Identification of Brachyspira species by cpn60 universal target sequencing is superior to NADH oxidase gene sequencing

Rohde, Judith; Rubin, Joseph E.; Kulathunga, D.G.R.S.; Hill, Janet E.; Habighorst-Blome, Kerstin; Hampson, David J.; La, Tom

doi:10.1016/j.vetmic.2019.108454

Cited by 6 publications

(5 citation statements)

References 23 publications

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“…Primers and probes were designed using CLC Main Workbench software 7.5.1 from alignments of available cpn60 sequences from the NCBI databank (Rohde et al, 2019). Additionally, cpn60 of nine clinical samples were partially sequenced ( Figure 1 -amplicon generated using primers cpn60_for and cpn60_rev.…”

Section: Development Of the 5-plex Qpcrmentioning

confidence: 99%

“…Recently, it has been shown that sequencing of chaperonin cpn60 is superior to nox sequencing and revealed more reliable species identification for some isolates (Rohde et al, 2019). Molecular chaperones are universally present in almost all eubacteria and archaea harboring phylogenetically more discriminative gene sequences for species identification than those of the traditionally used 16S rDNA target (Hill et al, 2004; Links et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

“…Recently, it has been shown that sequencing of chaperonin cpn60 is superior to nox sequencing and revealed more reliable species identification for some isolates (Rohde et al, 2019).…”

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confidence: 99%

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Novel multiplex TaqMan assay for differentiation of the four major pathogenic Brachyspira species in swine

Scherrer

Stephan

2021

MicrobiologyOpen

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A novel TaqMan 5‐plex real‐time PCR using a combination of locked nucleic acid‐modified (LNA)‐ and minor groove binding (MGB)‐conjugated DNA probes was developed for identification and differentiation between the four main pathogenic Brachyspira species in swine. B. hyodysenteriae, B. pilosicoli, and B. suanatina are identified using three hydrolysis probes targeting cpn60, while B. hampsonii is recognized by another nox specific probe. The assay also includes an exogenous internal control simultaneously verifying the PCR competency of the DNA samples. Validation of the novel assay was performed using DNA samples from 18 Brachyspira reference strains and 477 clinical samples obtained from porcine rectal swabs by comparing them with different PCR‐based methods targeting nox, 16S rDNA, and 23S rDNA. The specificity of the assay was 100% without cross‐reactivity or detection of different pathogens. Depending on the Brachyspira species, the limit of detection was between 10 and 20 genome equivalents with a cut‐off threshold cycle (Ct) value of 37. The developed highly sensitive and specific 5‐plex real‐time PCR assay is easy to implement in routine veterinary diagnostic laboratories and enables rapid differentiation between the main four pathogenic Brachyspira species recognized in pigs using a single‐tube approach.

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Section: Development Of the 5-plex Qpcrmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Novel multiplex TaqMan assay for differentiation of the four major pathogenic Brachyspira species in swine

Scherrer

Stephan

2021

MicrobiologyOpen

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“…During the process of collecting isolates for this study, six isolates that were identified as G. parasuis using the Howell et al primer pair [12,15] could not be serotyped and did not show any product in the LS-PCR. Their cpn60 universal target sequence was obtained and analyzed as described by Rohde et al to inspect their species assignment [18].…”

Section: Cpn60 Sequencingmentioning

confidence: 99%

Serotyping and pathotyping of Glaesserella parasuis isolated 2012–2019 in Germany comparing different PCR-based methods

Schuwerk

Hoeltig

Waldmann

et al. 2020

Vet Res

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Glaesserella parasuis is an important pathogen in swine production. It acts as a primary pathogen in systemic Glässer´s disease and as a secondary pathogen in Porcine Respiratory Disease Complex. In this study, a collection of 308 isolates from carrier animals and individuals with respiratory or Glässer´s disease isolated 2012–2019 in Germany was analysed. Isolates were characterized for serovar implementing two different PCR methods. Additionally, two different PCR methods for pathotyping isolates were applied to the collection and results compared. Serovar 6 (p < 0.0001) and 9 (p = 0.0007) were correlated with carrier isolates and serovar 4 was associated with isolates from animals with respiratory disease (p = 0.015). In systemic isolates, serovar 13 was most frequently detected (18.9%). Various other serovars were isolated from all sites and the ratio of serovar 5 to serovar 12 was approximately 1:2. These two serovars together represented 14.3% of the isolates; only serovar 4 was isolated more frequently (24.7%). The pathotyping method based on the leader sequence (LS = ESPR of vta) was easy to perform and corresponded well to the clinical background information. Of the carrier isolates 72% were identified as non-virulent while 91% of the systemic isolates were classified as virulent (p < 0.0001). Results of the pathotyping PCR based on 10 different marker genes overall were in good agreement with clinical metadata as well as with results of the LS-PCR. However, the pathotyping PCR was more complicated to perform and analyze. In conclusion, a combination of the serotyping multiplex-PCR and the LS-PCR could improve identification of clinically relevant G. parasuis isolates, especially from respiratory samples.

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“…cpn60 barcode sequences are excellent predictors of whole genome sequence relationships [3][4][5] and can be used for sub-species resolution of bacterial taxa [6][7][8][9]. The availability of "universal" PCR primers [10,11] targeting the cpn60 barcode and a manually curated database of reference sequences (cpnDB [12]) have made this sequence an attractive target for diagnostics [13][14][15], characterization of new species [16][17][18] and taxonomic profiling of a variety of microbial communities, including microbiota of human and animal body sites [19][20][21][22][23][24], and environmental samples [25][26][27].…”

Section: Introductionmentioning

confidence: 99%

Rapid and accurate taxonomic classification of cpn60 amplicon sequence variants

Ren

Hill

2023

Preprint

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The 'universal target' region of the gene encoding the 60 kDa chaperonin protein (cpn60, also known as groEL or hsp60) is a proven sequence barcode for bacteria and a useful target for marker gene amplicon-based studies of complex microbial communities. To date, identification of cpn60 sequence variants from microbiome studies has been accomplished by alignment of queries to a reference database. Naïve Bayesian classifiers including the RDP classifier offer an alternative identification method that provides variable rank classification and shorter analysis times. We curated a set of cpn60 barcode sequences to train the RDP classifier and tested its performance on data from previous human microbiome studies. Results showed that sequences accounting for 79%, 86% and 92% of the observations (read counts) in saliva, vagina and infant stool microbiome data sets were classified to the species rank. We also established a threshold confidence value of 0.6 at phylum rank for filtering non-target amplicon sequences from study data and demonstrated that trimming the training sequences to match the lengths of the queries is not needed for accurate cpn60 sequence classification. Successful implementation of a naïve Bayesian classifier for cpn60 sequences will facilitate future microbiome studies and open opportunities to integrate cpn60 amplicon sequence identification into existing analysis pipelines.

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Identification of Brachyspira species by cpn60 universal target sequencing is superior to NADH oxidase gene sequencing

Cited by 6 publications

References 23 publications

Novel multiplex TaqMan assay for differentiation of the four major pathogenic Brachyspira species in swine

Novel multiplex TaqMan assay for differentiation of the four major pathogenic Brachyspira species in swine

Serotyping and pathotyping of Glaesserella parasuis isolated 2012–2019 in Germany comparing different PCR-based methods

Rapid and accurate taxonomic classification of cpn60 amplicon sequence variants

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