Pyrosequencing 法を用いた臨床分離抗酸菌株に対する同定法の精度
Contents
1. COMMAS 57 6 DNA 16S rRNA gene 591 8555 1180 pyrosequencing PCR2. DDH DDH DNA 2 3 mm 15mL Tris EDTA buffer 10 mM Tris 0 1 mM EDTA pH 80 TE 500 uL 95C 10 10 vortex 12 000 rpm 5 1 5 mL pyrosequencing MGTT HW O3 15 mL 1 mL 12 000 rpm 15 TE 100 uL 95 C 30 vortex 12 000 rpm 1 PCR HC Takara Ex Taq
3. pyrosequenc ing o LL AAAA TTTT 1 1 pyrosequencing Runyon 24 3 No 98 No 66 No 64 py rosequencing 7 WE No 98 TI M colombiense M bouchedurhonense
4. 100 100 BACTEC MGIT960 KY AR OU ES lk 0 5 mg ml PNBA p nitrobenzoic acid 7H11 WALT L lC yE 24 Runyon pyrosequencing Runyon M tuberculosis complex 10 M avium 10 M intracellulare 10 M kansasi
5. CE f a H gt gt ee M TEL 072 252 3021 did FAX 072 251 1372 DNA ATP E mail dustin kch hosp go jp 26 VoL25 No l 2015 Pyrosequencing 27 E PCR 1 DNA 4 pyrose quencing 2011 1 12
6. Vol 25 No 1 2015 29 zE r1 30 189 A1oje1oqe IUMO Aq LJOS pagquoprun 10 Z AQL 99S x S189 amp 10je1oqe JUNOT Aq 93e ost pegnuoprun 107 AQLI 99S asuauoy npayonog WN LOTO esuemuiopoo 7Y osuauognpoeqonoq JA a1e nj 999 13UI W 991 ds JN asuamquio oo WN asuaypeALL TA J0Z0 ONT SISUOIST W ds eyja nureyns SA umonosue TA sruowmd Sujiou nb s 7gg7nZS TA eyjainueynsy x lduroO2 umre PF urnooe njolosuIed A uumnooe njolosuIed JA 9S09Jr79gEr7 TAI 3ua8 VN SOT se8 WW LIJSES JA aerguods IISesuey TA IISESUEX TA Bye Jnurens L wnAe JA uln op njJo OSb Ipd PY urn op nJo I9Sb Ipd TAI 9g 97r7Od JA umnomaosue W stuowjnd denjJ93ed ur W umnordsuoo JA umnnordsuoo NW 9Sn979ggrr JA rgg7nZS TJA e o rnure xns r 9SUgIqUio oo TAI um ooe ngor2s WN umnooe njolos NW unmulIS 1 d TA Sur uonboso1Aq Sj nsa1 soroods ParHUePt 10 uon TI AI xaIdur02 unne TA unooe njo2s TAI umneoemjoros Jy umnurrigolod W eogisse p UOAUNY VNd pre T de oruozuoqo nru q U9 0UIO I2 0 02G UISOUIOIYI UON U9SOUIOIQ2 UON U9SOUIOIQ2 UON U9SO0UIOIQ2 UON UdSOUIOIYO UON U9SOUIOIQ2 UON uonejuoulsIq Uoouws Uoouws Uoouws oouws Uoouws oouws Asojoydiour AuO 0 O LE 297 1 AOTD x 99 1 9 68 G b 18 MSHPLILYI Sa BIOSI euo Sa e OSI umrr 1oeqooKur pagnuoprun jo UONYLNJYU PI on u s8 pue eo
7. GR conflict of interest x ak 1 Heller L C M Jones R H Widen 2008 Comparison of DNA pyrosequencing with alternative methods for identification of mycobacteria J Clin Microbiol 46 2092 2094 2 Cristia Fernstr m M M Olofsson E Chryssanthou et al 2007 Pyrosequencing of a short hypervariable 16S rDNA fragment for the identification of nontu Vol25 Nol 2015 31 32 3 8 9 32 berculous mycobacteria a comparison with conven tional 16S rDNA sequencing and phenotyping AP MIS 115 1252 1259 Tuohy M J G S Hall M Sholtis et al 2005 Pyrose quencing as a tool for the identification of common isolates of Mycobacterium sp Diag Microbiol Infect Dis 51 245 250 Bao J R R N Master D A Schwab et al 2010 Identi fication of acid fast bacilli using pyrosequencing analysis Diag Microbiol Infect Dis 67 234 238 2013 WREE 62 246 251
8. T 94C 30 55C 30 72C 1 35 16S rRNA A B 285F 5 GAG AGT TTG ATCCTGGCT CAG 3 259R 5 TTT CACGAA CAA CGC GAC AA 3 PCR 7c PCR BigDye Terminator Ready Reaction Cycle Sequencing Kit Applied Bio systems Japan 16S rRNA BLAST Basic Local Alignment Search Tool 99 C Is Witt He UZ Pyrosequencing DNA pyrosequencing PyroMark PCR MOTT 16S Primers for VoL25 Nol 2015 27 28 Table 1 Clinical isolates identified by pyrosequencing correlating to conventional 16S rRNA gene sequencing and phe notypic identification N 100 Identified results of Conventional 16S rRNA gene routine laboratory tests N Pyrosequencing 16S rRNA gene sequencing BLAST results M tuberculosis complex 10 10 M tuberculosis complex 10 M tuberculosis complex M avium 10 10 M avium 10 M avium M intracellulare 10 10 M i
9. 2008 Mycobac terium lentiflavum 83 417 422 Murcia M L E Tortoli M C Menendez et al 2006 Mycobacterium colombiense sp nov a novel mem ber of the Mycobacterium avium complex and de scription of MAC X as a new ITS genetic variant Int J Syst Evol Microbiol 56 2049 2054 Salah I B C Cayrou D Raoult et al 2009 Mycobac terium marseillense sp nov Mycobacterium ti monense sp nov and Mycobacterium bouchedur honense sp nov members of the Mycobacterium avium complex Int J Syst Evol Microbiol 59 2803 2808 Durnez L P Suykerbuyk V Nicolas et al 2010 Ter Vol 25 No 1 2015 gt gt E 10 11 12 13 14 15 restrial small mammals as reservoirs of Mycobacte rium ulcerans in Benin Appl Environ Microbiol 76 4574 4577 Davies E J Wieboldt T Stanley et al 2012 Isolation and identification of Mycobacterium angelicum from a patient with type II respiratory failure sug gested reporting guidelines to molecular clinical labo ratories J Biomed Sci 69 134 136 Richter E E Tortoli A Fischer et al 2007 Myco bacterium alsiense a novel slowly growing species isolated from two patients with pulmonary disease J Clin Microbiol 45 3837 3839 van Ingen J S A Al Hajoj M Boeree et al 2009 My cobacterium riya
10. M colombiense 2 M colombiense 7 9 Wk No 66 IIHR M szulgai3 M angelicum M szulgai TL 1 Crocidura olivieri BIO M angelicum M szulgai L lt 142 OU M szulgai QO 3k M angelicum M alsiensis M ri yadhense 16S rRNA gene 10096 99 5 99 1 HBA CL Tsukamurella WeIVEW CMe CMR CE CHS WH No 64 Tsukamurella H
11. 3 AM scrofulaceum M paras crofulaceum pyrosequencing Z Runyon JHO I M kansasii M gas tri E ILRI C 5 2 3 OQ W M scrofulaceum M conspicuum M parascrofulaceum 5 Score1 000 No 85 7 peregrinum pyrosequencing M mageritense 3 No 64 No 66 No 98 Runyon pyrose quencing Runyon No 64 TV AA CHE Tsukamurella sp PPK No 66 23 III H EBE C432 M szulgai L lt M an gelicum FARK No 98 I TH CHS
12. O H AB gt E Lr 2015 Pyrosequencing 26 5 13 26 9 8 pyrosequencing NHO 100 pyrosequencing 93 pyrosequencing 7 3 M avium complex 1 M parascrofulaceum 2 pyrosequencing 1 M
13. pyrosequencing FFF Py roMarkQ24 pyrosequencing 40 bp FASTA Ribosomal Database Project under Favor ites http rdp cmemsu edu Score 0 990 1 000 pyrosequencing 76 73 pyrosequencing pyrosequencing ZORRAC LT S oE M kansasii10 pyrosequencing Pyrosequencing 29 Table 2 Sequence comparisons of 24 unidentified strains by commercial identification kits Identified results Pyrosequencing Conventional 16S rRNA gene sequencing No of routine 16S rRNA gene BLAST results laboratory tests Identification Score Identification Homology 64 JV Tsukamurella pulmonis 1 000
14. AT WORE CHA 5982 M fortuitum pyrosequencing iki pyrosequencing Runyon
15. Tsukamurella pulmonis 100 Tsukamurella spongiae Tsukamurella sp INBio 3020F 66 III M szulgai M angelicum 1 000 M szulgai M angelicum 100 100 M alsiensis M riyadhense 99 43 99 24 98 H M colombiense M sp LC6 1 000 M colombiense CECT 3035 99 81 99 81 M intracellulare M bouchedurhonense M bouchedurhonense 87 H M scrofulaceum M conspicuum 1 000 M scrofulaceum 100 M paraffinicum M simiae 62 IV M mucogenicum M phocaicum 1 000 M mucogenicum 99 8 63 IV M abscessus M massiliense 1 000 M abscessus M massiliense 100 M bolletii M chelonae M bolletii M chelonae 91 II M interjectum 1 000 M interjectum 99 8 67 82 III 2 isolates M shimoidei M triviale 1 000 M shimoidei 99 81 99 81 Mycobacterium sp 1777 80 99 II 2 isolates M intermedium M sp Y2 1 000 M intermedium 100 86 100 II 13 isolates M lentiflavum M sp 1 000 M lentiflavum 100 Discrepancies between identification by routine laboratory and genetic identification by pyrosequencing and conven tional sequencing MOTT 16S Primer 1 for Sequencing M conspicuum M kansasii M gastri 3 Vif MOTT 16S Primer 2 for Sequencing Ye M kansasii M gasg7 M xenopi 3 M xenopi M heckeshornense M terrae 2 M terrae M kumamotoense WAGER RIO M apscessus 10 M chelonae5 pyrosequencin
16. and both sequencing results See table 2 for the detailed explanation PCR DNA PCR 192 bp PCR PyroMarkQ24 Sepharose PCR 7096 Denaturation Wash buffer 1 Annealing buffer Pyro MarkQ24 MOTT 16S Primer 1 For Se quencing M kansasii MOTT 16S Primer 2 For Sequencing M kansasii amp rikpi oo SS D E RA COM 28 VoL25 No l 2015
17. dhense sp nov a non tuberculous species identified as Mycobacterium tuberculosis complex by a commercial line probe assay Int J Syst Evol Microbiol 59 1049 1053 Weon H Y S H Yoo R Anandham et al 2010 Tsu kamurella soli sp nov isolated from soil Int J Syst Evol Microbiol 60 1667 1671 Yassin A F F A Rainey H Brzezinka et al 1996 Tsukamurella pulmonis sp nov Int J Syst Bacteriol 46 429 436 Perez V A J Swigris S J Ruoss 2008 Coexistence of primary adenocarcinoma of the lung and Tsuka murella infection a case report and review of the lit erature J Med Case Reports 2 207 Pyrosequencing 33 Novelidentification of clinical mycobacterium isolates by pyrosequencing of 16S ribosomal RNA gene Shiomi Yoshida Motohisa Tomita DClinical Research Center National Hospital Organization Kinki chuo Chest Medical Center Osaka Japan 2Clinical Laboratory National Hospital Organization Kinki chuo Chest Medical Center Osaka Japan Conventional methods for identification of mycobacterium isolates are often inexact and time consuming Pyro sequencing of a short hypervariable 16S rRNA gene fragment is accurate rapid and effective We have retrospec tively evaluated the discriminative power of pyrosequencing for mycobacterium identification and compared with the results of the routine laboratory tests and conventional 16S rRNA gene sequencing Sanger sequencing A s
18. eries of 100 clinical isolates were investigated furthermore growth rate and pigment production were anno tated when species could not be resolved by pyrosequencing alone 93 of the identified isolates by commercial kits and phenotypic characterization were unambiguously identified by both Sanger sequencing and pyrosequencing An additional 3 isolates were directly identified to same species by Sanger sequencing and routine test but the discrepancies with pyrosequencing 1 M avium complex 2 M parascrofulaceum By both sequencing methods one isolate was identified as M mageritense however identified as M peregrinum by routine test The remain ing 3 isolates needed both sequencings because the discrepancies between Runyon classification IV III and ID and both sequencings were resulted Tsukamurella sp M szulgai or M angelicum and M colombiense or M bouchedurhonense We consider the pyrosequencing procedure to be a useful alternative for the identification of several mycobacterium species and a versatile tool for the characterization of clinical mycobacterium isolates At times it requires additional tests for definite species diagnosis and correct identification VoL25 No l 2015 33
19. g M apscessus complex O i fii M abscessus M massiliense M bolletii M chelo nae Runyon 24 pyrose quencing 100 93 Table 1 Table 2 Runyon 24 pyrosequencing 7 Table 3 3 pyrosequencing 1 No 83 M avium complex M avium complex pyrosequencing M colombiense M avium M intracellulare 2 No 74 No 75
20. i 10 M absces sus 10 M chelonae 5 M fortuitum 4 M gordonae 3 M szulgai 3 M xenopi 2 M terrae 2 M scrofulaceum 2 M marinum 1 M avium complex 1 M peregrinum 2 24 Runyon JE II HA 18 II A 3 IV W 3 TB M avium M intracellulare M avium complex M kansasi M gordonae
21. ntracellulare 10 M intracellulare M kansasii 10 10 M conspicuum M kansasii M gastri 10 M kansasii M gastri Primer 1 M kansasii M gastri Primer 2 M abscessus 10 10 M abscessus M massiliense M bolletii 10 M abscessus M massiliense M chelonae M bolletii M chelonae M chelonae 5 5 M abscessus M massiliense M bolletii 5 M abscessus M massiliense M chelonae M bolletii M chelonae M fortuitum 4 4M fortuitum 4 M fortuitum M gordonae 3 3 M sordonae 3 M sordonae M szulgai 3 3 M szulgai M angelicum 3 M szulgai M angelicum M xenopi 3 3 M xenopi M heckeshornense 3 M xenopi M heckeshornense M terrae 2 2M terrae M kumanmotoense 2 M terrae M kumanmotoense M scrofulaceum 2 2 M scrofulaceum M conspicuum 2 M parascrofulaceum M parascrofulaceum M kansasii M gastri M marinum 1 1 M marinum 1 M marinum M avium complex 1 1 M colombiense M avium M intracellulare 1 M avium complex M peregrinum 1 1 M peregrinum 1 M peregrinum M peregrinum 1 1 M peregrinum M mageritense M poriferae 1 M mageritense Unidentified isolates 24 24 identified included 3 discrepancies 24 identified included 3 discrepancies Total 100 93 100 7 discrepancies 93 100 7 discrepancies dentified species by routine laboratory tests with commercial kits pigmentation and growth rate Discrepancies of isolate between phenotypic identification by routine laboratory tests
22. pere grinum pyrosequencing M mageritense 3 Runyon pyrosequencing iX IV A Tsuka murella sp HI W l4 M szulgai L lt lk M angelicum II M colombiense L lt M bouchedurhonense Pyrosequencing pyrosequencing Key words Pyrosequencing 16S rRNA x Deb b J E AS B cy OHS 2001 0 94 2011 3 3596 20 100
23. rureqoorq IOJ SIMSI Jued zosiq AQEL Vol 25 No 1 2015 H 30 Pyrosequencing 31 M colombiense M intracellulare UL lt t M bouchedurhonense No 66 3 L M szulgai 3 PNBA IC BIJA WE No 66 M szulgai3 2 1 795 E pyrosequencing 93 24 pyrosequencing pyrosequencing lt 40 bp 16S rRNA gene pyrosequenc ing
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