, the specificity of the PCR assay showed that 4 reference strains and 7 field isolates belonging to avian mycoplasma species could be all detected. The sensitivity of the PCR assay was determined using pure cultured Mycoplasma spp. and Acholeplasma spp. with a range of 1 to 100 colony forming units/ml in 9 CFR Mycoplasma broth. To test the availability of the PCR assay for veterinary live viral vaccines, A. laidlawii was artificially inoculated into the swine transmissible gastroenteritis-rota virus combined vaccine and canine parvovirus vaccine, respectively and the sensitivity of the PCR assay was similar with the result of cultured samples. In this study, the PCR assays could be used as rapid and sensitive methods for the detection of mycoplasma in veterinary live viral vaccines.

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