Evaluation of four commercial real-time PCR assays for detection of Bordetella spp. in nasopharyngeal aspirates.
ABSTRACT We evaluated the performances of 4 commercial real-time PCR kits for Bordetella pertussis IS481 sequence detection in nasopharyngeal aspirates by comparison with an in-house real-time PCR assay. Among them, the Simplexa Bordetella pertussis/parapertussis assay (Focus Diagnostics), the SmartCycler Bordetella pertussis/parapertussis assay (Cepheid), and Bordetella R-gene (Argene) present sensitivities over 90%. One kit proved unsuitable for routine clinical use.
- SourceAvailable from: Udo Reischl[show abstract] [hide abstract]
ABSTRACT: Detection of Bordetella holmesii by a real-time PCR assay targeting IS481 of Bordetella pertussis is reported. Sequencing of IS481-specific PCR products from B. pertussis and B. holmesii isolates revealed sequence homology. Restriction fragment length polymorphism demonstrated a low copy number of IS481-like sequences in B. holmesii. These results, and culture of B. holmesii from patients with cough, suggest that the specificity and predictive value of IS481-based PCR assays for pertussis may be compromised.Journal of Clinical Microbiology 06/2001; 39(5):1963-6. · 4.07 Impact Factor
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ABSTRACT: We developed a single-vessel multiplex real-time PCR assay that detects Helicobacter pylori infection and identified the four existing alleles of the 23S rRNA genes of H. pylori--the wild-type sequence and the three mutations conferring clarithromycin resistance--using allele-specific Scorpion primers directly on biopsy specimens. The Scorpion primers combine a primer and a probe in a single molecule and are able to distinguish single-nucleotide polymorphism. Fluorescent signals, produced when the probes are annealed, are read in four channels by a SmartCycler thermocycler. The assay was first applied successfully on 4 reference and 61 clinical strains. MICs of clarithromycin were determined by the Etest method. A perfect concordance was obtained between Etest and Scorpion PCR. Mixed populations were better detected by Scorpion PCR. We examined 259 biopsies from 229 patients by culture, PCR-restriction fragment length polymorphism (RFLP), and Scorpion PCR. One biopsy, positive for culture, exhibited inhibitors for both PCR-RFLP and Scorpion PCR. Twelve biopsies were positive for PCR-RFLP and Scorpion PCR but negative for culture with concordant determination of mutations in the 23S rRNA genes by the two PCR assays. Three biopsies were positive for Scorpion PCR only. Compared to culture, the sensitivity of Scorpion PCR was 98.3% and the specificity was 92.5%. The Scorpion PCR assay provides a highly accurate, rapid, and precise method for the detection and determination of mutations conferring clarithromycin resistance to H. pylori.Journal of clinical microbiology 08/2008; 46(7):2320-6. · 4.16 Impact Factor
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ABSTRACT: A real-time PCR assay based on the TaqMan technology was developed for the detection of Bordetella pertussis and B. parapertussis in clinical samples. The assay was evaluated with 182 specimens from 153 patients with and without symptoms of pertussis. The analytical sensitivity ranged from 0.1 to 10 cfu for B. pertussis and B. parapertussis, respectively, and diagnostic sensitivity was 94.1% when culture was used as a reference. No sample from a patient without symptoms of pertussis was positive in PCR. Twenty-four of 28 patients who were negative by culture and positive by PCR assay met the CDC clinical case definition for pertussis; the remaining four patients had paroxysms of shorter duration. Intra- and inter-assay variation were <5% and results were available within 4 h.Journal of Medical Microbiology 05/2001; 50(5):436-40. · 2.30 Impact Factor
JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 2011, p. 3943–3946
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 49, No. 11
Evaluation of Four Commercial Real-Time PCR Assays for Detection
of Bordetella spp. in Nasopharyngeal Aspirates?
Philippe Lanotte,1,2Chloe ´ Plouzeau,3Christophe Burucoa,3,4Carole Gre ´laud,5Sophie Guillot,6
Nicole Guiso,6and Fabien Garnier5*
CHRU de Tours, Service de Bacte ´riologie-Virologie, Ho ˆpital Bretonneau, Tours, France1; Universite ´ Franc ¸ois Rabelais, Tours,
France2; CHU de Poitiers, Laboratoire de Bacte ´riologie, Ho ˆpital La Mile ´trie, Poitiers, France3; Universite ´ de
Poitiers, Poitiers, France4; CHU de Limoges, Laboratoire de Bacte ´riologie-Virologie-Hygie `ne, Limoges,
France5; and Institut Pasteur, Centre National de Re ´fe ´rence de la Coqueluche et
Autres Bordetelloses, Unite ´ de Pre ´vention et The ´rapie Mole ´culaires des
Maladies Humaines, 25 rue du Docteur Roux, 75015 Paris, France6
Received 16 February 2011/Returned for modification 19 May 2011/Accepted 6 September 2011
We evaluated the performances of 4 commercial real-time PCR kits for Bordetella pertussis IS481 sequence
detection in nasopharyngeal aspirates by comparison with an in-house real-time PCR assay. Among them, the
Simplexa Bordetella pertussis/parapertussis assay (Focus Diagnostics), the SmartCycler Bordetella pertussis/
parapertussis assay (Cepheid), and Bordetella R-gene (Argene) present sensitivities over 90%. One kit proved
unsuitable for routine clinical use.
Nucleic acid amplification tests (NAAT), including PCR
and, more recently, real-time PCR, overcome some of the
limitations of culture and serological methods for the diagnosis
of Bordetella infection (1, 6, 12). NAAT targets include IS481
for Bordetella pertussis (this gene is also present in B. holmesii
and sometimes in B. bronchiseptica) (12, 13), the pertussis toxin
promoter or porin gene BP3385 for B. pertussis (10), and
IS1001 for B. parapertussis (5, 13). NAAT methods are highly
sensitive and may be either genus or species specific, depend-
ing on the choice of primers and targets. Real-time PCR ac-
celerates the diagnostic process by combining amplification
and detection (12, 13). These methods have proved more sen-
sitive than the equivalent gel-based system (9, 12), and many
medical laboratories have developed in-house tests. A volun-
tary external molecular quality control procedure for these
in-house methods was set up in France in 2009 by the National
Reference Centre for Whooping Cough and Other Bordetello-
ses (Institut Pasteur, Paris) (3), following a study performed in
eight hospital laboratories throughout France to assess the
performance of in-house methods and adaptations of the tech-
niques developed by Reischl et al. (11), Ko ¨sters et al. (8), Caro
et al. (3), and Templeton et al. (13). Commercial molecular
diagnostic kits are now available but have never been compar-
The aim of this study was to compare the performances of 4
commercial real-time PCR assays for the detection of B. per-
tussis, using as a reference an in-house method evaluated dur-
ing the French external molecular quality control (3).
Eighty-one nonredundant nasopharyngeal aspirates (NPA)
from patients with suspected pertussis were tested in the bac-
teriology laboratories of three French teaching hospitals
(Tours, Poitiers, and Limoges). Sixty-six samples tested posi-
tive with our in-house PCR, while 15 had tested negative but
were from patients with a suspicion of pertussis. The samples
and DNA extracts were stored at ?20°C until use. DNA was
extracted with the Invisorb spin cell minikit (Invitek, Berlin,
Germany) in Tours and with the QIAamp DNA minikit (Qia-
gen) in Poitiers and Limoges by following the manufacturers’
instructions. The principles and procedures of the two extrac-
tion methods are very similar. Tenfold serial dilutions of DNA
from B. pertussis strain Tohama, containing 238 copies of
IS481, were used to determine the analytical sensitivity of each
method. DNA extracted from each of the 81 samples was
tested with the in-house real-time PCR assay using prim-
ers Bp481F (5?-CCGAACCGGATTTGAGAAAC-3?) and
Bp481R (5?-TAGGAAGGTCAATCGGGCAT-3?), which tar-
get a 100-bp fragment of IS481. Detection was based on hy-
bridization of the Bp481S probe (5?–6-carboxyfluorescein
tramethylrhodamine [TAMRA]–3?). Primers were purchased
from Eurogentec (Seraing, Belgium) and were used at a con-
centration of 10 ?mol/liter. Amplification was performed with
a SmartCycler device (Cepheid, Maurens-Scopont, France),
with 5 ?l of DNA, 12.5 ?l of premix Ex Taq (TaKaRa, Foster
City, CA), 4.2 ?l of water, 0.4 ?l of each primer, and 2.5 ?l of
Bp481S probe (1 ?M). The thermal cycling conditions were as
follows: 1 cycle of 15 s at 95°C, followed by 45 cycles of 5 s at
95°C and 10 s at 60°C. Each laboratory tested the specimens
collected in its host hospital and used the same protocols as the
other laboratories. The SmartCycler II apparatus (Cepheid)
was used for all assays.
The four commercial PCR kits, all based on TaqMan tech-
nology, were Bordetella R-gene (Argene, Verniolle, France),
the Simplexa Bordetella pertussis/parapertussis assay (Focus
Diagnostics; distributed in France by Eurobio, Courtabeuf,
France), the Bordetella pertussis real-time PCR kit (catalog no.
RD-0061-02 [Shanghai ZJ Bio-Tech; distributed in France by
* Corresponding author. Mailing address: Laboratoire de Bacte ´ri-
ologie-Virologie-Hygie `ne, Centre Hospitalier Universitaire de Li-
moges, 2 avenue Martin Luther King, 87042 Limoges Cedex, France.
Phone: 33 5 55 05 63 48. Fax: 33 5 55 05 67 22. E-mail: fabien.garnier
?Published ahead of print on 14 September 2011.
TABLE 1. Comparison of main parameters of 4 real-time PCR kits tested
Name of kit
No. of cycles
(no. of cycles
which kit is
Yes (10 ?l IC to
add in sample
ABI Prism, Rotor-Gene
Shanghai ZJ Bio-Tech
Bordetella pertussis real-
time PCR kit
Yes (1 ?l IC to
add in mix
in mix ?20
France by Eurobio)
Yes (5 ?l IC to
add in sample
aIC, internal control.
bAs recommended by the manufacturer of the SmartCycler apparatus.
3944NOTESJ. CLIN. MICROBIOL.
BioAdvance, Bussy-Saint-Martin, France]), and the SmartCy-
cler Bordetella pertussis/parapertussis assay (Cepheid). The dif-
ferent kits were used according to the recommendations of the
manufacturers, including cycle thresholds (CT) of 7 for the
Shanghai ZJ Bio-Tech kit, 15 and 30 for the Focus Diagnostics
kit, and 30 for the other two methods. Differences between the
methods are shown in Table 1.
The analytical sensitivity of each method, tested in duplicate,
is shown in Fig. 1. The CTvalues were similar for the different
methods with up to 104copies of IS481 per reaction, except
that the CTof the Shanghai ZJ Bio-Tech test was about 7
cycles above that of the other methods whatever the number of
copies. Only the in-house method and the Argene, Cepheid,
and Focus Diagnostics kits were positive with the dilution
containing 100 copies of IS481 per reaction.
Each clinical DNA specimen was tested with the in-house
method and with the 4 commercial kits. All the DNA speci-
mens were thawed and tested on the same day (no refreezing
occurred between the different assays). The results are shown
in Table 2. No false positives were noted for any assay. With
the in-house method as the gold standard, clinical sensitivity
was 97% with the Focus Diagnostics kit, 93.9% with the Ceph-
eid kit, 90.9% with the Argene kit, and 51.5% with the Shang-
hai ZJ Bio-Tech kit. The Focus Diagnostics kit had the lowest
mean CT(Table 2), which was not significantly different from
that of the in-house assay. The Argene and Cepheid kits had
slightly but not significantly higher mean CTvalues than the
in-house assay. In contrast, the Shangai ZJ Bio-Tech kit had
significantly higher mean CTvalues than the in-house assay. It
is noteworthy that the manufacturer of the Shanghai ZJ Bio-
Tech kit recommends using only half the amount of DNA used
in the other kits. The performance of the Focus Diagnostics kit
was unaffected when a CTof 15 rather than 30 was used (data
Internal controls are included to detect PCR inhibition. Be-
ta-globin was used for this purpose in the in-house method in
a separate vial. The Argene and Focus Diagnostics kits also
include extraction controls (Table 1). The list of apparatus on
which kits can be used is shown in Table 1. Two kits (the
Cepheid and Focus Diagnostics assays) are claimed to detect
B. pertussis and B. parapertussis in the same tube, while the
other two kits and the in-house method target only IS481. B.
holmesii, which also possesses IS481, cannot be distinguished
from B. pertussis with these kits. This species rarely induces
FIG. 1. Representation of the crossing threshold obtained for each commercial kit and the in-house real-time PCR for the detection of the
Bordetella IS481 sequence.
TABLE 2. Clinical sensitivities and mean CTvalues for real-time PCR kitsa
No. of positive specimens
identified/total no. of
Mean CT? SD
Focus Diagnostics kit
Shanghai ZJ Bio-Tech kit
25.5 ? 8.6
27.1 ? 9.5
25.8 ? 9.2
24.8 ? 8.8b
37.3 ? 8c
aData for the four real-time PCR kits tested are in comparison to results of the in-house real-time PCR assay for detection of the Bordetella IS481 sequence in 66
bAs recommended by the manufacturer, the CTretained for the comparison was 15.
cAs recommended by the manufacturer, the CTretained for the comparison was 7.
dDetermined by paired t test (2). NS, not significant.
VOL. 49, 2011NOTES 3945
pertussis-like symptoms and can be identified by real-time
PCR-based on the recA gene (7).
In conclusion, all four kits tested here were highly specific,
whereas their sensitivities were highly variable. The Argene,
Focus Diagnostics, and Cepheid kits performed as well as the
in-house method in terms of both analytical and clinical sen-
sitivities. These three kits include an internal control to detect
inhibitors, which is important in routine practice (4), but only
two of them, the Argene and Focus Diagnostics tests, validate
the extraction step. The Shanghai ZJ Bio-Tech kit needs to be
improved if it is to be used in routine clinical settings.
We thank Marie Odile Viaud, Lucie Yzon, and Catherine Louin for
their technical assistance.
The different kits were supplied free of charge by the different
corporations, which provided no other financial support for this study.
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