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Articles |
1
Centre de Recherche en Infectiologie de l’Université Laval, Ste-Foy, Québec, Canada, G1V 4G2.
2
Division de Microbiologie, Faculté de Medicine
and
3
Département de Biochimie, Faculté des
Sciences et de Génie, Université Laval, Ste-Foy,
Québec, Canada, G1K 7P4.
a Address correspondence to this author at: Centre de Recherche en Infectiologie de l’Université Laval, 2705 Boul. Laurier, Ste-Foy, Québec, Canada, G1V 4G2. Fax 418-654-2715.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: We have developed a PCR-based assay for the rapid detection of GBS. The cfb gene encoding the Christie-Atkins-Munch-Petersen (CAMP) factor was selected as the genetic target for the assay. The PCR primers were initially tested by a conventional PCR method followed by gel electrophoresis. The assay was then adapted for use with the LightCyclerTM. For this purpose, two fluorogenic adjacent hybridization probes complementary to the GBS-specific amplicon were designed and tested. In addition, a rapid sample-processing protocol was evaluated by colony-forming unit counting and PCR. A total of 15 vaginal samples were tested by both standard culture method and the two PCR assays.
Results: The conventional PCR assay was specific because it amplified only GBS DNA among 125 bacterial and fungal species tested, and was able to detect all 162 GBS isolates from various geographical areas. This PCR assay allowed detection of as few as one genome copy of GBS. The real-time PCR assay was comparable to conventional PCR assay in terms of sensitivity and specificity, but it was more rapid, requiring only ~30 min for amplification and computer-based data analysis. The presence of vaginal specimens had no detrimental effect on the sensitivity of the PCR with the sample preparation protocol used. All four GBS-positive samples identified by the standard culture method were detected by the two PCR assays.
Conclusion: These assays provide promising tools for the rapid detection and identification of GBS.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rapid tests have been developed, such as the rapid antigen-based tests, but these tests are neither sensitive nor specific enough to substitute for bacterial culture (6)(7)(8)(9). Hybridization-based methods have been used successfully for the detection of GBS from broth cultures (10)(11)(12), but they remain insufficiently sensitive for the detection and identification of GBS directly from clinical specimens of colonized women (10)(11). GBS-specific PCR-based assays have demonstrated better sensitivity, but they require complicated procedures that are not applicable to clinical use (13)(14)(15).
A real-time amplification-detection apparatus with air thermal cycling and fluorescently monitored product analysis (LightCyclerTM) in a closed-tube assay format recently has been developed (16)(17)(18). This new technology is particularly attractive because it is able to avoid carryover and requires ~30 min for completion of a 45-cycle PCR.
GBS can be presumptively identified by the Christie-Atkins-Munch-Petersen (CAMP) test, based on detection of a diffusible extracellular protein (CAMP factor) produced by the majority of GBS (19). The cfb gene encoding the CAMP factor is present in virtually every GBS isolate and is an obvious candidate for the development of a PCR assay for identification of GBS (20). In this study, a pair of GBS-specific PCR amplification primers were designed from the cfb gene and initially evaluated by conventional PCR using agarose gel electrophoresis. Subsequently, the assay was adapted for use with the LightCycler, which allowed for shorter running time and real-time detection of amplicons by using fluorescence measurements. These assays were shown to be specific and highly sensitive for the detection and identification of GBS.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A wide variety of gram-positive and gram-negative bacterial strains as
well as two fungal species obtained from the ATCC were used to test the
specificity of the PCR assays (Table 1).
All strains were grown on media under conditions that support optimal growth.
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DNA isolation
Genomic DNA from all strains tested was obtained using the G NOME
kit (Bio101) with modification. A RNase pretreatment was added before
quantification of genomic DNA. The concentrations of DNA preparations
were calculated by measuring the absorbance at 260 nm or by comparison
with DNA calibrators after agarose gel electrophoresis.
oligonucleotides
The cfb gene sequences available in GenBank
[Streptococcus agalactiae (X72754), S. uberis
(U34322), and S. pyogenes (AF079502)] were aligned with the
GCG package (Ver. 9.0; Genetics Computer Group) to compare the homology
of these cfb sequences. The cfb sequences from
two GBS strains described by Podbielski et al. (20) were
also used to identify regions conserved in GBS only. PCR primers (Table 2
) complementary to these conserved regions were analyzed using the Oligo
primer analysis software (Ver. 5.0; National Biosciences). Primers were
synthesized with a model 391 DNA synthesizer (Perkin-Elmer).
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Two pairs of fluorescently labeled adjacent hybridization probes
(STB-F/STB-C hybridizing to GBS-specific amplicons and IC-F/IC-C
hybridizing to the internal control amplicon) were synthesized and
HPLC-purified by Operon Technologies (Table 2
) and were designed to
meet the recommendations of the manufacturer (Idaho Technology)
(16). These adjacent probes, which are separated by one
nucleotide, allow fluorescence resonance energy transfer to generate an
increased fluorescence signal when hybridizing to their target
sequences. The probes STB-F and IC-F were labeled with fluorescein, and
STB-C and IC-C were labeled with Cy5TM (Amersham
Pharmacia Biotech). The Cy5-labeled probes contained a 3'-blocking
phosphate group to prevent extension of the probes during the PCR
reactions.
construction of the internal control
An internal control was constructed essentially as described
previously by Rosenstraus et al. (21). A 252-bp DNA fragment
consisting of a 206-bp sequence not found in GBS flanked by the
sequences of each of the two GBS-specific primers was used as a
template for the internal control. This fragment was cloned into the
pCR2.1 vector (Invitrogen). The recombinant plasmid, named pSTB, was
isolated from transformed Escherichia coli by the Qiagen
plasmid mini kit (Qiagen). The purified plasmid was then linearized
with EcoRI (New England Biolabs) and serially diluted. The
concentration of the linearized plasmid was optimized to permit
amplification of the 252-bp internal control product without
significant detrimental effect on the GBS-specific amplification.
PCR amplification
Relevant characteristics and optimal PCR conditions for the
GBS-specific conventional and real-time PCR assays are given in Table 3
. Strict precautions to prevent carryover of amplified DNA were
used (22). Pre- and post-PCR manipulations were conducted in
separate areas. Aerosol-resistant pipette tips were used to handle all
reagents and samples. Control reactions to which no DNA was added were
performed routinely to verify the absence of DNA carryover.
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Concomitant amplification of the internal control allowed verification of the efficiency of the PCR to ensure that there was no significant PCR inhibition by the test sample. For conventional PCR, the internal control was amplified simultaneously with the GBS genomic target. On the other hand, for real-time PCR, the control was amplified in a separate reaction vessel because only two fluorescent signals can be monitored in the same capillary with the LightCycler model used.
specificity and sensitivity tests
The specificity of the conventional PCR assay was verified using
purified genomic DNA (0.1 ng/reaction) from a battery of ATCC reference
strains representing 105 aerobic and 18 anaerobic bacterial species as
well as 2 fungal species (Table 1
). These microbial species included 28
species of streptococci and many members of the typical vaginal and
anal flora. The specificity of the real-time PCR assay was verified by
testing genomic DNA from bacterial species that are phylogenetically
close to GBS, including members of the genera Streptococcus,
Lactococcus, Enterococcus, Abiotrophia,
Peptostreptococcus, and Listeria. Some species
encountered in the typical vaginal flora were also tested (Table 1
). A
total of 162 clinical isolates of GBS from various origins were also
tested to further validate the GBS-specific conventional PCR assay by
performing amplifications from standardized bacterial suspensions
before adapting the assay to the LightCycler platform.
The detection limit (i.e., minimal number of genome copies that can be detected) of the PCR assays was determined by serial twofold dilutions of purified genomic DNA from five GBS ATCC strains. To evaluate the efficiency of the IDI DNA extraction kit (Infectio Diagnostics Inc.) to prevent PCR inhibition, three GBS-negative vaginal samples prepared for PCR using the IDI extraction kit were supplemented with various amounts of purified GBS genomic DNA (equivalent of 1–40 genome copies).
evaluation of sample preparation for pcr
The efficacy of the IDI DNA extraction kit to lyse GBS cells was
evaluated by comparing the minimal number of colony-forming units
(CFUs) detected with the preparations without pretreatment to those
prepared by using the IDI kit. The detection limits in CFUs were
determined using cultures of three GBS strains (ATCC 13813, 12400, and
27591) in the logarithmic phase of growth (absorbance at 600
nm, 
0.6) diluted 10-fold in phosphate-buffered saline. Each
10-fold dilution in phosphate-buffered saline was either added directly
to the PCR reaction mixture or processed using the IDI DNA extraction
kit before PCR amplification. The number of CFUs was estimated by
standard plating procedures.
clinical specimens and gbs-selective culture and pcr
Vaginal specimens were collected from 15 consenting pregnant women
admitted for delivery at the Centre Hospitalier Universitaire de
Québec, Pavillon Saint-François d’Assise, using a
polyurethane-tipped swab (CulturetteTM; Beckon
Dickinson) after excessive discharge was removed. The samples were
obtained either before or after rupture of amniotic membranes. Ampoules
containing 0.5 mL of Stuart’s bacterial transport medium were crushed
in the swab immediately after collection of samples. The swabs were
transported at ambient temperature to the Centre de Recherche en
Infectiologie de l’Université Laval and were tested within
24 h of collection. Upon receipt, the swabs were immersed in tubes
prefilled with 1 mL of selective Todd-Hewitt broth containing nalidixic
acid (15 mg/L) and gentamicin (8 mg/L; GNS broth) for at least
3 min. Approximately 0.5 mL of the specimen suspension and the tip of
the swab were added to GNS broth for identification of GBS by the
standard culture method recommended by the CDC (3). Vaginal
swabs were prepared for PCR amplification by using the IDI DNA
extraction kit.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) were chosen from GBS
unique regions selected from a multiple sequence alignment of the
cfb genes from GBS, S. uberis, and S.
pyogenes (data not shown). Sequence comparison of streptococcal
cfb genes showed that these three genes were fairly
divergent, with nucleotide identities ranging from 60.8% to 66.7%,
hence facilitating the design of specific oligonucleotides.
evaluation of the gbs-specific conventional pcr assay
The specificity of the assay was assessed using purified genomic
DNAs from the panel of gram-positive and gram-negative bacterial
species as well as fungal species listed in Table 1
. This assay was
specific because only DNAs from GBS strains could be amplified (Fig. 1
). Many members of the vaginal or anal flora tested
(23), including E. coli, Candida
albicans, Gardnerella vaginalis, enterococci,
coagulase-negative staphylococci, Lactobacillus spp.,
Peptostreptococcus spp., and Bacteriodes spp.
were not amplified by the GBS-specific PCR assay. Moreover, the PCR
assay was able to efficiently detect all 162 GBS strains used in this
study, including reference ATCC strains as well as clinical isolates of
both human and bovine origins, thereby showing a perfect correlation
with standard culture-based identification methods.
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The detection limit of the assay was determined using purified genomic DNA from the five ATCC strains of GBS. The detection limit for all five ATCC strains was one genome copy of GBS. When we used the IDI extraction kit, as few as one genome copy of GBS was also detected from all three GBS-negative vaginal samples to which genomic DNA of GBS had been added. In terms of CFUs, the PCR assay was able to detect 1–3 CFUs from mid-log phase cultures when the IDI DNA extraction kit was used compared with 10–24 CFUs with diluted cultures added directly to the PCR mixture without pretreatment. These results confirmed the high sensitivity of our GBS-specific PCR assay as well as the efficacy of the IDI kit for lysis of GBS cells and prevention of significant PCR inhibition.
During PCR amplification, the internal control template (i.e.,
linearized pSTB) integrated into all PCR reactions allowed verification
of the efficiency of all amplifications. The 252-bp PCR product for the
internal control was amplified by the GBS-specific primers. Thus, the
GBS-specific primer pair could amplify both the target genomic sequence
in GBS and the internal control template. This strategy allows
validation of the amplification primers, simplification of the assay,
and prevention of potential detrimental competition between different
PCR primer pairs. As expected, when GBS DNA was absent, the internal
control was always amplified efficiently (Fig. 1
). When GBS DNA was present, amplification of the
internal control was either lower or absent because of competitive
inhibition by amplification of the GBS genomic target. It is critical
that there be no significant competitive inhibition from the internal
control template to minimize a decrease in the sensitivity of the
assay.
evaluation of the gbs-specific real-time pcr assay
The specificity of the real-time PCR assay was also verified using
a battery of bacterial species, including streptococci (28 species),
enterococci, lactococci, and Peptostreptococcus spp., as
well as members of typical vaginal and anal flora (Table 1
). Only GBS
could be detected by the production of an increased fluorescence signal
that was interpreted as a positive PCR result. The fluorescence
resonance energy transfer signal for the internal control performed in
a separate capillary was detected for all PCR reactions, thereby
showing the absence of significant PCR inhibition. As with the
conventional PCR assay, the internal control signal progressively
decreased as the amount of GBS target DNA increased (data not shown).
The real-time PCR assay showed the same sensitivity as the conventional
PCR assay described above (Fig. 2
).
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identification of gbs colonization in pregnant women
Among 15 vaginal samples obtained from pregnant women at delivery,
4 were positive for GBS, whereas the other 11 samples were negative for
GBS as determined by both the standard culture method and the two PCR
assays. For conventional PCR, the time required for sample processing,
PCR amplification, and gel electrophoresis was ~100 min. On the other
hand, the time required for real-time PCR was ~45 min because thermal
cycling is much faster and amplicon detection is performed in real
time. Both PCR methods were able to identify GBS colonization in a much
shorter turnaround time than the gold standard culture method.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Currently, the gold standard method for detection of vaginal colonization with GBS is selective broth culture performed at 35–37 weeks of gestation, which is sensitive enough to allow detection of both light and heavy colonization, but identification results are not available until 48 h later (4). Therefore, these methods are not useful for identification of GBS at or near the time of delivery. Moreover, culturing at 35–37 weeks is not always indicative of carrier status at delivery because GBS colonization often is transient (3). Because rapid diagnosis of GBS colonization or infection is of importance in the prevention of neonatal sepsis and meningitis, many simple and rapid tests for GBS have been developed. The Gram stain smear is of limited clinical help in detecting GBS because of low sensitivity and poor specificity (6). Several special media have been introduced to rapidly detect GBS by pigment production (25)(26)(27). However, some GBS isolates lack the ability to produce pigment, and thus cannot be identified by these media. The sensitivities of latex agglutination tests and immunoassays for detection of GBS directly from clinical specimens vary from 19% to 82% when selective broth media are used as standards to recover GBS from specimens (9). Generally, these tests are not sufficiently sensitive for direct detection of GBS, and only women with heavy colonization can be readily identified by these methods (6)(7)(8).
In this study, we developed a conventional PCR assay that is rapid (~100 min), specific for GBS, and sensitive enough to detect a single genome copy of GBS. Importantly, there was no amplification with purified genomic DNA from 27 species of streptococci other than GBS and many members of the vaginal and anal flora. Furthermore, the assay was capable of efficiently amplifying DNA from 162 GBS strains from various geographic regions. Such an assay may be useful for the detection of GBS colonization directly from clinical specimens because of its high specificity and sensitivity. Although all GBS strains tested were positive for the CAMP test, the assays should be able to identify CAMP test-negative strains because the cfb gene is present in virtually all GBS isolates and is well conserved within this species according to phenotypic and molecular characterizations (19)(20).
Other molecular methods for identifying GBS have been described (10)(11)(12). The Accuprobe system (Gen-Probe) is suitable for the identification of GBS from cultures but is insufficiently sensitive for direct detection from the clinical specimens described (10)(12). PCR has become one of the most widely used methods in molecular diagnosis. However, little work has been done on detection of GBS. A PCR assay for detection of the C-protein gene in GBS has been developed. However, this assay allowed the detection of only 63% of the GBS strains tested and therefore is not suitable for screening GBS colonization or infection (15). Two nested PCR assays have been reported to detect GBS by amplifying the 16S rRNA gene (13) or the 16S-23S spacer region (14). These assays allowed detection, from cerebrospinal fluid samples, of ~100 and 5 CFUs of GBS, respectively. However, nested PCR protocols often are limited by the increased risk of PCR carryover.
Rapid thermal cycling for PCR amplification coupled with real-time fluorescence monitoring of the PCR reactions (17)(18) provides a new tool for rapid detection of microorganisms. The detection of PCR products is achieved by monitoring the change of fluorescence on the basis of fluorescence resonance energy transfer when the two hybridization probes anneal to the target DNA (GBS-specific amplicons in this study) in close proximity. Using this technology, Woo et al. (28) detected Leptospira genospecies DNA with 45-cycle amplification requiring only 18 min. Because amplification and detection occur in the same reaction vessel and no postamplification sample transfer is needed, the LightCycler platform greatly reduces the risk of carryover, making it more suitable for use in the routine clinical laboratory than conventional heating-block thermocyclers coupled with agarose gel electrophoresis.
To adapt our PCR assay to the LightCycler platform, two fluorescently labeled adjacent hybridization probes complementary to part of the amplicon were used for specific hybridization to the GBS-specific amplification product. Analysis of 14 cfb sequences from Podbielski et al. (20) and from our own laboratory (data not shown), representing the major serotypes of GBS, indicates that the target sequences for the adjacent probes are highly conserved. This ensures the ability of the two adjacent probes to hybridize to all GBS-specific amplicons. The additional specificity provided by the combination of species-specific primers with internal probes ensures direct detection and identification of GBS from anovaginal specimens in which the typical vaginal and anal floras are microbiologically extremely complex and not well known (23). The PCR assay with the LightCycler constantly detected as few as one to three genome copies as well as a comparable number of CFUs. Such sensitivity is suitable for direct detection of GBS from clinical specimens. This real-time PCR assay is able to quantify the GBS load of vaginal specimens by comparison to calibration curves with known amounts of GBS DNA or cells. However, no attempt to determine the degree of GBS colonization was made because both heavy and light colonization in mothers are associated with infantile GBS diseases (3).
The LightCycler (LC-32; Idaho Technology) platform used in this study allowed monitoring of only two fluorescence signals in each capillary. Therefore, a second capillary was needed to verify the presence of PCR inhibitors in the clinical samples by the use of the other pair of adjacent hybridization probes to target the internal control template. A new generation LightCycler instrument and software developed recently by Roche allow monitoring of three fluorescence signals in the same capillary, permitting the use of two pairs of adjacent probes within the same reaction vessel.
The IDI DNA extraction kit allows simple, rapid, and efficient release of GBS DNA from vaginal specimens. In fact, there was a perfect correlation between the results of direct detection of GBS by culture and by both PCR assays. Furthermore, as demonstrated by sensitivity tests with vaginal samples to which purified genomic DNA had been added, the IDI DNA extraction kit prevents significant PCR inhibition. In addition, sensitivity tests performed with GBS cultures showed that the IDI protocol assures efficient GBS cell lysis.
We have found that much higher PCR inhibition is encountered in vaginal samples from pregnant women compared with samples obtained from nonpregnant women (unpublished data). Although a limited number of specimens were tested, our results indicate that both PCR assays are suitable for GBS screening in pregnant women. A clinical study in progress (29) indicates that the IDI lysis protocol is also suitable for direct detection of GBS from vaginal/anal specimens, which is the sample type recommended by the CDC for the screening of GBS carriers (3).
Rapid and reliable detection of GBS colonization would benefit parturient mothers, especially those with poor prenatal care during pregnancy, and permit more effective prevention of GBS infections. Improved diagnostic tools for GBS, such as our PCR assay using the LightCycler, may lead to more rational use of antibiotics. Integration of rapid sample preparation with rapid amplification and detection technologies may help to improve the management and prevention of infectious diseases because clinicians will have rapidly in hand the clinical microbiology results.
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Acknowledgments |
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Footnotes |
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References |
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S. M. Borchardt, B. Foxman, D. O. Chaffin, C. E. Rubens, P. A. Tallman, S. D. Manning, C. J. Baker, and C. F. Marrs Comparison of DNA Dot Blot Hybridization and Lancefield Capillary Precipitin Methods for Group B Streptococcal Capsular Typing J. Clin. Microbiol., January 1, 2004; 42(1): 146 - 150. [Abstract] [Full Text] [PDF]
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 L. Pereira, E. Maidji, S. McDonagh, O. Genbacev, and S. Fisher Human Cytomegalovirus Transmission from the Uterus to the Placenta Correlates with the Presence of Pathogenic Bacteria and Maternal Immunity J. Virol., December 15, 2003; 77(24): 13301 - 13314. [Abstract] [Full Text] [PDF]
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P. Lapierre, A. Huletsky, V. Fortin, F. J. Picard, P. H. Roy, M. Ouellette, and M. G. Bergeron Real-Time PCR Assay for Detection of Fluoroquinolone Resistance Associated with grlA Mutations in Staphylococcus aureus J. Clin. Microbiol., July 1, 2003; 41(7): 3246 - 3251. [Abstract] [Full Text] [PDF]
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S. M. Hansen and U. B. S. Sorensen Method for Quantitative Detection and Presumptive Identification of Group B Streptococci on Primary Plating J. Clin. Microbiol., April 1, 2003; 41(4): 1399 - 1403. [Abstract] [Full Text] [PDF]
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S. D. Belanger, M. Boissinot, N. Clairoux, Francois. J. Picard, and M. G. Bergeron Rapid Detection of Clostridium difficile in Feces by Real-Time PCR J. Clin. Microbiol., February 1, 2003; 41(2): 730 - 734. [Abstract] [Full Text] [PDF]
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Y. Asai, T. Jinno, H. Igarashi, Y. Ohyama, and T. Ogawa Detection and Quantification of Oral Treponemes in Subgingival Plaque by Real-Time PCR J. Clin. Microbiol., September 1, 2002; 40(9): 3334 - 3340. [Abstract] [Full Text] [PDF]
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I. Meiri-Bendek, E. Lipkin, A. Friedmann, G. Leitner, A. Saran, S. Friedman, and Y. Kashi A PCR-Based Method for the Detectionof Streptococcus agalactiae in Milk J Dairy Sci, July 1, 2002; 85(7): 1717 - 1723. [Abstract] [Full Text] [PDF]
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S. D. Belanger, M. Boissinot, C. Menard, F. J. Picard, and M. G. Bergeron Rapid Detection of Shiga Toxin-Producing Bacteria in Feces by Multiplex PCR with Molecular Beacons on the Smart Cycler J. Clin. Microbiol., April 1, 2002; 40(4): 1436 - 1440. [Abstract] [Full Text] [PDF]
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F. Kong, S. Gowan, D. Martin, G. James, and G. L. Gilbert Molecular Profiles of Group B Streptococcal Surface Protein Antigen Genes: Relationship to Molecular Serotypes J. Clin. Microbiol., February 1, 2002; 40(2): 620 - 626. [Abstract] [Full Text] [PDF]
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F. Kong, S. Gowan, D. Martin, G. James, and G. L. Gilbert Serotype Identification of Group B Streptococci by PCR and Sequencing J. Clin. Microbiol., January 1, 2002; 40(1): 216 - 226. [Abstract] [Full Text] [PDF]
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A. Sekizawa, T. Kondo, M. Iwasaki, A. Watanabe, M. Jimbo, H. Saito, and T. Okai Accuracy of Fetal Gender Determination by Analysis of DNA in Maternal Plasma Clin. Chem., October 1, 2001; 47(10): 1856 - 1858. [Full Text] [PDF]
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F. Martineau, F. J. Picard, D. Ke, S. Paradis, P. H. Roy, M. Ouellette, and M. G. Bergeron Development of a PCR Assay for Identification of Staphylococci at Genus and Species Levels J. Clin. Microbiol., July 1, 2001; 39(7): 2541 - 2547. [Abstract] [Full Text] [PDF]
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R. T. Hayden, J. R. Uhl, X. Qian, M. K. Hopkins, M. C. Aubry, A. H. Limper, R. V. Lloyd, and F. R. Cockerill Direct Detection of Legionella Species from Bronchoalveolar Lavage and Open Lung Biopsy Specimens: Comparison of LightCycler PCR, In Situ Hybridization, Direct Fluorescence Antigen Detection, and Culture J. Clin. Microbiol., July 1, 2001; 39(7): 2618 - 2626. [Abstract] [Full Text] [PDF]
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H. H. Kessler, G. Muhlbauer, E. Stelzl, E. Daghofer, B. I. Santner, and E. Marth Fully Automated Nucleic Acid Extraction: MagNA Pure LC Clin. Chem., June 1, 2001; 47(6): 1124 - 1126. [Full Text] [PDF]
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J. Wilhelm, M. Hahn, and A. Pingoud Influence of DNA Target Melting Behavior on Real-Time PCR Quantification Clin. Chem., November 1, 2000; 46(11): 1738 - 1743. [Abstract] [Full Text] [PDF]
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 M. G. Bergeron, D. Ke, C. Menard, F. J. Francois, M. Gagnon, M. Bernier, M. Ouellette, P. H. Roy, S. Marcoux, and W. D. Fraser Rapid Detection of Group B Streptococci in Pregnant Women at Delivery N. Engl. J. Med., July 20, 2000; 343(3): 175 - 179. [Abstract] [Full Text] [PDF]
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H. H. Kessler, G. Mühlbauer, B. Rinner, E. Stelzl, A. Berger, H.-W. Dörr, B. Santner, E. Marth, and H. Rabenau Detection of Herpes Simplex Virus DNA by Real-Time PCR J. Clin. Microbiol., July 1, 2000; 38(7): 2638 - 2642. [Abstract] [Full Text]
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