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Fig. 1.
16S rRNA primer (primer A) P1 (primer B) 3
Fig. 2.
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Table 1.
*/
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
M. pneumoniae ‡ˆ*/ 15 */bc DNA 89op!qrc CP >$ TM >!?@
TM >
CP >
stuvw
5,120 ‰
640 ‰
y40 ‰Š1,280 ‰
2,560 ‰
80 ‰Š10,240 ‰
640 ‰
640 ‰
320 ‰
320 ‰
5,120 ‰
160 ‰
1,280 ‰
ND
5,120 ‰
ND
DNA 89<
DNA 89G
DNA 89<
DNA 89G
34.45
30.56
33.44
31.13
32.22
34.60
33.91
30.38
30.67
30.84
28.74
33.32
35.00
31.02
34.51
x35.00
29.64
31.80
30.25
32.91
30.87
34.73
32.42
29.72
29.72
26.76
31.95
31.98
28.82
31.58
84.90
84.78
84.85
84.65
84.99
85.42
84.95
84.69
84.94
84.96
84.79
84.95
85.34
84.84
85.03
85.07
84.65
84.61
84.59
85.16
84.66
84.84
84.69
84.82
84.75
84.68
84.84
84.80
84.67
84.72
CP >z crossing point{ |>$}~!€{
TM >z melting temperature{ }~‚ƒ!„…†{
Table 2.
M. pneumoniae Ka!)**/ 10 */bc DNA 89op!qrc CP >$ TM >?@
*/
stuvw
P
Q
R
S
T
U
V
W
X
Y
ND
y40
80
ND
ND
y40
y40
y40
y40
y40
CP >
TM >
DNA 89<
DNA 89G
DNA 89<
DNA 89G
x35.00
x35.00
x35.00
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x35.00
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x35.00
x35.00
x35.00
x35.00
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x35.00
77.26
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76.52
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CP >z crossing point{ |>$}~!€{
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15 */!0123456+ QIAamp DNA Mini
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Kit +7 DNA 89:;$ DNA 89+<
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10 */bc CP >$ TM >!
=:;! CP >$ TM >+?@AB+ Table
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DNA 89< 10 */k 8 */ 35 cy-
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30 &'ƒ‘’“ Vol. 19
No. 3
2009.
¡¢>H£… Mycoplasma pneumoniae Real-Time PCR @A
Table 3.
PCR CP ”•–—c˜™š
1 mg/ml
10 mg/ml
CEZ
CAM
MINO
LVFX
ST
VCM
25.96
24.62
25.51
25.63
24.55
24.79
25.46
23.68
24.45
24.78
23.93
24.34
25.20
153
(4) PCR PCR Table 3 PCR Table 4 CP CP !"#! 1 mg/ml "$ 10 mg/ml %
CP &'()*+ ,- ./01 2345+! CP 36
7
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M. pneumoniae
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PCR 2
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24.04
24.17
26.26
29.77
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Miyashita, N., H. Fukano, K. Mouri, et al. 2005.
Community-acquired pneumonia in Japan: A
prospective ambulatory and hospitalized patient study. J. Med. Microbiol. 54: 395῍400.
;< = >?@A BCDE FG 2006G HI
JK.LMNOPQRSMTQ>UVWXY
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a>bc ;< = def+ FG 2006G gh
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qrst uvw@A xy?z FG 2006G {
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65: 280῍286.
;< =G 2006G ghijklgJKSMM
NG mn 40: 223῍225.
Takiguchi, Y., N. Shikama, N. Aotsuka, et al.
2001. Fulminant Mycoplasma pneumoniae
pneumonia. Internal. Medicine 40: 345῍348.
Chan, E. D., C. H. Welsh. 1995. Eulminant Mycoplasma pneumoniae pneumonia. West. J. Med.
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Z#[\]^[\]ŽM o/h…kh
‘’“”^G 2007G ’•HIJK4nh…k
hG Z#[\]^ –—G
Kessler, H. H., D. E. Dodge, K. Pierer, et al. 1997.
11)
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Rapid detection of Mycoplasma pneumoniae by
an assay based on PCR and probe hybridization in a nonradioactive microwell plate format. J. Clin. Microbiol. 35: 1592῍1594.
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real-time PCR, real-time nucleic acid sequencebased amplification, conventional PCR, and serology for diagnosis of Mycoplasma pneumoniae. J. Clin. Microbiol. 41: 4366῍4371.
˜™ š ›œ  žy Ÿ FG 2007G g
hijklgŽ ¡45$¢£¤G Z#
[\]^` 45: 936῍942.
Raggam, R. B., E. Leitner, J. Berg, et al. 2005.
Single-run, parallel detection of DNA from
three pneumonia-producing bacteria by realtime polymerase chain reaction. J. Mol. Diagn.
7: 133῍138.
¥¦§¨© ªe « xy¬+ FG 2003. Mycoplasma pneumoniae &'­,®Š
PCRῌ¯q[\]ŽM°,±²³ῌG Z#´
n$^_` 51: 289῍299.
Loens, K., T. Beck, D. Ursi, et al. 2008. Development of real-time multiplex nucleic acid sequence-based amplification for detection of
Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. in respiratory specimens. J. Clin. Microbiol. 46: 185῍191.
Saito, R., Y. Misawa, K. Moriya, et al. 2005.
Development and evaluation of a loopmediated isothermal amplification assay for
rapid detection of Mycoplasma pneumoniae. J.
Med. Microbiol. 54: 1037῍1041.
Construction of Inspection System for M. pneumoniae Rapid Detection
Method in Outpatients, Using Real-Time PCR Method
Akihiro Nakamura, Noriyuki Abe, Hisashi Kono, Yoshiko Fujimoto, Saori Fukuda,
Noriko Hatanaka, Syuji Matsuo
Department of Clinical Bacteriology, Clinical Pathology, Tenri Hospital
Atypical pneumonia pathogens hold about 10 to 30µ among community-acquired pneumonia pathogens in Japan. The quick and accurate detection of atypical pneumonia pathogens, such as M. pneumoniae,
is di$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,
Z#·¸VWX_` Vol. 19
No. 3
2009. 33
156
which is equivalent to about 102ῌ103 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. 3
2009.