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Journal of Clinical Microbiology, November 2000, p. 4256-4259, Vol. 38, No. 11
Centre for Infectious Diseases and Microbiology, Institute
of Clinical Pathology and Medical Research, Westmead
Hospital,1 and National Centre for
Immunisation Research and Surveillance of Vaccine Preventable
Diseases, Royal Alexandra Hospital for
Children,2 Westmead, New South Wales 2145, Australia
Received 3 May 2000/Returned for modification 10 July 2000/Accepted 8 September 2000
We designed several new primers and modified previously described
species- and type-specific primers targeting the Mycoplasma pneumoniae P1 adhesin gene. Optimized thermal profiles allowed one-step or nested PCR to be completed in less than 1 h. In 10 patients with pneumonia, M. pneumoniae type 1 was
identified in 3 and type 2 in 7.
Mycoplasma pneumoniae is
a causative agent of tracheobronchitis and primary atypical pneumonia
in children (5) and one of the commonest causes of
community-acquired pneumonia in adults, ranging in severity from mild
to life-threatening (13, 25). M. pneumoniae also
causes extrapulmonary complications and infections, involving the heart
(16), central nervous system (3), and genitourinary tract (18).
Rapid confirmation of the diagnosis is important for clinical and
epidemiological reasons (14), but culture is slow,
technically demanding, and relatively insensitive (4, 18).
Currently, diagnosis of M. pneumoniae infection usually
relies on serology, which also has major limitations (4,
19). Recently, PCR has been accepted as a valuable method for
diagnosis of M. pneumoniae infections (1, 4).
M. pneumoniae can be separated into two types on the basis
of divergence of P1 gene sequence (21, 22, 23). PCR is the simplest and the most practical typing method (10). The
relationship between these types and virulence, epidemic activity,
reinfection, cross-protection or clinical severity, and complications
of M. pneumoniae infection requires further investigation
(10, 20).
In this study, we aimed to develop a faster and more practical PCR for
detection and typing of M. pneumoniae.
Reference strains used were M. pneumoniae M129 (ATCC 29342),
M. pneumoniae FH (ATCC 15531), and Mycoplasma
genitalium (ATCC 33530), which were purchased directly from the
American Type Culture Collection. Nasopharyngeal aspirates were
obtained from 176 children admitted to the hospital with pneumonia
during a 12-month period (April 1998 to March 1999). Specimens were
stored at The following reference sequences were used to improve and design new
primers: P1 adhesin gene sequences of M. pneumoniae type 1 (M129 [ATCC 29342]) (GenBank accession numbers M18639, M21519, and
M20916); P1 adhesin gene sequences of M. pneumoniae type 2 (TW 7-5 and FH [ATCC 15531]) sequences as previously described (21); and the MgPa adhesin gene sequence of M. genitalium (G-37 [ATCC 33530]) (GenBank accession number
M31431). The oligonucleotide primers used are listed in Table
1. The primer pairs used for initial
screening for M. pneumoniae in clinical specimens and for
optimizing thermal profiles in single-step and nested PCRs for
detection and typing of M. pneumoniae, as well as the
conditions used for PCRs, are shown in Tables
2 and 3.
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Copyright © 2000, American Society for Microbiology. All rights reserved.
Rapid-Cycle PCR for Detection and Typing of
Mycoplasma pneumoniae in Clinical Specimens
ABSTRACT
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70°C. Before processing, they were thawed and separated
into two portions, each about 200 to 500 µl. One portion (the
"original" specimen) was used directly for DNA extraction and PCR
examination, and the other was inoculated into 2 ml of SP4 broth
("broth-enhanced" specimen). After 24 h of incubation at
37°C, 500 µl of broth was used for DNA extraction. DNA was prepared
as previously described (6, 11, 26).
TABLE 1.
Oligonucleotide primers used in this study
TABLE 2.
Primer pairsa and conditions used
for one-step PCRs for the detection and typing of
M. pneumoniae
TABLE 3.
Primer pairs and conditions used for nested PCRs for the
detection and typing of M. pneumoniae
The 25-µl amplification reaction mixtures were used as previously described (11). For nested PCR, 1 to 5 µl of the first-step PCR product was used as the template in the second-step PCR reaction systems. All PCRs were performed in a Perkin-Elmer thermocycler 9600. Twelve microliters of PCR products was analyzed by electrophoresis on 2% agarose gels, which were stained with 0.5-µg/ml ethidium bromide. If all controls were satisfactory, a visible band of the appropriate size on UV translumination was accepted as evidence of the presence of M. pneumoniae DNA.
Unmodified primer pairs P4A-P4B (18) and MP-P11-MP-P12
(2) were used in parallel to screen all original and
broth-enhanced specimens for M. pneumoniae. The thermal
profiles used were as previously described (2, 18). M. pneumoniae DNA was detected in 10 of 176 (6%) specimens from
patients with pneumonia by using this single-round PCR
both specimen
types were positive in six cases, while original or broth-enhanced
specimens alone were positive in two cases each. Results were the same
with both sets of detection primers for all but one specimen, which was
positive only with primers P4A-P4B.
Nested PCR, including the use of type-specific inner primers, showed
that three specimens contained M. pneumoniae type 1, while
seven specimens contained type 2 P1 gene fragments. Amplicons (P1-40/MPAW2 or P1-40/MPAW1)containing highly heterologous regions (partial Rep MP4) of the P1 adhesin genefrom all M. pneumoniae-positive specimens were sequenced to confirm the PCR
typing results and the specificity of new type-specific primer pairs.
Sequences of all amplicons were identical with those of the
corresponding M. pneumoniae P1 adhesin gene sequences in
GenBank (M129, type 1) or published previously (TW 7-5 and FH, type 2)
(21).
It has been suggested that the times commonly used in PCR cycles are
often unnecessarily long, which may reduce the specificity and
sensitivity of reactions (8, 27). Rapid-cycle PCR can improve product specificity significantly (9). Newer types of thermocyclers have been used successfully for rapid-cycle PCR, with
total reaction times between 90 s and 20 min (7, 12, 17,
24). However, because conventional heat-block thermocyclers are
still most commonly used, our aim was to develop a faster PCR cycle
that could be used with this type of equipment (e.g., Perkin-Elmer
thermocycler 9600). To allow shorter ramp and/or incubation times and
increase the specificity, we modified the most commonly used primer
pairs targeting the P1 adhesin gene to increase their melting
temperatures (Tm) (
72°C), so that high annealing temperatures (70°C) could be used.
After clinical specimens containing M. pneumoniae (in one or both portions) had been identified by PCR as described above, both portions of those 10 specimens were used to optimize thermal profiles for the modified and new primers. The methods were adjusted to achieve sensitivities at least as high as those obtained by using unmodified primer pairs P4A-P4B (18) and MP-P11-MP-P12 (2), i.e., to give positive results in all known M. pneumoniae-positive specimens. This was achieved using denaturation and annealing and elongation at times and temperatures of 1 s at 96°C, 1 s at 70°C, and 1 to 10 s (depending on the amplicon length) at 70 to 74°C, respectively, in a Perkin-Elmer thermocycler 9600 (Tables 2 and 3).
With use of modified and new primers with optimized thermal profiles,
the one-step PCR could be finished within 40 min and the nested PCR
within 1 h (15). Thus, the whole procedureDNA preparation (<2 h), detection of M. pneumoniae in clinical
specimens (1 h), typing of M. pneumoniae for positive
specimens (1 h), and electrophoresis (<2 h for both detection and
typing)could be completed in less than one working day. We believe
that this is the fastest M. pneumoniae detection and typing
method reported so far. In future, more rapid DNA preparation and
automated amplicon detection systems (instead of traditional
electrophoresis) will allow PCR detection and typing time to be further reduced.
An algorithm was developed for clinical use of a rapid, nested PCR for
detection and typing of M. pneumoniae (Fig.
1). We used a single outer primer pair
(either P1-40-MPAW2 or P1-40-MPAW1) for the first round, followed by
species- and type-specific primers for the second round, as required.
The choice of primers was based on their sensitivity, band clarity, and
suitable amplicon size. Our typing results showed that contrary to
results in several other countries (20), type 2 was more
commonly implicated than type 1 in a group of Australian children with
pneumonia.
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ACKNOWLEDGMENTS |
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We thank Mark Wheeler for assistance with sequencing, Melanie Wong and Peter McIntyre for referring specimens and providing clinical data, Gregory James for helpful advice, and Zhengfang Ma for technical assistance and support.
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FOOTNOTES |
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* Corresponding author. Mailing address: Centre for Infectious Diseases and Microbiology, Institute of Clinical Pathology and Medical Research, Westmead Hospital, Darcy Rd., Westmead, New South Wales, 2145, Australia. Phone: (612) 9845 6255. Fax: (612) 9893 8659. E-mail: lyng{at}icpmr.wsahs.nsw.gov.au.
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Journal of Clinical Microbiology, November 2000, p. 4256-4259, Vol. 38, No. 11
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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