肺炎マ検出マニュアル2011改訂版_

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ときやまのかみしゃ
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1 Mycoplasma pneumoniae

2 1. Mycoplasma pneumoniae 2. 1) 2) 3) 4) 3. 1) M. pneumoniae 2) 3) 4) PCR LAMP 4. M. pneumoniae Hayflick

3 Mycoplasma pneumoniae M. pneumoniae Mollicutes ycoplasma 800kb G+C 40% M. pneumoniae X M. pneumoniae M. pneumoniae M. pneumoniae M. pneumoniae Community Acquired Respiratory Distress Syndrome toxin (CARDS ) 3

4 M. pneumoniae BSL2 P2 M. pneumoniae M. pneumoniae M. pneumoniae 37 4 M. pneumoniae M. pneumoniae DNA 4

5 M. pneumoniae WHO Hayflick 200 g/ml BD Universal Viral Transport for Viruses, Chlamydiae, Mycoplasmas and Ureaplasmas Hayflick 5

6 DNA 0.45 m M. pneumoniae 0.45 m l DNA PA PA -MYCO II X M. pneumoniae PA 4 6

7 320 CF 4 64 M. pneumoniae DNA M. pneumoniae X M. pneumoniae M. pneumoniae 4 7

8 M. pneumoniae M. pneumoniae M. pneumoniae M. pneumoniae Hayflick PPLO 1 PPLO 21 g 5 g (0.5%) 20 mg (0.002%) 750 ml ml (15 %) 25 % 100 ml (10 %) G % 10 ml PPLO Broth w/o CV, Difco , Becton Dickinson 25 % G 50 g/ml 3 ml 20 8

9 Hayflick PPLO 1 PPLO 35 g 750 ml ml (15 %) 25 % 100 ml (10 %) G 100 PPLO Agar, Difco , Becton Dickinson 4-5mm 9 cm 25ml 4 G 50µg/ml M. pneumoniae M. pneumoniae Hayflick 0.001% M. pneumoniae PPLO 1 ml 9

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.

10 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

11 25 % M. pneumoniae g 1 Nippon Beet Sugar MFG. Co. Ltd 3N NaOH 8000 x g 8000 x g 25 cm 2 50ml g xg 20 NaOH ph

12 50ml m Gibco M. pneumoniae 0.2 ml 0.1 ml PPLO 37 ph 5% 5% 5% 37 M. pneumoniae

13 M. pneumoniae 1 3 M. pneumoniae 3 10 M. pneumoniae PPLO Hayflick µl PPLO PPLO 0.1mm M. pneumoniae 13

14 Dienes Dienes II 2.5 g 1.25 g 10 g 0.25 g 0.25 g 100 ml 2 3ml Hayflick m PPLO M. pneumoniae M. pneumoniae 14

15 M. pneumoniae M. pneumoniae M. genitalium M. genitalium PBS 100 7ml PBS 2 3. M.pneumoniae Hayflick Hayflick M. pneumoniae Metabolic inhibition 15

16 test M. pneumoniae PCR PCR M. pneumoniae 80 (DMSO) 2 1 : PPLO broth w/o CV 2 10% 12 10% M. pneumoniae 16

17 M. pneumoniae ph 1 2 M. pneumoniae ph 17

18 M. pneumoniae PA CF IC ELISA EIA IFA MI PA CF IgM PA M. pneumoniae (IHA) -MYCO II 3 IgM IgG 18

19 CF CF IgG CF CF CF x 32 IC Meridian Diagnostics, Immuno Card Mycoplasma Test M. pneumonia 5 IgM ELISA ELISA M. pneumoniae 19

20 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

21 MI M. pneumoniae M. pneumoniae MI M. pneumoniae M. pneumoniae M. pneumoniae M. pneumoniae M. pneumoniae DFA M. pneumoniae M. pneumoniae 21

22 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 x g Triton-X100 TE buffer 10mM Tris-HCl, 1mM EDTA, ph l 95 5 DNA 1 µg/ml DNA DNA QIAGEN QIAamp DNA Mini Kit 22

23 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

24 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 st PCR 0.8% 24

25 DNA 1st PCR 1 l 2nd PCR PCR 5 10µl µg/ml UV PCR DNA 25

26 LAMP LAMP LAMP Loopamp P ( ) Loopamp Loopamp Loopamp DNA 95 5 UV 26

27 M. pneumoniae β M. pneumoniae 23S rrna V 8 M. pneumoniae M. pneumoniae 1 1 Hayflick 2 M. pneumoniae CFU / ml l 96 ( ) M. pneumoniae 100 l 200 l 27

28 100 l M. pneumoniae 100 l M. pneumoniae 37 2 M. pneumoniae MIC; g / ml ph MIC ph MIC ph M. pneumoniae MIC 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

29 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 (MN23SDVR) 5 - GTCCTCGCTTCGGTCCTCTCG -3 ( ) 2 PCR Solution Premix Taq 1 (MN23SDVF 10 M) 2 (MN23SDVR 10 M) DNA

30 PCR First PCR 927bp Second PCR 23S rrna b) Second PCR bp 1( MN23SF1937): 5 -ACTATAACGGTCCTAAGGTA (MN23SR2128): 5 -ACCTATTCTCTACATGATAA PCR Solution Premix Taq 1 (MN23SF M) 2 (MN23SR M) DNA PCR

31 c) Second PCR bp 1(MN23SF2577): 5 -TACGTGAGTTGGGTTCAAA-3 ( ) 2(MN23SR2664): (M23SDVR ) 5 -GTCCTCGCTTCGGTCCTCTCG-3 ( ) 2 PCR Solution Premix Taq 1 (MN23SF M) 2 (MN23SR M) DNA PCR a) BceAI BbsI BsaI b) Second PCR A2063C A2063G BceAI (1U/PCR 1µl) BbsI (5U/PCR 1µl) A2064G BsaI 1U/PCR 1µl b) BsmFI c) Second PCR C2617G BsmFI (2U/PCR 1µl)

32 PCR 15 4 Nusive3:1 2) 23S rrna V 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 M. pneumonie M129 (accession no.x68422) 32

33 M. pneumoniae M. pneumoniae 1 6 M. pneumoniae M. pneumoniae 33

34 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 M. pneumoniae DNA PCR 2.5 kb PCR PCR HaeIII 2 34

35 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

36 M. pneumoniae p1 PCR HaeIII ADH1-ADH2 ADH3-ADH4 I II II' I II II' bp Nested PCR M. pneumoniae PCR-RFLP M. pneumoniae p1 PCR DNA PCR M. pneumoniae PCR-RFLP Nested PCR M. pneumoniae DNA DNA 36

37 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 M. pneumoniae DNA PCR 4 kb PCR 0.8% PCR 1st PCR 2nd PCR 1st PCR RepMP4 RepMP2/3 2 PCR 37

38 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 st 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

39 Nested PCR-RFLP PCR-RFLP 39

40 Hayflick PPLO 1 PPLO 21 g 5 g (0.5%) 20 mg (0.002%) 750 ml 1N HCl ph ml (15 %) 25 % 100 ml (10 %) G % 10 ml PPLO Broth w/o CV, Difco , Becton Dickinson G 50µg/ml Mycoplasma PPLO PPLO 2 37 M. pneumoniae M. orale 40

41 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

42 Mycoplasma pneumoniae 42

43 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, 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 : , 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 : , 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 : , :. 77(5) : 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: ,

44 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: , 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: , 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 J. Clin. Microbiol. 49: , Matsuoka M. et al :Characterization and molecular analysis of macrolideresistant Mycoplasma pneumoniae clinical isolates obtained in Japan. Antimicrob. Agents Chemother. 48: , 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, 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):

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2 3 4 TTT TCT TAT TGT TTC TCC TAC TGC TTA TCA TAA TGA TTG TCG TAG TGG CTT CCT CAT CGT CTC CCC CAC CGC CTA CCA CAA CGA CTG CCG CAG CGG ATT ACT AAT AGT ATC ACC AAC AGC ATA ACA AAA AGA ATG ACG AAG AGG GTT