Development of two real-time PCR assays for the detection of Mycoplasma hyopneumoniae in clinical samples

Dubosson, Christoph R.; Conzelmann, Claudia; Miserez, R.; Boerlin, Patrick; Frey, Joachim; Zimmermann, Werner; Häni, H; Kuhnert, Peter

doi:10.1016/j.vetmic.2004.05.007

Cited by 89 publications

(67 citation statements)

References 23 publications

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“…Based on the different combination of REP/ABC results which were included as a complement in this study, we hypothesized earlier that different strains are present in Switzerland [3]. This study further supports this, since all three combinations were found.…”

Section: Discussionsupporting

confidence: 81%

“…Further, single case samples of 23 farms from routine diagnosis, indicated by "P", were included. Most of the samples were collected in the context of a study on diagnosis of EP and are described there [3]. Only samples which were previously shown positive for M. hyopneumoniae in the real-time PCR were used for amplification of a part of the p146 gene.…”

Section: Samplesmentioning

confidence: 99%

“…Detection of the REP and ABC target genes by PCR was described previously [3]. For this study a multiplex assay on the ABI 7500 (Applied Biosystems) was used.…”

Section: Multiplex Real-time Pcrmentioning

confidence: 99%

“…We recently published a highly specific and sensitive real-time PCR targeting two different sequences of M. hyopneumoniae, one a repetitive element (REP) the other a putative ABCtransporter gene (ABC) [3]. Analysis of clinical samples from lung or nasal swabs of infected herds showed that about 60% of the samples were positive for both, REP and ABC, 30% were only positive for ABC and 10% gave a result with REP alone [3,17]. This indicates that different M. hyopneumoniae strains are circulating in the swine population.…”

Section: Introductionmentioning

confidence: 99%

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Diversity ofMycoplasma hyopneumoniaein pig farms revealed by direct molecular typing of clinical material

et al. 2007

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-Mycoplasma hyopneumoniae is the etiological agent of enzootic pneumonia in swine.Various reports indicate that different strains are circulating in the swine population. We investigated the variety of M. hyopneumoniae strains by a newly developed genetic typing method based on the polyserine repeat motif of the LppS homolog P146. PCR amplification using M. hyopneumoniae specific, conserved primers flanking the region encoding the repeat motif, followed by sequencing and cluster analysis was carried out. The study included strains isolated from different geographic regions as well as lysates from lung swabs from a series of pig farms in Switzerland. High diversity of M. hyopneumoniae was observed but farms being in close geographic or operative contact generally seemed to be affected by the same strains. Moreover, analysis of multiple samples from single pig farms indicated that these harbored the same, farm-specific strain. The results indicate that multiple strains of M. hyopneumoniae are found in the swine population but that specific strains or clones are responsible for local outbreaks. The method presented is a highly reproducible epidemiologic tool allowing direct typing of M. hyopneumoniae from clinical material without prior isolation and cultivation of strains. enzootic pneumonia / epidemiology / LppS / P146

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Section: Discussionsupporting

confidence: 81%

Section: Samplesmentioning

confidence: 99%

“…Detection of the REP and ABC target genes by PCR was described previously [3]. For this study a multiplex assay on the ABI 7500 (Applied Biosystems) was used.…”

Section: Multiplex Real-time Pcrmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Diversity ofMycoplasma hyopneumoniaein pig farms revealed by direct molecular typing of clinical material

et al. 2007

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“…According to Marois et al (2010), because of the great variability reported among M. hyopneumoniae strains, PCR assays using only one DNA target per reaction could have reduced performance. Nevertheless, a triplex RT-PCR developed by these authors showed approximately the same detection limit as other RT-PCR assays (DUBOSSON et al, 2004;STRAIT et al, 2008), which detect four targets independently. In our study, including more than one DNA target of the microorganism in the assay increased the number of positive samples detected, which is in agreement with Marois et al (2010).…”

Section: Discussionmentioning

confidence: 81%

Detection, quantification and genetic variability of Mycoplasma hyopneumoniae from apparently healthy and pneumonic swine

Pulgarón¹,

Marques

Campos

et al. 2015

Braz. J. Vet. Res. Anim. Sci.

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Molecular differences among Mycoplasma hyopneumoniae strains present in pneumonic lungs of swine have been largely studied. However, no comparative studies concerning the strains present in apparently healthy pigs have been carried out. This study aimed to detect, quantify and perform molecular analysis of M. hyopneumoniae strains in pig lungs with and without pneumonic lesions. The detection of M. hyopneumoniae was performed using multiplex PCR (YAMAGUTI, 2008), real-time PCR (STRAIT et al., 2008) and multiple VNTR amplification (VRANCKX et al., 2011). Molecular characterization of the strains was achieved by analysis of the VNTR copy number in P97R1, P146R3, H2R1 and H4. M. hyopneumoniae was detected in samples from healthy and pneumonic pigs and the amount of M. hyopneumoniae positive samples detected varied with the type of assay. The greater number of positive samples was identified by the multiple VNTR amplification combined with capillary electrophoresis. Using real-time PCR, 4.9*10 4 M. hyopneumoniae genome copies/mL was detected in apparently healthy lungs. A mean quantity of 3.9*10 6 M. hyopneumoniae genome copies/mL was detected in pneumonic lungs. The analysis of VNTR copy number demonstrated a high genetic variability of the M. hyopneumoniae strains present in apparently healthy and pneumonic lungs. Strains having 3 VNTR copy number in P97R1, were detected only in pneumonic lungs and strains having 40 and 43 VNTR copy number in P146R3 were detected only in apparently healthy lungs. Despite the genetic variability of M. hyopneumoniae, predominant strains in the swine farms could be identified.Keywords: M. hyopneumoniae. Enzootic pneumonia. Healthy. Quantification. VNTR. ResumoAs diferenças moleculares entre as estirpes de Mycoplasma hyopneumoniae presentes em pulmões de suínos com pneumonia têm sido estudadas. Porém, estudos comparativos relativos às estirpes presentes nos suínos aparentemente saudáveis não foram levados a cabo. O objetivo do estudo foi a detecção, quantificação e análise molecular de M. hyopneumoniae nos pulmões suínos com e sem lesões pneumônicas. Para a detecção de M. hyopneumoniae usaramse o PCR Múltiplo (YAMAGUTI, 2008), o PCR a Tempo Real (STRAIT et al., 2008) e a amplificação de múltiplo VNTR (VRANCKX et al., 2011). A caracterização molecular das estirpes foi realizada mediante a análise do número de cópias de VNTR em P97R1, P146R3, H2R1 e H4. O M. hyopneumoniae foi detectado em amostras de suínos saudáveis e pneumônicos e a quantidade de M. hyopneumoniae nas amostras positivas variou com o tipo de ensaio. O maior número de amostras positivas foi identificado pela amplificação de múltiplas VNTR combinado com a eletroforese de capilares. Usando o PCR a Tempo Real, 4.9*10 4 cópias de genoma/mL de M. hyopneumoniae foram detectadas em pulmões aparentemente saudáveis. Uma quantidade média de 3.9*10 6 cópias de genoma/mL de M. hyopneumoniae foi detectada em pulmões pneumônicos. A análise do número de cópias de VNTR demonstrou uma elevada variabilidade genética das estirpes d...

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Mycoplasma

Brown¹,

May²,

Bradbury³

et al. 2015

Bergey's Manual of Systematics of Archaea and Bacteria

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My.co.plas'ma. Gr. masc. n. myces a fungus; Gr. neut. n. plasma something formed or molded, a form; N.L. neut. n. Mycoplasma fungus form. Tenericutes / Mollicutes / Mycoplasmatales / Mycoplasmataceae / Mycoplasma Pleomorphic cells, 300–800 nm in diameter, varying in shape from spherical, ovoid or flask‐shaped, or twisted rods, to slender branched filaments ranging in length from 50 to 500 nm. Cells lack a cell wall and are bounded by a single plasma membrane. Gram‐stain‐negative due to the absence of a cell wall. Some have a complex internal cytoskeleton. Some have a specific tip structure that mediates attachment to host cells or other surfaces. Usually nonmotile , but gliding motility has been demonstrated in some species. Aerobic or facultatively anaerobic . Optimum growth at 37°C is common, but permissive growth temperatures range from 20 to 45°C. Chemo‐organotrophic , usually using either sugars or arginine as the major energy source. Require cholesterol or related sterols for growth. Colonies are usually less than 1 mm in diameter. The typical colony has a fried‐egg appearance . The genome size of species examined ranges from 580 kbp to about 1350 kbp . The codon UGA encodes tryptophan in all species examined. Commensals or pathogens in a wide range of vertebrate hosts. DNA G + C content ( mol %): 23–40. Type species : Mycoplasma mycoides (Borrel, Dujardin‐Beaumetz, Jeantet and Jouan 1910) Freundt 1955, 73 ( Asterococcus mycoides Borrel, Dujardin‐Beaumetz, Jeantet and Jouan 1910, 179).

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Development of two real-time PCR assays for the detection of Mycoplasma hyopneumoniae in clinical samples

Cited by 89 publications

References 23 publications

Diversity ofMycoplasma hyopneumoniaein pig farms revealed by direct molecular typing of clinical material

Diversity ofMycoplasma hyopneumoniaein pig farms revealed by direct molecular typing of clinical material

Detection, quantification and genetic variability of Mycoplasma hyopneumoniae from apparently healthy and pneumonic swine

Mycoplasma

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