Mycoplasma pneumoniae 23 9 1
1. Mycoplasma pneumoniae 2. 1) 2) 3) 4) 3. 1) M. pneumoniae 2) 3) 4) PCR LAMP 4. M. pneumoniae Hayflick 5. 6. 2
Mycoplasma pneumoniae M. pneumoniae Mollicutes ycoplasma 800kb G+C 40% M. pneumoniae 30 40 4 X M. pneumoniae M. pneumoniae M. pneumoniae M. pneumoniae Community Acquired Respiratory Distress Syndrome toxin (CARDS ) 3
M. pneumoniae BSL2 P2 M. pneumoniae M. pneumoniae M. pneumoniae 37 4 M. pneumoniae M. pneumoniae DNA 4
M. pneumoniae 4 80 20 WHO Hayflick 200 g/ml BD Universal Viral Transport for Viruses, Chlamydiae, Mycoplasmas and Ureaplasmas Hayflick 5
DNA 0.45 m M. pneumoniae 0.45 m 10 500 l DNA PA PA -MYCO II X M. pneumoniae PA 4 6
320 CF 4 64 M. pneumoniae DNA M. pneumoniae X M. pneumoniae M. pneumoniae 4 7
M. pneumoniae M. pneumoniae M. pneumoniae M. pneumoniae Hayflick PPLO 1 PPLO 21 g 5 g (0.5%) 20 mg (0.002%) 750 ml 50 55 30 150 ml (15 %) 25 % 100 ml (10 %) G 100 2.5 % 10 ml PPLO Broth w/o CV, Difco 255420, Becton Dickinson 25 % G 50 g/ml 3 ml 20 8
Hayflick PPLO 1 PPLO 35 g 750 ml 50 55 30 150 ml (15 %) 25 % 100 ml (10 %) G 100 PPLO Agar, Difco 241210, Becton Dickinson 4-5mm 9 cm 25ml 4 G 50µg/ml M. pneumoniae M. pneumoniae Hayflick 0.001% M. pneumoniae PPLO 1 ml 9
を入れて固めた後 メチレンブルー含有培地を 2 ml を重層して二層 培地とする この培地では液層と寒天層の境界の酸化還元電位が M. pneumoniae の増殖に適し 分離率が向上するとされている 二層培 地の有効使用期間は 作製後 冷蔵保存で２週間程度である 図１ 図 1 二層培地 Hayflick 変法液体培地は ph 指示薬として添加したフェノールレ ッドによって赤色だが M. pneumoniae が増殖すると ph が低下し黄 色に変色する 選択培地用にメチレンブルーを添加すると Hayflick 変法培地は茶色がかった色になる これに M. pneumoniae が増殖する と緑黄色になる 図２ 図２ メチレンブルーの有無による Hayflick 変法培地の色調変化の例 10
25 % M. pneumoniae 1 1.5 1 500 g 1 Nippon Beet Sugar MFG. Co. Ltd 3N NaOH 8000 x g 8000 x g 25 cm 2 50ml 1.5 500 g 1.5 20 3 4 4 8000-10000 xg 20 NaOH ph 7.6 11
50ml 20 0.45 m Gibco 55 30 M. pneumoniae 0.2 ml 0.1 ml PPLO 37 ph 5% 5% 5% 37 M. pneumoniae 2 4 80 12
M. pneumoniae 1 3 M. pneumoniae 3 10 M. pneumoniae PPLO Hayflick 10-3 10-6 10 µl PPLO 1 4 6 PPLO 0.1mm 40 100 M. pneumoniae 13
Dienes Dienes II 2.5 g 1.25 g 10 g 0.25 g 0.25 g 100 ml 2 3ml Hayflick 25 0.45 m PPLO M. pneumoniae M. pneumoniae 14
M. pneumoniae M. pneumoniae M. genitalium M. genitalium PBS 100 7ml 37 30 1 PBS 2 3. M.pneumoniae Hayflick Hayflick M. pneumoniae Metabolic inhibition 15
test M. pneumoniae PCR PCR M. pneumoniae 80 (DMSO) 2 1 : 1 80 2 PPLO broth w/o CV 2 10% 12 10% M. pneumoniae 16
M. pneumoniae ph 1 2 M. pneumoniae ph 17
M. pneumoniae PA CF IC ELISA EIA IFA MI PA CF IgM PA M. pneumoniae (IHA) -MYCO II 3 IgM IgG 18
CF CF IgG 4 64 2 CF CF CF x 32 IC Meridian Diagnostics, Immuno Card Mycoplasma Test M. pneumonia 5 IgM http://www.tfb-net.com/iryou/products/01_03.html ELISA ELISA M. pneumoniae 19
M. pneumoniae ELISA P1 M. pneumoniae ELISA 4 IFA M. pneumoniae M. pneumoniae M. pneumoniae M. pneumoniae Ig M. pneumoniae 4 O Rh(-) I IgM 1 2 30 40 20
MI M. pneumoniae M. pneumoniae MI M. pneumoniae M. pneumoniae M. pneumoniae M. pneumoniae M. pneumoniae DFA M. pneumoniae M. pneumoniae 21
M. pneumoniae PCR PCR M. pneumoniae DNA PCR PCR M. pneumoniae P1 p1 nested PCR nested PCR p1 PCR DNA DNA M. pneumoniae DNA DNA 1. DNA 1 2 ml 15000 x g 10 0.1 Triton-X100 TE buffer 10mM Tris-HCl, 1mM EDTA, ph 8.0 50 l 95 5 DNA 1 µg/ml 55 15 DNA DNA QIAGEN QIAamp DNA Mini Kit 22
2. DNA PCR M. pneumoniae DNA QIAGEN PCR p1 1st PCR ADH2F: 5 -GGC AGT GGC AGT CAA CAA ACC ACG TAT-3 ADH2R: 5 -GAA CTT AGC GCC AGC AAC TGC CAT-3 2nd PCR ADH/3F : 5 -GAA CCG AAG CGG CTT TGA CCG CAT -3 ADH/3R: 5 -GTT GAC CAT GCC TGA GAA CAG TAA -3 1st PCR 1451 bp 2nd PCR 1324 bp PCR M. pneumoniae M. pneumoniae p1 nested PCR 23
PCR 1st 2nd PCR DNA 10ng 1µg 5µl dntp 1.25 mm 16µl F 10 pmol/ml 2µl R 10 pmol/ml 2µl Taq DNA 1 U/µl 2µl MgCl 2 25 mmol/l 8µl 10 * 10µl 55µl 100µl *10 PCR PCR PCR Premix Taq EX DNA PCR M. pneumoniae DNA PCR PCR 1st, 2nd PCR 94 1 94 1 55 1 35 72 1 72 5 1st PCR 0.8% 24
DNA 1st PCR 1 l 2nd PCR PCR 5 10µl 0.8 0.5µg/ml UV PCR DNA 25
LAMP http://loopamp.eiken.co.jp/ LAMP LAMP Loopamp P ( ) Loopamp Loopamp Loopamp DNA 95 5 UV 26
M. pneumoniae β M. pneumoniae 23S rrna V 8 M. pneumoniae M. pneumoniae 1 1 Hayflick 2 M. pneumoniae 10 4 10 5 CFU / ml 3 2 4 100 l 96 ( ) M. pneumoniae 100 l 200 l 27
100 l M. pneumoniae 100 l M. pneumoniae 37 2 M. pneumoniae MIC; g / ml ph MIC ph7.2 8.2 MIC ph M. pneumoniae MIC 10 4 10 5 CFU/ml MIC M. pneumoniae MIC MIC M. pneumoniae MIC MIC MIC MIC M. pneumoniae MIC M. pneumoniae M. pneumoniae 23S rrna PCR-RFLP ( 15) PCR ( 16 17) 28
1) PCR-RFLP PCR-RFLP ( 15) M. pneumoniae 23S rrna A2063C 2063 adenine cytosine A2063G A2064G C2617G Nested PCR DNA a) M. pneumoniae 0.5ml b) 15,000rpm 20 c) 20µ TE d) 99 5 e) Nested PCR a) First PCR 23S rrna V 927bp 1 (MN23SDVF) 5 - GCAGTGAAGAACGAGGGG -3 1758-1775 2 (MN23SDVR) 5 - GTCCTCGCTTCGGTCCTCTCG -3 (2664-2684) 2 PCR Solution Premix Taq 1 (MN23SDVF 10 M) 2 (MN23SDVR 10 M) DNA 25 17 3 3 2 50 29
PCR 94 2 94 45 55 1 30 72 1.2 72 5 First PCR 927bp Second PCR 23S rrna 2063 2064 2617 b) Second PCR-1 2063 2064 210bp 1( MN23SF1937): 5 -ACTATAACGGTCCTAAGGTA-3 1918-1937 2(MN23SR2128): 5 -ACCTATTCTCTACATGATAA-3 2128-2177 2 PCR Solution Premix Taq 1 (MN23SF1937 10 M) 2 (MN23SR2128 10 M) DNA 25 17 3 3 2 50 PCR 94 2 94 45 55 1 30 72 1.2 72 5 30
c) Second PCR-2 2617 108bp 1(MN23SF2577): 5 -TACGTGAGTTGGGTTCAAA-3 (2577-2595) 2(MN23SR2664): (M23SDVR ) 5 -GTCCTCGCTTCGGTCCTCTCG-3 (2664-2684) 2 PCR Solution Premix Taq 1 (MN23SF2557 10 M) 2 (MN23SR2664 10 M) DNA 25 17 3 3 2 50 PCR 94 2 94 45 55 1 30 72 1.2 72 5 a) BceAI BbsI BsaI b) Second PCR A2063C A2063G BceAI (1U/PCR 1µl) BbsI (5U/PCR 1µl) 37 2 50 A2064G BsaI 1U/PCR 1µl 50 2 50 b) BsmFI c) Second PCR C2617G BsmFI (2U/PCR 1µl) 37 2 50 31
PCR 15 4 Nusive3:1 2) 23S rrna V 2063 2064 2617 PCR-RFLP A2064C PCR-RFLP M. pneumoniae DNA PCR DNA PCR-RFLP PCR PCR-RFLP First PCR PCR 927bp DNA MiniElute PCR purification kit (Qiagen) BigDye Terminater V3.1 cycle sequencing kit(applied Biosystems) ABI Prism3100 genetic analyzer (Applied Biosystems) PCR-RFLP 2063,2064 MN23SDVF MN23SDVR MN23SF1973 PCR DNA BLAST http://blast.ncbi.nlm.nih.gov/blast.cgi M. pneumonie M129 (accession no.x68422) 32
M. pneumoniae M. pneumoniae 1 6 M. pneumoniae M. pneumoniae 33
M. pneumoniae PCR-RFLP M. pneumoniae PCR-RFLP M. pneumoniae p1 P1 RepMP2/3 RepMP4 PCR ADH1 ADH2 RepMP4 ADH3 ADH4 RepMP2/3 RepMP4 RepMP2/3 1 RepMP4 ADH1 : CTGCCTTGTCCAAGTCCACT ADH2 : AACCTTGTCGGGAAGAGCTG 2 RepMP2/3 ADH3 : CGAGTTTGCTGCTAACGAGT ADH4 : CTTGACTGATACCTGTGCGG PCR 94 1 94 1 55 1 30 72 2.5 72 10 M. pneumoniae DNA PCR 2.5 kb PCR PCR HaeIII 2 34
M 100 bp I II II IIa 7 M. pneumoniae p1 PCR-RFLP 1 I 2 II 3 ADH3-ADH4 I II RFLP IIa 3 II IIa IIb IIc I 1 14 RFLP p1 35
M. pneumoniae p1 PCR HaeIII ADH1-ADH2 ADH3-ADH4 I II II' I II II' 510 939 939 824 824 989 396 396 396 579 440 440 316 313 313 313 313 313 253 240 240 305 183 183 250 237 237 153 153 153 240 91 91 104 119 119 130 17 17 55 119 112 91 31 112 104 31 10 104 39 17 8 55 31 39 10 31 8 10 8 bp Nested PCR M. pneumoniae PCR-RFLP M. pneumoniae p1 PCR DNA PCR M. pneumoniae PCR-RFLP Nested PCR M. pneumoniae DNA DNA 36
1st PCR 1st PCR ADH1 ADH4 p1 1st PCR ADH1 : CTGCCTTGTCCAAGTCCACT ADH4 : CTTGACTGATACCTGTGCGG PCR 1st PCR DNA 10ng 1 g F 10 pmol/ l R 10 pmol/ l EX Taq DNA premix 5 l 2 l 2 l 25 l 16 l 50 l 1st PCR 98 10 98 10 65 30 30 72 4 72 10 M. pneumoniae DNA PCR 4 kb PCR 0.8% PCR 1st PCR 2nd PCR 1st PCR RepMP4 RepMP2/3 2 PCR 37
1 RepMP4 ADH1a: AAGTCCACTTGGATTCTCATCCTCACCGCC ADH2a: GGAAGAGCTGCTAACAATTCCGGATTGAGA 2 RepMP2/3 ADH3a: GCTAACGAGTACGAGCGCTTTAACCAGAAG ADH4a: ACCTGTGCGGTTAATGATTTCCTTAAAGACA PCR 2nd PCR DNA 10ng 1 g F 10 pmol/ l R 10 pmol/ l EX Taq DNA premix 5 l 2 l 2 l 25 l 16 l 50 l 2nd PCR 98 10 98 10 65 30 18 72 2 72 10 1st PCR PCR 2.5 kb PCR 0.8% PCR 2 µl Nested PCR M. pneumoniae HaeIII RFLP 2nd PCR RepMP4 RepMP2/3 Hae III 2% 38
Nested PCR-RFLP PCR-RFLP 39
Hayflick PPLO 1 PPLO 21 g 5 g (0.5%) 20 mg (0.002%) 750 ml 1N HCl ph 50 55 30 150 ml (15 %) 25 % 100 ml (10 %) G 100 2.5 % 10 ml PPLO Broth w/o CV, Difco 255420, Becton Dickinson G 50µg/ml Mycoplasma PPLO PPLO 2 37 M. pneumoniae M. orale 40
M. fermentans M. penetrans M. pneumoniae M. orale M. pneumoniae M. pneumoniae M. orale M. salivarium M. fermentans M. hominis M. genitalium M. penetrans M. amphoriforme Acholeplasma laidlawii 41
Mycoplasma pneumoniae 42
. 1988. 1980. 1974. Waites, K. B., Bébéar, C. M., Robertson, J. A., Talkington, D. F. and Kenny, G. E. : Laboratory diagnosis of mycoplasmal infections in Cumitech-Cumulatie techniques and procedures in clinical microbiology, edited by Nolte, F. S. 2001, ASM press, Washington, DC, U.S.A. 5. Mycoplasma,, 2001, 27 650-653 6. Kenri T, Taniguchi R, Sasaki Y et al. : Identification of a new variable sequence in the P1 cytadhesin gene of Mycoplasma pneumoniae: evidence for the generation of antigenic variation by DNA recombination between repetitive sequences. Infect Immun 67 : 4557-4562, 1999. 7. Dorigo-Zetsma JW, Wilbrink B, Dankert J et al. : Mycoplasma pneumoniae P1 Type 1- and Type 2-Specific Sequences within the P1 Cytadhesin Gene of Individual Strains. Infect Immun 69 : 5612-5618, 2001. 8. Okazaki N, Narita M, Yamada S, Izumikawa K, Umetsu M, Kenri T, Sasaki Y, Arakawa Y, Sasaki T.: Characteristics of macrolide-resistant Mycoplasma pneumoniae strains isolated from patients and induced with erythromycin in vitro. Microbiol Immunol 45 : 617-620, 2001 9. :. 77(5) : 310-315 2003 10. Matthias F.C.Beersma, et al : Evaluation of 12 Commercial Tests and the Complement Fixation Test for Mycoplasma pneumoniae- Specific Immunoglobulin G (IgG) and IgM Antibodies, with PCR Used as the Gold Standard. J. Clin. Microbiol. 43: 2277-2285, 2005 43
11. Thacker, W. L., and D. F. Talkington. Analysis of complement fixation and commercial enzyme immunoassays for detection of antibodies to Mycoplasma pneumoniae in human serum. Clin. Diagn. Lab. Immunol. 7:778 780, 2000. 12.. 19 27-35 2007 13. Kenri T, Okazaki N, Yamazaki T, Narita M, Izumikawa K, Matsuoka M, Suzuki S, Horino A, Sasaki T. Genotyping analysis of Mycoplasma pneumoniae clinical strains in Japan between 1995 and 2005: type shift phenomenon of M. pneumoniae clinical strains. J. Med. Microbiol. 57: 469-475, 2008. 14. Zhao F, Cao B, Li J, Song S, Tao X, Yin Y, He L, Zhang J. Sequence analysis of the p1 adhesin gene of Mycoplasma pneumoniae in clinical isolates collected in Beijing in 2008 to 2009. J. Clin. Microbiol. 49: 3000-3003, 2011. 15. Matsuoka M. et al :Characterization and molecular analysis of macrolideresistant Mycoplasma pneumoniae clinical isolates obtained in Japan. Antimicrob. Agents Chemother. 48:4624 4630, 2004. 16. Morozumi M, et al.:acute Respiratory Diseases Study Group. Increased macrolide resistance of Mycoplasma pneumoniae in pediatric patients with community-acquired pneumonia. Antimicrob. Agents Chemother. 52:348-50,2008. 17 Peuchant O,et al.: Increased macrolide resistance of Mycoplasma pneumoniae in France directly detected in clinical specimens by real-time PCR and melting curve analysis. J Antimicrob Chemother. 64(1):52-8.2009 44
253-0087 1-3-1 E-mail: 208-0011 4-7-1 E-mail: horino@nih.go.jp kenri@nih.go.jp yuko@nih.go.jp 45