Development of a multiplex real-time quantitative PCR assay to detect Chlamydia pneumoniae, Legionella pneumophila and Mycoplasma pneumoniae in respiratory tract secretions

Welti, Martine; Jaton, Katia; Altwegg, Martin; Sahli, Roland; Wenger, A.; Billé, Jacques

doi:10.1016/s0732-8893(02)00484-4

Cited by 155 publications

(116 citation statements)

References 28 publications

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“…This standardization allows us to run all the different RT-PCRs in batches in 384 multi-well plates. Thus, currently, we are able to detect w65 different pathogens using similar homemade TaqMan PCRs, including several intracellular bacteria such as C. trachomatis (Jaton et al, 2006), C. pneumoniae (Welti et al, 2003) and Coxiella burnetii (Jaton et al, 2013). Branley et al (2008) have developed a PCR targeting the 16S rRNA gene, but using SYBR Green technology, that presented a T m lower than the target T m of our platform (60 uC) (Branley et al, 2008).…”

Section: Discussionmentioning

confidence: 99%

Improving the molecular diagnosis of Chlamydia psittaci and Chlamydia abortus infection with a species-specific duplex real-time PCR

Opota

Jaton

Branley

et al. 2015

Journal of Medical Microbiology

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Chlamydia psittaci and Chlamydia abortus are closely related intracellular bacteria exhibiting different tissue tropism that may cause severe but distinct infection in humans. C. psittaci causes psittacosis, a respiratory zoonotic infection transmitted by birds. C. abortus is an abortigenic agent in small ruminants, which can also colonize the human placenta and lead to foetal death and miscarriage. Infections caused by C. psittaci and C. abortus are underestimated mainly due to diagnosis difficulties resulting from their strict intracellular growth. We developed a duplex real-time PCR to detect and distinguish these two bacteria in clinical samples. The first PCR (PCR1) targeted a sequence of the 16S-23S rRNA operon allowing the detection of both C. psittaci and C. abortus. The second PCR (PCR2) targeted the coding DNA sequence CPSIT_0607 unique to C. psittaci. The two PCRs showed 100 % detection for ¢10 DNA copies per reaction (1000 copies ml 21 ). Using a set of 120 samples, including bacterial reference strains, clinical specimens and infected cell culture material, we monitored 100 % sensitivity and 100 % specificity for the detection of C. psittaci and C. abortus for PCR1. When PCR1 was positive, PCR2 could discriminate C. psittaci from C. abortus with a positive predictive value of 100 % and a negative predictive value of 88 %. In conclusion, this new duplex PCR represents a low-cost and time-saving method with high-throughput potential, expected to improve the routine diagnosis of psittacosis and pregnancy complication in largescale screening programs and also during outbreaks.

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Section: Discussionmentioning

confidence: 99%

Improving the molecular diagnosis of Chlamydia psittaci and Chlamydia abortus infection with a species-specific duplex real-time PCR

Opota

Jaton

Branley

et al. 2015

Journal of Medical Microbiology

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“…Atypical respiratory pathogens have been identified by several previous mPCR protocols [34][35][36][37]. However, the present PCR protocol is unique, as it is constructed for identification of two typical and two atypical pathogens, probably the four most common aetiologies of CAP [38].…”

Section: Discussionmentioning

confidence: 99%

Evaluation of a multiplex PCR for bacterial pathogens applied to bronchoalveolar lavage

Strålin

Korsgaard²,

Olcén³

2006

Eur Respir J

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The present study assessed the diagnostic usefulness of a multiplex PCR (mPCR) for Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae and Chlamydophila pneumoniae applied to bronchoalveolar lavage (BAL).Fibreoptic bronchoscopy was performed on 156 hospitalised adult patients with lower respiratory tract infection (LRTI) and 36 controls. BAL fluid was analysed with bacterial culture and mPCR.By conventional diagnostic methods, S. pneumoniae, H. influenzae, M. pneumoniae and C. pneumoniae were aetiological agents in 14, 21, 3.2 and 0% of the LRTI patients, respectively. These pathogens were identified by BAL mPCR in 28, 47, 3.2 and 0.6% of cases, respectively, yielding sensitivities of 86% for S. pneumoniae, 88% for H. influenzae, 100% for M. pneumoniae and 0% for C. pneumoniae, and specificities of 81, 64, 100 and 99% for S. pneumoniae, H. influenzae, M. pneumoniae and C. pneumoniae, respectively. Of the 103 patients who had taken antibiotics prior to bronchoscopy, S. pneumoniae was identified by culture in 2.9% and by mPCR in 31%. Among the controls, mPCR identified S. pneumoniae in 11% and H. influenzae in 39%.In lower respiratory tract infection patients, bronchoalveolar lavage multiplex PCR can be useful for identification of Streptococcus pneumoniae, Mycoplasma pneumoniae and Chlamydophila pneumoniae. The method appears to be particularly useful in patients treated with antibiotics.

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“…The technique has been used to detect Cp. pneumoniae [10,16,23,33], C. trachomatis [19], Cp. felis [12,13,20] and Cp.…”

Section: Discussionmentioning

confidence: 99%

Development of a Chlamydophila psittaci species-specific and genotype-specific real-time PCR

et al. 2005

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-A Chlamydophila psittaci species-specific real-time PCR targeting the rDNA ribosomal spacer was developed as well as a genotype-specific real-time PCR targeting the Cp. psittaci outer membrane protein A (ompA) gene. The SYBR Green-based species-specific real-time PCR detected Cp. psittaci genotypes A to F, and the recently discovered E/B genotype. The genotype-specific real-time PCR could easily distinguish genotypes C, D, F by use of TaqMan probes. Genotypes A, B and E could not be distinguished from each other by simply using TaqMan probes. For this purpose, non-fluorescent competitor oligonucleotides, had to be used next to the TaqMan probes. Genotype E/B could only be detected by use of a minor groove binder (MGB) probe. Both real-time PCR assays allowed reproducible, sensitive (10 rDNA or ompA copies/µL DNA extract) and specific detection of Cp. psittaci DNA. The genotype-specific real-time PCR was compared to ompA sequencing and ompA restriction fragment length polymorphism (RFLP) analysis using five Cp. psittaci field isolates (99, 61/8, 7344/2, 8615/1 and 7778B15) each consisting of two different genotypes. The currently developed real-time PCR assays were used in a case study on a veterinary school and a turkey farm. In the veterinary school, Cp. psittaci genotypes D, E/B and F infection were detected in all five groups of turkeys, and one veterinarian who was taking care of all these turkeys. On the turkey farm, the presence of two Cp. psittaci genotype B infection waves was demonstrated in one randomly selected turkey, the first wave at the age of 6 weeks, and the second at the age of 12 weeks.

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Development of a multiplex real-time quantitative PCR assay to detect Chlamydia pneumoniae, Legionella pneumophila and Mycoplasma pneumoniae in respiratory tract secretions

Cited by 155 publications

References 28 publications

Improving the molecular diagnosis of Chlamydia psittaci and Chlamydia abortus infection with a species-specific duplex real-time PCR

Improving the molecular diagnosis of Chlamydia psittaci and Chlamydia abortus infection with a species-specific duplex real-time PCR

Evaluation of a multiplex PCR for bacterial pathogens applied to bronchoalveolar lavage

Development of a Chlamydophila psittaci species-specific and genotype-specific real-time PCR

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