- Email: [email protected]
Development of a loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive identification of Arcanobacterium pluranimalium
Accepted Manuscript Development of a loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive identification of Arcanobacterium pluranimalium A. Abdulmawjood, J. Wickhorst, O. Sammra, C. Lämmler, G. Foster, P.N. Wragg, E. Prenger-Berninghoff, G. Klein PII:
S0890-8508(15)30016-5
DOI:
10.1016/j.mcp.2015.06.003
Reference:
YMCPR 1144
To appear in:
Molecular and Cellular Probes
Received Date: 18 May 2015 Revised Date:
12 June 2015
Accepted Date: 12 June 2015
Please cite this article as: Abdulmawjood A, Wickhorst J, Sammra O, Lämmler C, Foster G, Wragg PN, Prenger-Berninghoff E, Klein G, Development of a loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive identification of Arcanobacterium pluranimalium, Molecular and Cellular Probes (2015), doi: 10.1016/j.mcp.2015.06.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Development of a loop-mediated isothermal amplification (LAMP) assay for
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rapid and sensitive identification of Arcanobacterium pluranimalium
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A. Abdulmawjooda, J. Wickhorstb, O. Sammrab, C. Lämmlerb, G. Fosterc, P. N.
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Wraggd, E. Prenger-Berninghoffe, G. Kleina
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a
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Hannover
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Bischofsholer Damm 15, 30173 Hannover, Germany
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b
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Schubertstr 81, 35392 Gießen, Germany c
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IV2 4JZ, UK
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d
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Merrythought, Calthwaite, Penrith CA11 9RR, UK
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e
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Frankfurterstr. 85-91, 35392 Gießen, Germany
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Institut für Pharmakologie und Toxikologie, Justus-Liebig-Universität Gießen,
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SAC Consulting Veterinary Services, Drummondhill, Stratherrick Road, Inverness
Animal Health and Veterinary Laboratories Agency, Penrith Regional Laboratory,
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Institut für Hygiene und Infektionskrankheiten der Tiere, Justus-Liebig-Universität,
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Institute of Food Quality and Food Safety,University of Veterinary Medicine
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Abstract
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In the present study 28 Arcanobacterium pluranimalium strains isolated from
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various origins could successfully be identified with a newly designed loop-mediated
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isothermal
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pluranimaliumlysin. No comparable reaction could be observed with control strains
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representing five species of genus Arcanobacterium and five species of genus
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Trueperella. The presented pla LAMP assay might allow a reliable and low cost
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identification of this bacterial pathogen also in laboratories with less specified
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equipment.
(LAMP)
assay
based
on
gene
pla
encoding
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amplification
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Keywords: Arcanobacterium pluranimalium, loop-mediated isothermal amplification,
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pluranimaliumlysin, pla.
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1. Introduction Genus Arcanobacterium was originally described for the generally human pathogenic
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organism Arcanobacterium haemolyticum [1]. According to Yassin et al. [2] this
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genus consists of three additional species namely Arcanobacterium hippocoleae,
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Arcanobacterium phocae and Arcanobacterium pluranimalium. More recently
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Arcanobacterium canis, and Arcanobacterium phocisimile were described as novel
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species of this genus [3,4]. The closely related species Arcanobacterium pyogenes,
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Arcanobacterium
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bialowiezense and Arcanobacterium bonasi were reclassified to the newly described
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genus Trueperella as Trueperella pyogenes, Trueperella abortisuis, Trueperella
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bernardiae, Trueperella bialowiezensis and Trueperella bonasi [2]. However, both
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genera
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(http://www.ncbi.nlm.nih.gov/taxonomy).
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Arcanobacterium pluranimalium, initially described for isolates cultured from a
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harbour porpoise and a fallow deer, respectively [5], could also be isolated from a
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dog with pyoderma [6], from ovine specimen on 33 occasions [7], from bovine
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mastitis [7,8] and more recently also from a juvenile giraffe following necropsy [9].
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The identification of A. pluranimalium was performed phenotypically, partly by Matrix-
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assisted Laser Desorption Ionization Time of Flight (MALDI TOF) analysis and 16S
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rDNA sequencing and by Moser et al. [8] and Balbutskaya et al. [10] by sequencing
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gene pla encoding pluranimaliumlysin. This novel target gene appears to be highly
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specific and could be used for genotypic identification of this species. However,
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MALDI TOF and sequencing of bacterial target genes require specialized equipment
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which is not always available in diagnostic laboratories and clinics. However, for
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diagnostic laboratories without specific equipment other rapid, sensitive and cost effective
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methods are required to complement the generally first choice sequencing of species specific
Arcanobacterium
bernardiae,
Arcanobacterium
to
the
family
Actinomycetaceae
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ACCEPTED MANUSCRIPT target genes. A promising candidate for reliable identification of bacterial pathogens
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appears to be an isothermal DNA amplification called loop-mediated isothermal
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amplification (LAMP) [11].
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The present study was designed to investigate a newly developed LAMP assay
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targeting the pluranimaliumlysin encoding gene pla for identification of novel and
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previously described A. pluranimalium strains of various origins.
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2.
Materials and Methods
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2.1.
Bacterial strains
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The strains used in the present study included the reference strain A. pluranimalium
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DSM 13483, other presently available type strains and other strains of genus
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Arcanobacterium (n=8) and Trueperella (n=10) obtained from the strain collection of
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the Institute of Pharmacology and Toxicology at Justus Liebig University Giessen,
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Germany, and field strains of A. pluranimalium strains from ovine (n=23), bovine
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(n=3) [7,10] origin and from a juvenile giraffe (n=1) [9], (Table 2).
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2.2.
DNA extraction and template preparation
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The bacterial DNA was isolated using the DNeasy tissue isolation kit (Qiagen,
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Germany) according to the manufacturer’s instructions. Briefly, 3 colonies of freshly
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cultivated bacteria on sheep blood agar (Oxoid Deutschland GmbH, Wesel,
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Germany) for(48h at 37°C in a candle jar) were lyzed, as recommended by Qiagen
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for gram positive bacteria.The DNA was eluted with 200 µl elution buffer.
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In parallel, as a rapid and simple processing step, one freshly cultivated bacterial
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colony was picked up, suspended into a tube containing 500 µl HYPLEX® LPTV
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buffer tube (Amplex Diagnostics, Germany) and boiled for 10 min using a heatblock.
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Subsequently 3 µl of this suspension was used as template.
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2.3.
Design of oligonucleotide primers for LAMP assay
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The development of the oligonucleotide primers for the LAMP assay was based on
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the sequence of gene pla encoding pluranimaliumlysin of A. pluranimalium DSM
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13483 reference strain published in the National Center for Biotechnology (NCBI)
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GenBank under accession no. FR745890.1
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A set of six oligonucleotide primers, including two outer primers (forward primer
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Pluran-F3 and backward primer Pluran-B3), two inner primers (forward inner primer
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Pluran-FIP and backward inner primer Pluran-BIP), and two loop primers (forward
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loop primer Pluran-LoopF and backward loop primer Pluran-LoopB), were designed
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using LAMP Designer software, ver. 1.10 (PREMIER Biosoft, USA) (Table 1). These
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primers were selected to be specific for A. pluranimalium pla gene. The
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oligonucleotide primers were synthesized by Eurofins MWG Operon (Germany). For
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investigating the specificity the oligonucleotide sequences were submitted to the
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NCBI GenBank using LAMP Designer software.
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2.4.
LAMP reaction and amplification condition
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The LAMP reaction was carried out with a total volume of 25 µl of the reaction
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mixture containing 0.5 µl each of Pluran-F3 and Pluran-B3 primer (25 pmol/µl)
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equivalent to 0.5 µM final concentration, 2.0 µl each of Pluran-FIP and Pluran-BIP
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primer (25 pmol/µl) equivalent to 2 µM final concentration, 1.0 µl each of Pluran-
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LoopF and Pluran-LoopB primer (25 pmol/µl) equivalent to 1 µM final concentration
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and 15 µl Isothermal Master Mix Iso-001 (Optigene, UK). Subsequently 3 µl DNA was
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added as a template. The LAMP assay was run at 72°C for 30 min with a melting
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curve analysis step (annealing curve 98°-80°C ramping at 0.1 per min) in a portable
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ACCEPTED MANUSCRIPT real-time fluorometer (Genie II®, Optigene) according to the manufacturers
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instructions.
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The LAMP reaction was also implemented in 1.5 ml tubes using a heatblock at 72°C
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for 30 min. The presence of LAMP products was determined by electrophoresis of 10
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µl of the reaction products in a 2% agarose gel (PeQlab, Germany), with Trisacetate-
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electrophoresis buffer (TAE, pH 7.8) and a 100-bp DNA ladder (Roche Diagnostic,
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Germany) as molecular marker.
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2.5.
Analytic sensitivity of the LAMP assay and limit of detection (LOD)
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For the analytic sensitivity LAMP reaction of a serially diluted DNA isolated from
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reference strain A. pluranimalium DSM 13483 was performed using the conditions
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mentioned above. Serial dilutions (10-1-10-4) were prepared using Tris buffer (TE, pH
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8.0). The amount of DNA was estimated by using QubiTM Fluorometer (Invitrogen,
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Germany). In addition, the LOD was estimated by preparation of a serial dilutions (10-
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1
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the dilution steps was isolated using HYPLEX® LPTV buffer.
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2.6.
Positive and negative predictive values (PPV and NPV)
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The PPV was calculated as: (number of true positives)/(number of true positives +
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number of false positives) × 100, and the NPV was calculated as: (number of true
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negatives)/(number of true negatives + number of false negatives) × 100. The
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accuracy was calculated as: (number of true positives + number of true
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negatives)/(total number of samples) × 100.
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3.
Results and Discussion 6
ACCEPTED MANUSCRIPT The application of LAMP assays for detection of food borne bacterial pathogens and
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toxicants as well as mycotoxin producing food borne fungi has been reviewed by
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Niessen et al. [12]. This technique has also been used to identify Leptospira spp.
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[13], Erysipelothrix rhusiopathiae [14], Streptococcus equi subsp. zooepidemicus [15]
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and for identification of ostrich meat [16]. All these authors describe LAMP assays as
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an alternative to the use of PCR-based methods with a high sensitivity and
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specificity, a shorter reaction time and a comparably low susceptibility for inhibitors.
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The newly developed LAMP assay of the present study, targeting pluranimaliumlysin
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encoding gene pla, provided a rapid and reliable identification of all 27 A.
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pluranimalium field strains isolated from various origins and the type strain A.
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pluranimalium DSM 13483. This could be demonstrated using a heatblock and
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subsequent detection of the LAMP product in an agarose gel (Figure 1) or by using a
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real-time fluorometer. Melting curve analysis, which is termed by Genie II® as anneal
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curve analysis, revealed no significant differences among the different A.
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pluranimalium strains. The melting temperature of the A. pluranimalium specific
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amplicon was 87.40°C (± 0.16 °C) (Figure 2a and b).
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The amplification rate (detection probability) and the analytical sensitivity of the
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LAMP assay were calculated on the basis of a dilution row with DNA ranging from 5
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ng/µl (100) to 0.5 pg/ µl (10-4) of A. pluranimalium DNA yielding an analytical
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sensitivity of the LAMP assay at 5 pg/reaction. The detection probability was
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determined to be 80% (four of five were positive). This concentration could be
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visualized by a detection time of 24:37 (± 4:19) min (Table 3). The LOD for this assay
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was as little as 2 CFU / reaction of A. pluranimalium (without enrichment step) with a
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detection probability of 100 %. This was detectable after 23:09 (± 4:14) min (Table 4).
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This results demonstrated the high sensitivety of this assay. However, using the
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agarose gel electrophoresis detection method, aerosolized products, which might be
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caused by multiple handling of the amplicons, might lead to an elevated risk of cross
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contamination of subsequent samples (i.e. false-positive results).
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The control strains (n=18) representing the further 5 species of
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Arcanobacterium and the 5 species of genus Trueperella revealed no cross
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reactions, indicating that the pla LAMP assay of the present study is a highly specific
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tool for detection of A. pluranimalium suitable for use in laboratories that lack more
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specialised equipment. Comparable to the pla LAMP assay of the present study,
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Zhang et al. [17] used the T. pyogenes pyolysin encoding gene plo, which is well
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known as a universal target gene for molecular identification of T. pyogenes ([18-21],
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for development of a LAMP assay yielding a specific reaction for T. pyogenes.
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However, these authors used only a limited number of T. pyogenes strains and no
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control strains of closely related species of genus Trueperella and Arcanobacterium.
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The calculated PPV and NPV were 100% with the investigated strains. The pla LAMP
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assay of the present study allowed a correct identification of A. pluranimalium using
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extracted DNA of the bacterial strains or by suspension of the bacteria in HYPLEX®
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LPTV buffer proposed by the manufacturer. By using the real-time fluorometer the
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latter allowed an identification of A. pluranimalium within 30 min. The LAMP assay
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could also be performed by using a heat block available in many laboratories. The
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use of the pla LAMP assay of the present study might improve the identification of A.
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pluranimalium in future and might help to elucidate the role this bacterial pathogen
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plays in infections of animals and possibly in humans.
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ACCEPTED MANUSCRIPT Figure 1. Agarose gel electrophoresis of A. pluranimalium LAMP products. The positive reaction appeared as a ladder-like pattern (lane 1-3) ; lane 4 and 5 represent negative strains, lane 6 a non template control; lane M = 100 bp DNA ladder (Roche).
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Figure 2. Typical amplification signal of five A. pluranimalium LAMP products and a negative control (a). The melting curve (anneal reaction) of the same amplicons (b).
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ACCEPTED MANUSCRIPT ____________________________________________________
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*Corresponding author:
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PD Dr. med. vet. Amir Abdulmawjood
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Institute of Food Quality and Food Safety
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University of Veterinary Medicine Hannover
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Bischofsholer Damm 15, 30173 Hannover, Germany
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Fax: +49-(0)511-953-7694
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Email: [email protected]
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References [1] Collins MD, Jones D, Schofield GM. (1982) Reclassification of 'Corynebacterium haemolyticum' (MacLean, Liebow & Rosenberg) in the genus Arcanobacterium gen.nov. as Arcanobacterium haemolyticum nom.rev., comb.nov. Journal of general microbiology. 128: 1279-1281.
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[2] Yassin AF, Hupfer H, Siering C, Schumann P. (2011) Comparative chemotaxonomic and phylogenetic studies on the genus Arcanobacterium Collins et al. 1982 emend. Lehnen et al. 2006: proposal for Trueperella gen. nov. and emended description of the genus Arcanobacterium. International journal of systematic and evolutionary microbiology. 61: 1265-1274.
209 210 211 212 213
[3] Hijazin M, Prenger-Berninghoff E, Sammra O, Alber J, Lämmler C, Kämpfer P, et al. (2012) Arcanobacterium canis sp. nov., isolated from otitis externa of a dog, and emended description of the genus Arcanobacterium Collins et al. 1983 emend. Yassin et al. 2011. International journal of systematic and evolutionary microbiology. 62: 2201-2205.
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[4] Hijazin M, Sammra O, Ülbegi-Mohyla H, Nagib S, Alber J, Lämmler C, et al. (2013) Arcanobacterium phocisimile sp. nov., isolated from harbour seals. International journal of systematic and evolutionary microbiology. 63: 2019-2024.
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[5] Lawson PA, Falsen E, Foster G, Eriksson E, Weiss N, Collins MD. (2001) Arcanobacterium pluranimalium sp. nov., isolated from porpoise and deer. International journal of systematic and evolutionary microbiology. 51: 55-59.
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[6] Ülbegi-Mohyla H, Hassan AA, Alber J, Lämmler C, Prenger-Berninghoff E, Weiss R, et al. (2010) Identification of Arcanobacterium pluranimalium isolated from a dog by phenotypic properties and by PCR mediated characterization of various molecular targets. Veterinary microbiology. 142: 458-460.
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[7] Foster G, Hunt B. (2011) Distribution of Arcanobacterium pluranimalium in animals examined in veterinary laboratories in the United Kingdom. Journal of veterinary diagnostic investigation. 23: 962-964.
227 228 229 230
[8] Moser A, Stephan R, Sager J, Corti S, Lehner A. (2013) Arcanobacterium pluranimalium leading to a bovine mastitis: species identification by a newly developed pla gene based PCR. Schweizer Archiv für Tierheilkunde. 155: 373-375.
231 232 233 234
[9] Risse K, Schlez K, Eisenberg T, Geiger C, Balbutskaya A, Sammra O, et al. (2014) Phenotypical and genotypical properties of an Arcanobacterium pluranimalium strain isolated from a juvenile giraffe (Giraffa camelopardalis reticulata). Journal of veterinary medicine. 2014: 1-5.
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[10] Balbutskaya A, Sammra O, Nagib S, Hijazin M, Alber J, Lämmler C, et al. (2014) Identification of Arcanobacterium pluranimalium by matrix-assisted laser desorption ionization-time of flight mass spectrometry and, as novel target, by sequencing pluranimaliumlysin encoding gene pla. Veterinary microbiology. 168: 428-431.
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ACCEPTED MANUSCRIPT [11] Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. (2000) Loop-mediated isothermal amplification of DNA. Nucleic acids research. 28: e63.
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[12] Niessen L, Luo J, Denschlag C, Vogel RF. (2013) The application of loop-mediated isothermal amplification (LAMP) in food testing for bacterial pathogens and fungal contaminants. Food microbiology. 36: 191-206.
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[13] Koizumi N, Nakajima C, Harunari T, Tanikawa T, Tokiwa T, Uchimura E, et al. (2012) A new loop-mediated isothermal amplification method for rapid, simple, and sensitive detection of Leptospira spp. in urine. Journal of clinical microbiology. 50: 2072-2074.
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[14] Yamazaki Y, Oba E, Kashiwagi N, Sugita K, Shiiba K, Baba Y, et al. (2014) Development of a loop-mediated isothermal amplification assay for rapid and simple detection of Erysipelothrix rhusiopathiae. Letters in applied microbiology. 58: 362-369.
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[15] Kinoshita Y, Niwa H, Katayama Y. (2014) Development of a loopmediated isothermal amplification method for detecting Streptococcus equi subsp. zooepidemicus and analysis of its use with three simple methods of extracting DNA from equine respiratory tract specimens. The Journal of veterinary medical science. 76: 1271-1275.
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[16] Abdulmawjood A, Grabowski N, Fohler S, Kittler S, Nagengast H, Klein G. (2014) Development of loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive identification of ostrich meat. PloS one. 9: e100717.
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[17] Zhang W, Meng X, Wang J. (2013) Sensitive and rapid detection of Trueperella pyogenes using loop-mediated isothermal amplification method. Journal of microbiological methods. 93: 124-126.
265 266 267 268
[18] Billington SJ, Jost BH, Cuevas WA, Bright KR, Songer JG. (1997) The Arcanobacterium (Actinomyces) pyogenes hemolysin, pyolysin, is a novel member of the thiol-activated cytolysin family. Journal of bacteriology. 179: 61006106.
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[19] Ertaş HB, Kiliç A, Özbey G, Muz A. (2005) Isolation of Arcanobacterium (Actinomyces) pyogenes from abscessed cattle kidney and identification by PCR. Turkish Journal of veterinary and animal sciences. 29: 455-459.
272 273 274 275
[20] Ülbegi-Mohyla H, Hijazin M, Alber J, Lämmler C, Hassan AA, Abdulmawjood A, et al. (2010) Identification of Arcanobacterium pyogenes isolated by post mortem examinations of a bearded dragon and a gecko by phenotypic and genotypic properties. Journal of veterinary science. 11: 265-267.
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[21] Hijazin M, Ülbegi-Mohyla H, Alber J, Lämmler C, Hassan AA, Abdulmawjood A, et al. (2011) Molecular identification and further characterization of Arcanobacterium pyogenes isolated from bovine mastitis and from various other origins. Journal of dairy science. 94: 1813-9.
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ACCEPTED MANUSCRIPT Table 1. Oligonucleotide primer sequences used for the A. pluranimalium LAMP assay
Sequence
Primer length
Melting temperature
Pluran-F3
5`-GCT CCA GAA GTT ATC AGT GTT-`3
21 bp
55.9°C
Pluran-B3
5`-TTC GAA GTT CAC GTT GAG TT-`3
20 bp
53.2°C
Pluran-FIP
5`-TAA CTC CTT GGT CAA CGG TGC TCT ACC AGG ATT GAC GCA-`3
39 bp
73.7°C
Pluran-BIP
5`-GAC GGA TCG CCA TGA CAA GTG AGT TAA CCA TAG ACT CGT CG-`3
41 bp
74.4°C
Pluran-LoopF
5`-CGA AGG ATT TGG TAC GGT CA-`3
20 bp
57.3°C
Pluran-LoopB
5`-GCA CGC AGC CAA GAT TTC-`3
18 bp
56.0°C
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Primer name
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Table 2. Inclusivity and exclusivity test of the A. pluranimalium LAMP assay based on pla gene
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for pluranimaliumlysin using A. pluranimalium and non- A. pluranimalium strains.
Strain number/Origin
Reference
Detection time mm:ss
Melting temperature
1
A. pluranimalium
DSM 13483T/harbour purpoise
Lawson et al., 2001
11:00
87.71
2
A. pluranimalium
S601620-01-1/sheep
Foster and Hunt, 2011
10:15
87.71
3
A. pluranimalium
S214961-11-1/sheep
Balbutskaya et al., 2014 10:15
87.71
4
A. pluranimalium
S100813-01-1/sheep
Foster and Hunt, 2011
11:00
87.56
5
A. pluranimalium
S214809-11-1/sheep
Balbutskaya et al., 2014 10:15
87.56
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Species
A. pluranimalium
S704129-03-1/sheep
Foster and Hunt, 2011
12:45
87.46
A. pluranimalium
S604731-06-1/sheep
Foster and Hunt, 2011
13:00
87.45
8
A. pluranimalium
S702414-02-1/sheep
Foster and Hunt, 2011
13:15
87.51
9
A. pluranimalium
S215069-11-1/sheep
Balbutskaya et al., 2014 13:00
87.51
10
A. pluranimalium
S420838-12-1/sheep
Balbutskaya et al., 2014 09:45
87.51
11
A. pluranimalium
S1316-98-1/sheep
Foster and Hunt, 2011
10:45
87.51
12
A. pluranimalium
S600993-00-1/sheep
Foster and Hunt, 2011
13:45
87.40
13
A. pluranimalium
S603257-04-1/sheep
Foster and Hunt, 2011
14,15
87.22
14
A. pluranimalium
S603973-05-1/sheep
Foster and Hunt, 2011
14:15
87.37
15
A. pluranimalium
S005507-05-1/sheep
Foster and Hunt, 2011
16:15
87.27
16
A. pluranimalium
S704483-04-1/sheep
Foster and Hunt, 2011
13:30
87.27
17
A. pluranimalium
S603952-05-1/sheep
Foster and Hunt, 2011
14:45
87.26
18
A. pluranimalium
S607005-08-1/sheep
Foster and Hunt, 2011
13:15
87.12
19
A. pluranimalium
S609650-12-1/sheep
Balbutskaya et al., 2014 11:45
87.31
20
A. pluranimalium
S004572-04-1/sheep
Foster and Hunt, 2011
14:15
87.41
21
A. pluranimalium
S111911-12-1/sheep
Foster and Hunt, 2011
13:15
87.17
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A. pluranimalium
S501967-00-1/sheep
Foster and Hunt, 2011
14:00
87.17
A. pluranimalium
S601452-01-2/sheep
Foster and Hunt, 2011
13:45
87.41
24
A. pluranimalium
S603814-04-1/sheep
Foster and Hunt, 2011
15:15
87.27
25
A. pluranimalium
24-C359-11-10/bovine
Balbutskaya et al., 2014 14:00
87.27
26
A. pluranimalium
D13-0340-5-11/bovine
Balbutskaya et al., 2014 11:15
87.26
27
A. pluranimalium
D12-0439-1-1-1/bovine
Balbutskaya et al., 2014 12:30
87.31
28
A. pluranimalium
4868/giraffe
Risse et al., 2014
87.56
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13:45
Non- A. pluranimalium strains 1
A. canis
DSM 25104 T
2
A. canis
ZT 11003002
3
A. haemolyticum
DSM 20595T
4
A. hippocoleae
DSM 15539T
5
A. phocae
DSM 10002T
6
A. phocae
DSM 10003
7
A. phocisimile
DSM 26142T
8
A. phocisimile
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14
9
T. abortisuis
DSM 19515T
10
T. bernardiae
DSM 9152 T
11
T. bialowiezensis
DSM 17162T
12
T. bonasi
DSM 17163T
13
T. pyogenes
DSM 20630T
14
T. pyogenes
DSM 20594
15
T. pyogenes
59/11
16
T. pyogenes
336/1
17
T. pyogenes
1065/41
18
T. pyogenes
506/74
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Table 3: Analytical sensitivity of the LAMP assay using serial dilutions of DNA of A.
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pluranimalium DSM 13483
Dilution DNA amount step pg/µl
Detection time (h:mm:ss) run 1
run 2
run 3
run 4
Mean (Sd ±) run 5
Detection probability
500
0:14:15 0:21:45 0:16:30 0:17:30 0:15:30
0:17:06 (0:02:52)
100 %
10
-2
50
0:29:30 0:29:00 0:17:15 0:18:15 0:28:45
0:24:33 (0:06:13)
100 %
10
-3
5
0:26:15 0:26:15 0:18:15 0:27:45
0:24:37 (0:04:19)
80 %
-
0%
10-4
0.5
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Table 4: Limit of detection (LOD) of the LAMP assay using serial dilutions of A.
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pluranimalium DSM 13483 reference strain.
Detection time (h:mm:ss)
cfu / reaction
Mean (Sd ±)
Detection probability
run 2
run 3
run 4
run 5
2.2 x10
10:45
13:00
15:30
16:15
11:30
13:24 (02:10)
100 %
2.2 x10
2
15:30
17:00
17:30
18:30
13:15
16:21 (01:50)
100 %
2.2 x10
1
18:00
18:45
20:15
22:15
15:30
18:57 (02:15)
100 %
2.2 x100
19:45
28:45
27:00
22:45
17:30
23:09 (04:14)
100 %
2.2 x10-1
-
-
-
24:30
19:30
22:00 (02:30)
40 %
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1000 bp
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Figure 1. Agarose gel electrophoresis of A. pluranimalium LAMP products. The positive reaction appeared as a ladder-like pattern (lane 1-3) ; lane 4 and 5 represent
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negative strains, lane 6 a non template control; lane M = 100 bp DNA ladder (Roche).
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(a)
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24-C359-11-10 S604731-06-1 S600993-00-1 S603257-04-1 Positive control Negative control
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Figure 2. Typical amplification signal of five A. pluranimalium LAMP products and a negative control (a). The melting curve (anneal reaction) of the same amplicons (b).
ACCEPTED MANUSCRIPT Highlights:
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o A real-time fluorogenic LAMP assay for detection of Arcanobacterium pluranimalium was developed o This assay detects 2 cfu / reaction within 30 min with high specificity and sensitivity. o The used method has advantages of simplicity and inexpensiveness of operation.
S0890-8508(15)30016-5
DOI:
10.1016/j.mcp.2015.06.003
Reference:
YMCPR 1144
To appear in:
Molecular and Cellular Probes
Received Date: 18 May 2015 Revised Date:
12 June 2015
Accepted Date: 12 June 2015
Please cite this article as: Abdulmawjood A, Wickhorst J, Sammra O, Lämmler C, Foster G, Wragg PN, Prenger-Berninghoff E, Klein G, Development of a loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive identification of Arcanobacterium pluranimalium, Molecular and Cellular Probes (2015), doi: 10.1016/j.mcp.2015.06.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Development of a loop-mediated isothermal amplification (LAMP) assay for
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rapid and sensitive identification of Arcanobacterium pluranimalium
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A. Abdulmawjooda, J. Wickhorstb, O. Sammrab, C. Lämmlerb, G. Fosterc, P. N.
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Wraggd, E. Prenger-Berninghoffe, G. Kleina
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a
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Hannover
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Bischofsholer Damm 15, 30173 Hannover, Germany
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b
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Schubertstr 81, 35392 Gießen, Germany c
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IV2 4JZ, UK
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d
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Merrythought, Calthwaite, Penrith CA11 9RR, UK
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e
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Frankfurterstr. 85-91, 35392 Gießen, Germany
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Institut für Pharmakologie und Toxikologie, Justus-Liebig-Universität Gießen,
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SAC Consulting Veterinary Services, Drummondhill, Stratherrick Road, Inverness
Animal Health and Veterinary Laboratories Agency, Penrith Regional Laboratory,
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Institut für Hygiene und Infektionskrankheiten der Tiere, Justus-Liebig-Universität,
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Institute of Food Quality and Food Safety,University of Veterinary Medicine
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Abstract
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In the present study 28 Arcanobacterium pluranimalium strains isolated from
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various origins could successfully be identified with a newly designed loop-mediated
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isothermal
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pluranimaliumlysin. No comparable reaction could be observed with control strains
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representing five species of genus Arcanobacterium and five species of genus
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Trueperella. The presented pla LAMP assay might allow a reliable and low cost
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identification of this bacterial pathogen also in laboratories with less specified
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equipment.
(LAMP)
assay
based
on
gene
pla
encoding
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amplification
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Keywords: Arcanobacterium pluranimalium, loop-mediated isothermal amplification,
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pluranimaliumlysin, pla.
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1. Introduction Genus Arcanobacterium was originally described for the generally human pathogenic
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organism Arcanobacterium haemolyticum [1]. According to Yassin et al. [2] this
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genus consists of three additional species namely Arcanobacterium hippocoleae,
36
Arcanobacterium phocae and Arcanobacterium pluranimalium. More recently
37
Arcanobacterium canis, and Arcanobacterium phocisimile were described as novel
38
species of this genus [3,4]. The closely related species Arcanobacterium pyogenes,
39
Arcanobacterium
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bialowiezense and Arcanobacterium bonasi were reclassified to the newly described
41
genus Trueperella as Trueperella pyogenes, Trueperella abortisuis, Trueperella
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bernardiae, Trueperella bialowiezensis and Trueperella bonasi [2]. However, both
43
genera
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(http://www.ncbi.nlm.nih.gov/taxonomy).
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Arcanobacterium pluranimalium, initially described for isolates cultured from a
46
harbour porpoise and a fallow deer, respectively [5], could also be isolated from a
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dog with pyoderma [6], from ovine specimen on 33 occasions [7], from bovine
48
mastitis [7,8] and more recently also from a juvenile giraffe following necropsy [9].
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The identification of A. pluranimalium was performed phenotypically, partly by Matrix-
50
assisted Laser Desorption Ionization Time of Flight (MALDI TOF) analysis and 16S
51
rDNA sequencing and by Moser et al. [8] and Balbutskaya et al. [10] by sequencing
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gene pla encoding pluranimaliumlysin. This novel target gene appears to be highly
53
specific and could be used for genotypic identification of this species. However,
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MALDI TOF and sequencing of bacterial target genes require specialized equipment
55
which is not always available in diagnostic laboratories and clinics. However, for
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diagnostic laboratories without specific equipment other rapid, sensitive and cost effective
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methods are required to complement the generally first choice sequencing of species specific
Arcanobacterium
bernardiae,
Arcanobacterium
to
the
family
Actinomycetaceae
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abortisuis,
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ACCEPTED MANUSCRIPT target genes. A promising candidate for reliable identification of bacterial pathogens
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appears to be an isothermal DNA amplification called loop-mediated isothermal
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amplification (LAMP) [11].
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The present study was designed to investigate a newly developed LAMP assay
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targeting the pluranimaliumlysin encoding gene pla for identification of novel and
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previously described A. pluranimalium strains of various origins.
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2.
Materials and Methods
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2.1.
Bacterial strains
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The strains used in the present study included the reference strain A. pluranimalium
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DSM 13483, other presently available type strains and other strains of genus
69
Arcanobacterium (n=8) and Trueperella (n=10) obtained from the strain collection of
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the Institute of Pharmacology and Toxicology at Justus Liebig University Giessen,
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Germany, and field strains of A. pluranimalium strains from ovine (n=23), bovine
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(n=3) [7,10] origin and from a juvenile giraffe (n=1) [9], (Table 2).
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2.2.
DNA extraction and template preparation
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The bacterial DNA was isolated using the DNeasy tissue isolation kit (Qiagen,
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Germany) according to the manufacturer’s instructions. Briefly, 3 colonies of freshly
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cultivated bacteria on sheep blood agar (Oxoid Deutschland GmbH, Wesel,
78
Germany) for(48h at 37°C in a candle jar) were lyzed, as recommended by Qiagen
79
for gram positive bacteria.The DNA was eluted with 200 µl elution buffer.
80
In parallel, as a rapid and simple processing step, one freshly cultivated bacterial
81
colony was picked up, suspended into a tube containing 500 µl HYPLEX® LPTV
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buffer tube (Amplex Diagnostics, Germany) and boiled for 10 min using a heatblock.
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Subsequently 3 µl of this suspension was used as template.
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2.3.
Design of oligonucleotide primers for LAMP assay
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The development of the oligonucleotide primers for the LAMP assay was based on
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the sequence of gene pla encoding pluranimaliumlysin of A. pluranimalium DSM
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13483 reference strain published in the National Center for Biotechnology (NCBI)
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GenBank under accession no. FR745890.1
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A set of six oligonucleotide primers, including two outer primers (forward primer
91
Pluran-F3 and backward primer Pluran-B3), two inner primers (forward inner primer
92
Pluran-FIP and backward inner primer Pluran-BIP), and two loop primers (forward
93
loop primer Pluran-LoopF and backward loop primer Pluran-LoopB), were designed
94
using LAMP Designer software, ver. 1.10 (PREMIER Biosoft, USA) (Table 1). These
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primers were selected to be specific for A. pluranimalium pla gene. The
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oligonucleotide primers were synthesized by Eurofins MWG Operon (Germany). For
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investigating the specificity the oligonucleotide sequences were submitted to the
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NCBI GenBank using LAMP Designer software.
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2.4.
LAMP reaction and amplification condition
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The LAMP reaction was carried out with a total volume of 25 µl of the reaction
102
mixture containing 0.5 µl each of Pluran-F3 and Pluran-B3 primer (25 pmol/µl)
103
equivalent to 0.5 µM final concentration, 2.0 µl each of Pluran-FIP and Pluran-BIP
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primer (25 pmol/µl) equivalent to 2 µM final concentration, 1.0 µl each of Pluran-
105
LoopF and Pluran-LoopB primer (25 pmol/µl) equivalent to 1 µM final concentration
106
and 15 µl Isothermal Master Mix Iso-001 (Optigene, UK). Subsequently 3 µl DNA was
107
added as a template. The LAMP assay was run at 72°C for 30 min with a melting
108
curve analysis step (annealing curve 98°-80°C ramping at 0.1 per min) in a portable
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110
instructions.
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The LAMP reaction was also implemented in 1.5 ml tubes using a heatblock at 72°C
112
for 30 min. The presence of LAMP products was determined by electrophoresis of 10
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µl of the reaction products in a 2% agarose gel (PeQlab, Germany), with Trisacetate-
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electrophoresis buffer (TAE, pH 7.8) and a 100-bp DNA ladder (Roche Diagnostic,
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Germany) as molecular marker.
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2.5.
Analytic sensitivity of the LAMP assay and limit of detection (LOD)
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For the analytic sensitivity LAMP reaction of a serially diluted DNA isolated from
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reference strain A. pluranimalium DSM 13483 was performed using the conditions
121
mentioned above. Serial dilutions (10-1-10-4) were prepared using Tris buffer (TE, pH
122
8.0). The amount of DNA was estimated by using QubiTM Fluorometer (Invitrogen,
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Germany). In addition, the LOD was estimated by preparation of a serial dilutions (10-
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1
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the dilution steps was isolated using HYPLEX® LPTV buffer.
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-10-6) of the reference strain. The cfu was subsequently estimated and the DNA of
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2.6.
Positive and negative predictive values (PPV and NPV)
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The PPV was calculated as: (number of true positives)/(number of true positives +
129
number of false positives) × 100, and the NPV was calculated as: (number of true
130
negatives)/(number of true negatives + number of false negatives) × 100. The
131
accuracy was calculated as: (number of true positives + number of true
132
negatives)/(total number of samples) × 100.
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3.
Results and Discussion 6
ACCEPTED MANUSCRIPT The application of LAMP assays for detection of food borne bacterial pathogens and
136
toxicants as well as mycotoxin producing food borne fungi has been reviewed by
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Niessen et al. [12]. This technique has also been used to identify Leptospira spp.
138
[13], Erysipelothrix rhusiopathiae [14], Streptococcus equi subsp. zooepidemicus [15]
139
and for identification of ostrich meat [16]. All these authors describe LAMP assays as
140
an alternative to the use of PCR-based methods with a high sensitivity and
141
specificity, a shorter reaction time and a comparably low susceptibility for inhibitors.
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The newly developed LAMP assay of the present study, targeting pluranimaliumlysin
143
encoding gene pla, provided a rapid and reliable identification of all 27 A.
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pluranimalium field strains isolated from various origins and the type strain A.
145
pluranimalium DSM 13483. This could be demonstrated using a heatblock and
146
subsequent detection of the LAMP product in an agarose gel (Figure 1) or by using a
147
real-time fluorometer. Melting curve analysis, which is termed by Genie II® as anneal
148
curve analysis, revealed no significant differences among the different A.
149
pluranimalium strains. The melting temperature of the A. pluranimalium specific
150
amplicon was 87.40°C (± 0.16 °C) (Figure 2a and b).
151
The amplification rate (detection probability) and the analytical sensitivity of the
152
LAMP assay were calculated on the basis of a dilution row with DNA ranging from 5
153
ng/µl (100) to 0.5 pg/ µl (10-4) of A. pluranimalium DNA yielding an analytical
154
sensitivity of the LAMP assay at 5 pg/reaction. The detection probability was
155
determined to be 80% (four of five were positive). This concentration could be
156
visualized by a detection time of 24:37 (± 4:19) min (Table 3). The LOD for this assay
157
was as little as 2 CFU / reaction of A. pluranimalium (without enrichment step) with a
158
detection probability of 100 %. This was detectable after 23:09 (± 4:14) min (Table 4).
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This results demonstrated the high sensitivety of this assay. However, using the
160
agarose gel electrophoresis detection method, aerosolized products, which might be
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caused by multiple handling of the amplicons, might lead to an elevated risk of cross
162
contamination of subsequent samples (i.e. false-positive results).
163
The control strains (n=18) representing the further 5 species of
164
Arcanobacterium and the 5 species of genus Trueperella revealed no cross
165
reactions, indicating that the pla LAMP assay of the present study is a highly specific
166
tool for detection of A. pluranimalium suitable for use in laboratories that lack more
167
specialised equipment. Comparable to the pla LAMP assay of the present study,
168
Zhang et al. [17] used the T. pyogenes pyolysin encoding gene plo, which is well
169
known as a universal target gene for molecular identification of T. pyogenes ([18-21],
170
for development of a LAMP assay yielding a specific reaction for T. pyogenes.
171
However, these authors used only a limited number of T. pyogenes strains and no
172
control strains of closely related species of genus Trueperella and Arcanobacterium.
173
The calculated PPV and NPV were 100% with the investigated strains. The pla LAMP
174
assay of the present study allowed a correct identification of A. pluranimalium using
175
extracted DNA of the bacterial strains or by suspension of the bacteria in HYPLEX®
176
LPTV buffer proposed by the manufacturer. By using the real-time fluorometer the
177
latter allowed an identification of A. pluranimalium within 30 min. The LAMP assay
178
could also be performed by using a heat block available in many laboratories. The
179
use of the pla LAMP assay of the present study might improve the identification of A.
180
pluranimalium in future and might help to elucidate the role this bacterial pathogen
181
plays in infections of animals and possibly in humans.
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genus
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ACCEPTED MANUSCRIPT Figure 1. Agarose gel electrophoresis of A. pluranimalium LAMP products. The positive reaction appeared as a ladder-like pattern (lane 1-3) ; lane 4 and 5 represent negative strains, lane 6 a non template control; lane M = 100 bp DNA ladder (Roche).
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Figure 2. Typical amplification signal of five A. pluranimalium LAMP products and a negative control (a). The melting curve (anneal reaction) of the same amplicons (b).
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ACCEPTED MANUSCRIPT ____________________________________________________
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*Corresponding author:
192
PD Dr. med. vet. Amir Abdulmawjood
193
Institute of Food Quality and Food Safety
194
University of Veterinary Medicine Hannover
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Bischofsholer Damm 15, 30173 Hannover, Germany
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Fax: +49-(0)511-953-7694
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Email: [email protected]
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References [1] Collins MD, Jones D, Schofield GM. (1982) Reclassification of 'Corynebacterium haemolyticum' (MacLean, Liebow & Rosenberg) in the genus Arcanobacterium gen.nov. as Arcanobacterium haemolyticum nom.rev., comb.nov. Journal of general microbiology. 128: 1279-1281.
204 205 206 207 208
[2] Yassin AF, Hupfer H, Siering C, Schumann P. (2011) Comparative chemotaxonomic and phylogenetic studies on the genus Arcanobacterium Collins et al. 1982 emend. Lehnen et al. 2006: proposal for Trueperella gen. nov. and emended description of the genus Arcanobacterium. International journal of systematic and evolutionary microbiology. 61: 1265-1274.
209 210 211 212 213
[3] Hijazin M, Prenger-Berninghoff E, Sammra O, Alber J, Lämmler C, Kämpfer P, et al. (2012) Arcanobacterium canis sp. nov., isolated from otitis externa of a dog, and emended description of the genus Arcanobacterium Collins et al. 1983 emend. Yassin et al. 2011. International journal of systematic and evolutionary microbiology. 62: 2201-2205.
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[4] Hijazin M, Sammra O, Ülbegi-Mohyla H, Nagib S, Alber J, Lämmler C, et al. (2013) Arcanobacterium phocisimile sp. nov., isolated from harbour seals. International journal of systematic and evolutionary microbiology. 63: 2019-2024.
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[5] Lawson PA, Falsen E, Foster G, Eriksson E, Weiss N, Collins MD. (2001) Arcanobacterium pluranimalium sp. nov., isolated from porpoise and deer. International journal of systematic and evolutionary microbiology. 51: 55-59.
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[6] Ülbegi-Mohyla H, Hassan AA, Alber J, Lämmler C, Prenger-Berninghoff E, Weiss R, et al. (2010) Identification of Arcanobacterium pluranimalium isolated from a dog by phenotypic properties and by PCR mediated characterization of various molecular targets. Veterinary microbiology. 142: 458-460.
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[7] Foster G, Hunt B. (2011) Distribution of Arcanobacterium pluranimalium in animals examined in veterinary laboratories in the United Kingdom. Journal of veterinary diagnostic investigation. 23: 962-964.
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[8] Moser A, Stephan R, Sager J, Corti S, Lehner A. (2013) Arcanobacterium pluranimalium leading to a bovine mastitis: species identification by a newly developed pla gene based PCR. Schweizer Archiv für Tierheilkunde. 155: 373-375.
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[9] Risse K, Schlez K, Eisenberg T, Geiger C, Balbutskaya A, Sammra O, et al. (2014) Phenotypical and genotypical properties of an Arcanobacterium pluranimalium strain isolated from a juvenile giraffe (Giraffa camelopardalis reticulata). Journal of veterinary medicine. 2014: 1-5.
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[10] Balbutskaya A, Sammra O, Nagib S, Hijazin M, Alber J, Lämmler C, et al. (2014) Identification of Arcanobacterium pluranimalium by matrix-assisted laser desorption ionization-time of flight mass spectrometry and, as novel target, by sequencing pluranimaliumlysin encoding gene pla. Veterinary microbiology. 168: 428-431.
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ACCEPTED MANUSCRIPT [11] Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. (2000) Loop-mediated isothermal amplification of DNA. Nucleic acids research. 28: e63.
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[12] Niessen L, Luo J, Denschlag C, Vogel RF. (2013) The application of loop-mediated isothermal amplification (LAMP) in food testing for bacterial pathogens and fungal contaminants. Food microbiology. 36: 191-206.
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[13] Koizumi N, Nakajima C, Harunari T, Tanikawa T, Tokiwa T, Uchimura E, et al. (2012) A new loop-mediated isothermal amplification method for rapid, simple, and sensitive detection of Leptospira spp. in urine. Journal of clinical microbiology. 50: 2072-2074.
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[14] Yamazaki Y, Oba E, Kashiwagi N, Sugita K, Shiiba K, Baba Y, et al. (2014) Development of a loop-mediated isothermal amplification assay for rapid and simple detection of Erysipelothrix rhusiopathiae. Letters in applied microbiology. 58: 362-369.
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[15] Kinoshita Y, Niwa H, Katayama Y. (2014) Development of a loopmediated isothermal amplification method for detecting Streptococcus equi subsp. zooepidemicus and analysis of its use with three simple methods of extracting DNA from equine respiratory tract specimens. The Journal of veterinary medical science. 76: 1271-1275.
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[16] Abdulmawjood A, Grabowski N, Fohler S, Kittler S, Nagengast H, Klein G. (2014) Development of loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive identification of ostrich meat. PloS one. 9: e100717.
262 263 264
[17] Zhang W, Meng X, Wang J. (2013) Sensitive and rapid detection of Trueperella pyogenes using loop-mediated isothermal amplification method. Journal of microbiological methods. 93: 124-126.
265 266 267 268
[18] Billington SJ, Jost BH, Cuevas WA, Bright KR, Songer JG. (1997) The Arcanobacterium (Actinomyces) pyogenes hemolysin, pyolysin, is a novel member of the thiol-activated cytolysin family. Journal of bacteriology. 179: 61006106.
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[19] Ertaş HB, Kiliç A, Özbey G, Muz A. (2005) Isolation of Arcanobacterium (Actinomyces) pyogenes from abscessed cattle kidney and identification by PCR. Turkish Journal of veterinary and animal sciences. 29: 455-459.
272 273 274 275
[20] Ülbegi-Mohyla H, Hijazin M, Alber J, Lämmler C, Hassan AA, Abdulmawjood A, et al. (2010) Identification of Arcanobacterium pyogenes isolated by post mortem examinations of a bearded dragon and a gecko by phenotypic and genotypic properties. Journal of veterinary science. 11: 265-267.
276 277 278 279
[21] Hijazin M, Ülbegi-Mohyla H, Alber J, Lämmler C, Hassan AA, Abdulmawjood A, et al. (2011) Molecular identification and further characterization of Arcanobacterium pyogenes isolated from bovine mastitis and from various other origins. Journal of dairy science. 94: 1813-9.
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ACCEPTED MANUSCRIPT Table 1. Oligonucleotide primer sequences used for the A. pluranimalium LAMP assay
Sequence
Primer length
Melting temperature
Pluran-F3
5`-GCT CCA GAA GTT ATC AGT GTT-`3
21 bp
55.9°C
Pluran-B3
5`-TTC GAA GTT CAC GTT GAG TT-`3
20 bp
53.2°C
Pluran-FIP
5`-TAA CTC CTT GGT CAA CGG TGC TCT ACC AGG ATT GAC GCA-`3
39 bp
73.7°C
Pluran-BIP
5`-GAC GGA TCG CCA TGA CAA GTG AGT TAA CCA TAG ACT CGT CG-`3
41 bp
74.4°C
Pluran-LoopF
5`-CGA AGG ATT TGG TAC GGT CA-`3
20 bp
57.3°C
Pluran-LoopB
5`-GCA CGC AGC CAA GAT TTC-`3
18 bp
56.0°C
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Primer name
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Table 2. Inclusivity and exclusivity test of the A. pluranimalium LAMP assay based on pla gene
285
for pluranimaliumlysin using A. pluranimalium and non- A. pluranimalium strains.
Strain number/Origin
Reference
Detection time mm:ss
Melting temperature
1
A. pluranimalium
DSM 13483T/harbour purpoise
Lawson et al., 2001
11:00
87.71
2
A. pluranimalium
S601620-01-1/sheep
Foster and Hunt, 2011
10:15
87.71
3
A. pluranimalium
S214961-11-1/sheep
Balbutskaya et al., 2014 10:15
87.71
4
A. pluranimalium
S100813-01-1/sheep
Foster and Hunt, 2011
11:00
87.56
5
A. pluranimalium
S214809-11-1/sheep
Balbutskaya et al., 2014 10:15
87.56
RI PT
Species
A. pluranimalium
S704129-03-1/sheep
Foster and Hunt, 2011
12:45
87.46
A. pluranimalium
S604731-06-1/sheep
Foster and Hunt, 2011
13:00
87.45
8
A. pluranimalium
S702414-02-1/sheep
Foster and Hunt, 2011
13:15
87.51
9
A. pluranimalium
S215069-11-1/sheep
Balbutskaya et al., 2014 13:00
87.51
10
A. pluranimalium
S420838-12-1/sheep
Balbutskaya et al., 2014 09:45
87.51
11
A. pluranimalium
S1316-98-1/sheep
Foster and Hunt, 2011
10:45
87.51
12
A. pluranimalium
S600993-00-1/sheep
Foster and Hunt, 2011
13:45
87.40
13
A. pluranimalium
S603257-04-1/sheep
Foster and Hunt, 2011
14,15
87.22
14
A. pluranimalium
S603973-05-1/sheep
Foster and Hunt, 2011
14:15
87.37
15
A. pluranimalium
S005507-05-1/sheep
Foster and Hunt, 2011
16:15
87.27
16
A. pluranimalium
S704483-04-1/sheep
Foster and Hunt, 2011
13:30
87.27
17
A. pluranimalium
S603952-05-1/sheep
Foster and Hunt, 2011
14:45
87.26
18
A. pluranimalium
S607005-08-1/sheep
Foster and Hunt, 2011
13:15
87.12
19
A. pluranimalium
S609650-12-1/sheep
Balbutskaya et al., 2014 11:45
87.31
20
A. pluranimalium
S004572-04-1/sheep
Foster and Hunt, 2011
14:15
87.41
21
A. pluranimalium
S111911-12-1/sheep
Foster and Hunt, 2011
13:15
87.17
TE D
M AN U
SC
6 7
A. pluranimalium
S501967-00-1/sheep
Foster and Hunt, 2011
14:00
87.17
A. pluranimalium
S601452-01-2/sheep
Foster and Hunt, 2011
13:45
87.41
24
A. pluranimalium
S603814-04-1/sheep
Foster and Hunt, 2011
15:15
87.27
25
A. pluranimalium
24-C359-11-10/bovine
Balbutskaya et al., 2014 14:00
87.27
26
A. pluranimalium
D13-0340-5-11/bovine
Balbutskaya et al., 2014 11:15
87.26
27
A. pluranimalium
D12-0439-1-1-1/bovine
Balbutskaya et al., 2014 12:30
87.31
28
A. pluranimalium
4868/giraffe
Risse et al., 2014
87.56
AC C
EP
22 23
13:45
Non- A. pluranimalium strains 1
A. canis
DSM 25104 T
2
A. canis
ZT 11003002
3
A. haemolyticum
DSM 20595T
4
A. hippocoleae
DSM 15539T
5
A. phocae
DSM 10002T
6
A. phocae
DSM 10003
7
A. phocisimile
DSM 26142T
8
A. phocisimile
112
14
9
T. abortisuis
DSM 19515T
10
T. bernardiae
DSM 9152 T
11
T. bialowiezensis
DSM 17162T
12
T. bonasi
DSM 17163T
13
T. pyogenes
DSM 20630T
14
T. pyogenes
DSM 20594
15
T. pyogenes
59/11
16
T. pyogenes
336/1
17
T. pyogenes
1065/41
18
T. pyogenes
506/74
RI PT
ACCEPTED MANUSCRIPT
SC
286 287 288 289
M AN U
290
AC C
EP
TE D
291
15
ACCEPTED MANUSCRIPT 292
Table 3: Analytical sensitivity of the LAMP assay using serial dilutions of DNA of A.
293
pluranimalium DSM 13483
Dilution DNA amount step pg/µl
Detection time (h:mm:ss) run 1
run 2
run 3
run 4
Mean (Sd ±) run 5
Detection probability
500
0:14:15 0:21:45 0:16:30 0:17:30 0:15:30
0:17:06 (0:02:52)
100 %
10
-2
50
0:29:30 0:29:00 0:17:15 0:18:15 0:28:45
0:24:33 (0:06:13)
100 %
10
-3
5
0:26:15 0:26:15 0:18:15 0:27:45
0:24:37 (0:04:19)
80 %
-
0%
10-4
0.5
-
-
-
-
SC
294
-
-
RI PT
10
-1
295
AC C
EP
TE D
M AN U
296
16
ACCEPTED MANUSCRIPT 297
Table 4: Limit of detection (LOD) of the LAMP assay using serial dilutions of A.
298
pluranimalium DSM 13483 reference strain.
Detection time (h:mm:ss)
cfu / reaction
Mean (Sd ±)
Detection probability
run 2
run 3
run 4
run 5
2.2 x10
10:45
13:00
15:30
16:15
11:30
13:24 (02:10)
100 %
2.2 x10
2
15:30
17:00
17:30
18:30
13:15
16:21 (01:50)
100 %
2.2 x10
1
18:00
18:45
20:15
22:15
15:30
18:57 (02:15)
100 %
2.2 x100
19:45
28:45
27:00
22:45
17:30
23:09 (04:14)
100 %
2.2 x10-1
-
-
-
24:30
19:30
22:00 (02:30)
40 %
M AN U
300
SC
299
RI PT
run 1 3
AC C
EP
TE D
301
17
1
2
3
4
5
6
M
SC
M
RI PT
ACCEPTED MANUSCRIPT
1000 bp
M AN U
500 bp
TE D
Figure 1. Agarose gel electrophoresis of A. pluranimalium LAMP products. The positive reaction appeared as a ladder-like pattern (lane 1-3) ; lane 4 and 5 represent
AC C
EP
negative strains, lane 6 a non template control; lane M = 100 bp DNA ladder (Roche).
ACCEPTED MANUSCRIPT
SC
RI PT
(a)
M AN U
24-C359-11-10 S604731-06-1 S600993-00-1 S603257-04-1 Positive control Negative control
AC C
EP
TE D
(b)
Figure 2. Typical amplification signal of five A. pluranimalium LAMP products and a negative control (a). The melting curve (anneal reaction) of the same amplicons (b).
ACCEPTED MANUSCRIPT Highlights:
AC C
EP
TE D
M AN U
SC
RI PT
o A real-time fluorogenic LAMP assay for detection of Arcanobacterium pluranimalium was developed o This assay detects 2 cfu / reaction within 30 min with high specificity and sensitivity. o The used method has advantages of simplicity and inexpensiveness of operation.