Real-Time PCR Mycoplasma pneumoniae

Transcript

1 Real-Time PCR Mycoplasma pneumoniae Mycoplasma pneumoniae real-time PCR M. pneumoniae 3 SYBR Green I Light Cycler 2.0 system (Roche) PCR 45 PCR primer M. pneumoniae 16S rrna 3.09 fg/ml M. pneumoniae DNA M. pneumoniae 15 DNA crossing point values DNA PCR Key words: real-time PCR, Mycoplasma pneumoniae, 16S rrna gene Mycoplasma pneumoniae ) M. pneumoniae 2) M. pneumoniae ( ) 200 TEL: FAX: nakamurium a802@yahoo.co.jp 3 7) M. pneumoniae Streptococcus pneumoniae Haemophilus influenzae M. pneumoniae Chlamydia pneumoniae M. pneumoniae C. pneumoniae b- 26 Vol. 19 No

2 Mycoplasma pneumoniae Real-Time PCR 149 M. pneumoniae ) M. pneumoniae M. pneumoniae PCR 45 (1) Roche Real-time PCR Light Cycler 2.0 system LightCycler FastStart DNA Master SYBR Green I FastStart Taq DNA Polymerase, reaction bu#er, dntp mix, SYBR Green I 10 mm MgCl2 SYBR Green I (2) Primer 16S rrna primer 9) forward primer 5 cgccagcttgtaaaagtgagc-3 reverse primer 5 tagcaacacgtttttaaatattacc-3 primer A P1 primer 10) forward primer 5 -attcccgaacaaaataatga-3 reverse primer 5 -gtttgacaaagtc-cgtgaag-3 primer B 2 primer primer dimer primer DNA primer A 190 bp, primer B 151 bp (3) PCR PCR PCR 20 ml DW 14.0 ml, MgCl ml 3.0 mm Forward-primer 0.2 ml 0.5 mm Reverse-primer 0.2 ml 0.5 mm LightCycler FastStart DNA Master SYBR Green I2.0 ml sample 2.0 ml 20 ml PCR primer dimer Touch down PCR 95 7 min Hot start 95 1s s (step size 0.5 /cycle) 72 8 s 1 cycle 14 s 40 cycle s (step size 0.2 /cycle) s PCR 28 min (4) DNA DNA 500 ml 1 12,000 G 15 s 200 ml sample 15 s, 30 s, 1 min, 2 min 2min DNA sample M. pneumoniae PA M. pneumoniae 15 QIAamp DNA Mini Kit (QIAGEN) DNA sample sample PCR crossing point values CP M. pneumoniae 10 (5) PCR DNA PCR DNA 3 direct DNA sequence (6) PCR DNA PCR CEZ, CAM, MINO, LVFX, ST VCM 1 mg/ml 10 mg/ml 500 ml MacF0.5 M. pneumoniae 1 ml, 5 ml 10 ml 500 ml Vol. 19 No

3 150 Fig S rrna primer (primer A) P1 (primer B) 3 Fig S rrna primer (primer A) P1 primer (primer B) 28 Vol. 19 No

4 Mycoplasma pneumoniae Real-Time PCR 151 Fig. 3. PCR 12,000 G 2 min 12,000 G 15 s 12,000 G 30 s 12,000 G 1 min 12,000 G 2 min (1) Primer M. pneumoniae DNA 2 primer primer PCR Fig. 1 primer A TM 84.8 primer B TM 88.5 primer primer A primer B primer dimer 2 primer 3 Fig. 1 primer primer dimer M. pneumoniae DNA 2 primer RNA MS2 (Roche) DNA PCR Fig. 2 primer A 3.09 fg/ml primer B 30.9 fg/ml PCR primer 16S rrna primer A (2) PCR DNA Primer A PCR DNA 3 direct DNA sequence M. pneumoniae 16S rrna (3) DNA PCR Fig. 3 Vol. 19 No

5 152 Table 1. M. pneumoniae 15 DNA CP TM CP TM DNA DNA DNA DNA A 5, B C 40 1, D 2, E 80 10, F G H I J 5, K L 1, M ND N 5, O ND CP crossing point TM melting temperature Table 2. M. pneumoniae 10 DNA CP TM CP TM DNA DNA DNA DNA P ND Q R S ND T ND U 40 V W X Y CP crossing point TM melting temperature 12,000 G 15 s M. pneumoniae 15 QIAamp DNA Mini Kit DNA DNA CP TM Table 1 DNA CP A, E, G H 4 11 DNA CP DNA DNA CP 3.00 F 3.73 M 3.02 DNA CP TM M. pneumoniae 10 CP TM Table 2 DNA DNA cycle TM Vol. 19 No

6 Mycoplasma pneumoniae Real-Time PCR 153 Table 3. PCR CP 1 mg/ml 10 mg/ml CEZ CAM MINO LVFX ST VCM Table 4. PCR CP ml ml ml (4) PCR PCR Table 3 PCR Table 4 CP CP 1 mg/ml 10 mg/ml CP CP (5) M. pneumoniae M. pneumoniae Fig. 4 M. pneumoniae PCR Fig. 4. Mycoplasma Vol. 19 No

7 154 Di#-Quik (SYSMEX) PCR PCR LightCycler PCR 45 M. pneumoniae 11) real-time PCR LAMP NASBA M. pneumoniae 12 16) real-time PCR M. pneumoniae real-time PCR LightCycler PCR PCR 45 real-time PCR SYBR Green I, Hybridization Probe, Simple Probe TaqMan Probe PCR SYBR Green I SYBR Green I primer M. pneumoniae 16S rrna 3.09 fg/ml M. pneumoniae DNA 12,000 G 2 min sample 12,000 G 15 s M. pneumoniae 15 DNA 3 DNA CP DNA M. pneumoniae 10 DNA 35 cycle TM DNA PCR DNA PCR M. pneumoniae positive control 8) PCR LAMP NASBA M. pneumoniae C. pneumoniae 1 Multiplex PCR 32 Vol. 19 No

8 Mycoplasma pneumoniae Real-Time PCR 155 1) Miyashita, N., H. Fukano, K. Mouri, et al Community-acquired pneumonia in Japan: A prospective ambulatory and hospitalized patient study. J. Med. Microbiol. 54: ) : ) : ) : ) : ) Takiguchi, Y., N. Shikama, N. Aotsuka, et al Fulminant Mycoplasma pneumoniae pneumonia. Internal. Medicine 40: ) Chan, E. D., C. H. Welsh Eulminant Mycoplasma pneumoniae pneumonia. West. J. Med. 162: ) ) Kessler, H. H., D. E. Dodge, K. Pierer, et al Rapid detection of Mycoplasma pneumoniae by an assay based on PCR and probe hybridization in a nonradioactive microwell plate format. J. Clin. Microbiol. 35: ) Templeton, K. E., S. A. Scheltinga, A. W. Gra#elman, et al Comparison and evaluation of real-time PCR, real-time nucleic acid sequencebased amplification, conventional PCR, and serology for diagnosis of Mycoplasma pneumoniae. J. Clin. Microbiol. 41: ) : ) Raggam, R. B., E. Leitner, J. Berg, et al Single-run, parallel detection of DNA from three pneumonia-producing bacteria by realtime polymerase chain reaction. J. Mol. Diagn. 7: ) Mycoplasma pneumoniae PCR 51: ) Loens, K., T. Beck, D. Ursi, et al Development of real-time multiplex nucleic acid sequence-based amplification for detection of Mycoplasma pneumoniae, Chlamydophila pneumoniae,and Legionella spp. in respiratory specimens. J. Clin. Microbiol. 46: ) Saito, R., Y. Misawa, K. Moriya, et al Development and evaluation of a loopmediated isothermal amplification assay for rapid detection of Mycoplasma pneumoniae. J. Med. Microbiol. 54: Construction of Inspection System for M. pneumoniae Rapid Detection Method in Outpatients, Using Real-Time PCR Method Akihiro Nakamura, Noriyuki Abe, Hisashi Kono, Yoshiko Fujimoto, Saori Fukuda, Noriko Hatanaka, Syuji Matsuo Department of Clinical Bacteriology, Clinical Pathology, Tenri Hospital Atypical pneumonia pathogens hold about 10 to 30 among community-acquired pneumonia pathogens in Japan. The quick and accurate detection of atypical pneumonia pathogens, such as M. pneumoniae, is di$cult by the routine outpatient-based laboratory inspections. We established the M. pneumoniae rapid detection method in outpatients, using real-time PCR method. For the purpose of establishing the inspection methods, we focused on three points as follows. First of all we used SYBR Green I as a fluorescence detection format and 2.0 Light Cycler system, which enabled us to make up a 45-minutes rapid detection method from specimen presentation to report of results. The primer on M. pneumoniae 16S rrna gene was used for this PCR method, and sensitivity for M. pneumoniae in the minimum was showed to be 3.09 fg/ml, Vol. 19 No

9 156 which is equivalent to about copies in number. It seems that we could be able to su$ciently cope with an acute phase of M. pneumoniae pneumonia, although in which period numbers of bacteria could be abundant. Next, we tried to omit the DNA extraction used in common genetic diagnostic examinations. We compared crossing point values between two situations, one is performing DNA extraction and the other is not, in 15 cases diagnosed as M. pneumoniae pneumonia. As a result, the PCR sensitivity did not show any estrangement in both methods. Finally we examined about PCR inhibitors. Antibacterial agents did not influenced the PCR process, but blood did lower the sensitivity of the PCR. We believe that introduction of this genetic methodology into routine bacteriological examinations, such as Gram staining, bacteriological culture tests and serological examination, can contribute to quicker and accurate bacteriological diagnosis. 34 Vol. 19 No