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Journal of Clinical Microbiology, June 2001, p. 2367-2368, Vol. 39, No. 6
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.6.2367-2368.2001
LETTERS TO THE EDITOR
Improved Primer Design for Multiplex PCR Analysis of
Vancomycin-Resistant Enterococcus spp.
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LETTER |
Kariyama and colleagues (5) recently described a
multiplex PCR method for the detection of the vanA,
vanB, vanC1, and vanC2/C3 genes, in
addition to primers for the detection of the ddl genes of
Enterococcus faecalis and E. faecium and of 16S
rRNA (PCR control). However, the vanB primers used by these
authors were derived from the sequence of the vanB1 subtype
(1) and are incapable of detecting vanB2 or
vanB3 subtypes, as previously described (2, 4,
7) and recently demonstrated in our laboratories.
Calgary Laboratory Services (CLS) recently implemented the PCR scheme
described by Kariyama et al. (5). Three enterococcal strains which displayed a vancomycin-resistant enterococci phenotype were negative for the van genes using this methodology even
though the ddlE. faecalis and 16S rRNA genes
were amplified. PCR using the primer set for vanB alone
yielded an amplicon of 1.1 kb for all three strains, which was larger
than the expected 433 bp. The isolates were subsequently referred to
the National Microbiology Laboratory (NML), Winnipeg, Manitoba, for
further analysis. The NML has implemented a multiplex assay which uses novel, unpublished primers for the detection of vanB genes,
the ddl genes of E. faecalis and E. faecium, and a published set of primers for the vanA
gene (3). Using this assay, all three strains produced the
E. faecalis-specific product and the vanB product. In order to explain the discordant results, the NML sequenced the 1.1-kb product produced using the Kariyama et al. (5)
primer set. Sequence analysis revealed that the reverse primer bound correctly. However, the forward primer did not appear to anneal at the
site 5' GTG ACA AAC CGG AGG CGA GGA 3' since the corresponding sequence
of the vanB2 gene was 5' GTG ACA AGC CGG AGG CGG GTG 3'.
There are four nucleotide changes, with three of them at the 3-prime
end, which likely results in no primer binding. This is predicted to
also occur with the vanB3 gene (5' GTG ACA AGC CGG AGA CGG
GTG 3'). Further sequence analysis revealed that the forward primer is
binding to the upstream vanH gene at the following site: 5'
GGA TGT GTT GGA GGG CGA GGA 3'. Note that the last 8 bp of this region
and the forward primer used by Kariyama et al. (5) are
identical. This primer binding would result in an amplicon of 1,086 bp,
which is consistent with the PCR results.
The NML currently uses in-house primers developed from consensus
vanB sequences (forward, 5' AAG CTA TGC AAG AAG CCA TG 3', and reverse, 5' CCG ACA ATC AAA TCA TCC TC 3') capable of detecting all
vanB subtypes, with an amplicon size of approximately 536 bp. The multiplex assay described by Kariyama and colleagues
(5) was easy to perform and reliably detected
vanA, vanC1, vanC2/vanC3, and species-specific genes for E. faecalis and E. faecium from other isolates tested at both CLS and NML. However,
if one were to substitute the vanB primers used by Kariyama
et al. (5) with those developed by NML, the multiplex
assay would be capable of detecting all vanB subtypes. Other
vanB consensus primers have been developed (2);
however, these primers cannot be used in the multiplex assay described
by Kariyama et al. (5) since the vanC2 or
vanC3 amplicon sizes would be identical.
In summary, the presence of vanB subtypes necessitates the
use of consensus PCR primers for rapid and reliable detection. While
the work was in progress Kawalec et al. (6) described similar results in a vanB2 E. faecium strain and determined
that mispriming within vanH was responsible, though they did
not precisely determine the anomalous primer binding site.
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FOOTNOTES |
*
Phone: (403) 209-4675
Fax: (403) 209-5378
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2000.
Simple and reliable multiplex PCR assay for surveillance isolates of vancomycin-resistant enterococci.
J. Clin. Microbiol.
38:3092-3095[Abstract/Free Full Text].
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Kawalec, M.,
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39:811-815[Abstract/Free Full Text].
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Patel, R.,
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DNA sequence variation within vanA, vanB, vanC-1, and vanC-2/3 genes of clinical Enterococcus isolates.
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42:202-205[Abstract/Free Full Text].
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| | | | |
Sameer Elsayed*
Nina Hamilton
Calgary Laboratory Services
1638 10th Ave. SW
Calgary, Alberta, Canada T3C 0J5
|
| | | | |
David Boyd
Michael Mulvey
National Microbiology Laboratory
1015 Arlington St.
Winnipeg, Manitoba, Canada R3E 3R2
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| |
AUTHORS' REPLY |
We thank Elsayed and colleagues for their implementation of our
multiplex PCR scheme (2) and the primer design for
consensus vanB sequences. We have one comment for their
letter. The first sentence written by Elsayed and colleagues does not
correctly describe our primers. The sentence "in addition to primers
for the detection of the ddl genes of E. faecalis
and E. faecium" should be changed to "in addition to
primers for species-specific genes of E. faecalis and
E. faecium." In our article (2), the primer
set for E. faecalis amplifies the ddl gene, but
the primer set for E. faecium does not amplify the
ddl gene (1).
We performed the multiplex PCR assay (2) by using a novel
combination of primer sets which included a primer set for consensus vanB sequences (referred to here as vanB
consensus primer set) designed by Elsayed and colleagues. We found that
E. faecalis-specific amplification was inhibited by a novel
primer combination with the vanB consensus primer set if we
used the same primer concentrations as previously described
(2). Then, we optimized the vanB consensus primer concentration, which was found to be one-half of the
concentration previously described (2). As shown in Fig.
1, the best result was observed when the
novel primer combination was added to the reaction mixtures at 5 pmol
each of vanA, E. faecalis-specific primers; 2.5 pmol each of the vanC1, vanC2/C3,
rrs primers; and 1.25 pmol each of the vanB,
E. faecium-specific primers.
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FIG. 1.
Gel image generated by the Agilent 2100 bioanalyzer of
amplified vanA, vanB, vanC1,
vanC2/C3, E. faecalis-specific, E. faecium-specific, and rrs genes by the optimized
multiplex PCR assay (2) containing a novel primer
combination as described above. Lanes: M, DNA ladder; 1, an E. faecalis vanA isolate; 2, an E. faecalis vanA isolate;
3, an E. faecalis vanB isolate; 4, an E. faecalis
vanB isolate; 5, an E. faecium vanA isolate; 6, an
E. faecium vanA isolate; 7, an E. faecium vanB
isolate; 8, an E. faecium vanB isolate; 9, an E. gallinarum vanC1 isolate; 10, an E. gallinarum vanC1
and vanA isolate; 11, an E. gallinarum vanC1 and
vanB isolate; 12, an E. casseliflavus or E. flavescens vanC2 or vanC3 isolate.
|
|
Using the novel primer combination, vancomycin-resistant enterococci
(low- to high-level resistance) from our collection were tested. For
all isolates, PCR products with two or three intense bands on agarose
gels were generated as shown previously (2). Of 139 isolates tested, 14 E. faecalis (vanA) isolates,
29 E. faecalis (vanB) isolates, 42 E. faecium (vanA) isolates, 9 E. faecium
(vanB) isolates, 24 E. gallinarum
(vanC1) isolates, 2 E. gallinarum
(vanC1 and vanA) isolates, 2 E. gallinarum (vanC1 and vanB) isolates, and 17 E. casseliflavus or E. flavescens
(vanC2 or vanC3) isolates were clearly
identified. The results indicated that we missed four isolates
possessing a vanB2 or vanB3 gene previously.
Figure 1 shows a gel image of PCR products obtained by the Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, Calif.) which utilizes
chip-based nucleic acid separation technology (3). The
LabChip7500 (Caliper Technologies, Mountain View, Calif.) was used to
analyze PCR products. This instrument improves DNA analysis compared
with agarose gel electrophoresis and is suitable for routine
laboratories (3).
We appreciate Elsayed and colleagues, who designed the improved primer
for consensus vanB sequences and gave us an opportunity to
show new data obtained by the Agilent 2100 bioanalyzer. In summary, the
novel primer combination using the optimized primer concentration
described above is more reliable for the detection of
vancomycin-resistant enterococci, since this combination detects all
vanB subtypes.
| |
REFERENCES |
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Cheng, S.,
F. K. McCleskey,
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1997.
A PCR assay for identification of Enterococcus faecium.
J. Clin. Microbiol.
35:1248-1250[Abstract].
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| 2.
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Kariyama, R.,
R. Mitsuhata,
J. W. Chow,
D. B. Clewell, and H. Kumon.
2000.
Simple and reliable multiplex PCR assay for surveillance isolates of vancomycin-resistant enterococci.
J. Clin. Microbiol.
38:3092-3095.
|
| 3.
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Nachamkin, I.,
N. J. Panaro,
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Agilent 2100 bioanalyzer for restriction fragment length polymorphism analysis of the Campylobacter jejuni flagellin gene.
J. Clin. Microbiol.
39:754-757[Abstract/Free Full Text].
|
| | | | |
Reiko Kariyama
Ritsuko Mitsuhata
Hiromi Kumon
Section of Infection Control
Okayama University Hospital and
Department of Urology
Okayama University Medical School
2-5-1 Shikata
Okayama 700-8558, Japan
|
Journal of Clinical Microbiology, June 2001, p. 2367-2368, Vol. 39, No. 6
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.6.2367-2368.2001
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