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Development and Comparison of a Real-Time PCR Assay for Detection Of Frosth et al. Acta Veterinaria Scandinavica 2012, 54:6 http://www.actavetscand.com/content/54/1/6 RESEARCH Open Access Development and comparison of a real-time PCR assay for detection of Dichelobacter nodosus with culturing and conventional PCR: harmonisation between three laboratories Sara Frosth1*, Jannice S Slettemeås2, Hannah J Jørgensen2, Øystein Angen3 and Anna Aspán1 Abstract Background: Ovine footrot is a contagious disease with worldwide occurrence in sheep. The main causative agent is the fastidious bacterium Dichelobacter nodosus. In Scandinavia, footrot was first diagnosed in Sweden in 2004 and later also in Norway and Denmark. Clinical examination of sheep feet is fundamental to diagnosis of footrot, but D. nodosus should also be detected to confirm the diagnosis. PCR-based detection using conventional PCR has been used at our institutes, but the method was laborious and there was a need for a faster, easier-to-interpret method. The aim of this study was to develop a TaqMan-based real-time PCR assay for detection of D. nodosus and to compare its performance with culturing and conventional PCR. Methods: A D. nodosus-specific TaqMan based real-time PCR assay targeting the 16S rRNA gene was designed. The inclusivity and exclusivity (specificity) of the assay was tested using 55 bacterial and two fungal strains. To evaluate the sensitivity and harmonisation of results between different laboratories, aliquots of a single DNA preparation were analysed at three Scandinavian laboratories. The developed real-time PCR assay was compared to culturing by analysing 126 samples, and to a conventional PCR method by analysing 224 samples. A selection of PCR-products was cloned and sequenced in order to verify that they had been identified correctly. Results: The developed assay had a detection limit of 3.9 fg of D. nodosus genomic DNA. This result was obtained at all three laboratories and corresponds to approximately three copies of the D. nodosus genome per reaction. The assay showed 100% inclusivity and 100% exclusivity for the strains tested. The real-time PCR assay found 54.8% more positive samples than by culturing and 8% more than conventional PCR. Conclusions: The developed real-time PCR assay has good specificity and sensitivity for detection of D. nodosus, and the results are easy to interpret. The method is less time-consuming than either culturing or conventional PCR. Background disease may also progress to severe necrotic separation Footrot is a contagious bacterial disease that affects the of the claw capsule from underlying tissues. Severity of feet of sheep, and it has been reported in many coun- the disease depends on the breed of sheep, climatic con- tries [1]. The fastidious and anaerobic bacterium, Diche- ditions, management factors and virulence of the infect- lobacter nodosus, is the main causative agent of ovine ing D. nodosus strain. footrot [2]. Ovine footrot was first diagnosed in Sweden in 2004 In its mildest form, footrot manifests itself as a slight [3], and in 2008 it was detected for the first time in 60 inflammation of the interdigital skin of sheep, but the years in Norway [4]. In 2009, the disease was diagnosed in Denmark by culture and PCR [5], however a report on clinical disease among Danish sheep was published * Correspondence: [email protected] in 1988 [6]. The emergence of ovine footrot is a 1Department of Bacteriology, National Veterinary Institute, SE-751 89 Uppsala, Sweden Full list of author information is available at the end of the article © 2012 Frosth et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Frosth et al. Acta Veterinaria Scandinavica 2012, 54:6 Page 2 of 7 http://www.actavetscand.com/content/54/1/6 challenge for the diagnostic laboratories and for the Each 20-μl PCR reaction mixture contained 1× Per- sheep industries in all three Scandinavian countries. feCTa qPCR FastMix, UNG, Low Rox (Quanta BioS- Clinical examination of sheep feet is fundamental to ciences Inc., Gaithersburg, MD, USA), 0.1 mg/ml BSA diagnosis of footrot, but detection of D. nodosus should (Sigma-Aldrich, St Louis, MO, USA), 0.4 μM of each also be used to confirm the diagnosis. The presence of primer, 0.15 μM of the TaqMan-probe and 2.5 μlof the typical Gram-negative rods in lesion material can be template DNA. The PCR amplification was performed confirmed by microscopy, culturing, or PCR. Cultivation in an Applied Biosystems 7500 Fast Real-Time PCR Sys- of the bacterium is time consuming and laborious, and tem (Applied Biosystems, Carlsbad, CA, USA), a Strata- it is an advantage to also use PCR-based detection of D. gene Mx3005P real-time PCR thermocycler (Agilent nodosus. Technologies Inc., Santa Clara, CA, USA) or in a Cor- A PCR method for specific detection of D. nodosus bett Rotor-Gene 6000 (Qiagen, Hilden, Germany) with was developed in 1993 by La Fontaine et al. [7], and in an initial denaturation step at 95°C for 10 min, followed 2005 the method was improved by Moore et al. [8] for by 45 cycles of 95°C for 3 s and 60°C for 30 s. DNA detection of D. nodosus from clinical swabs. Previously, extracted from the type strain of D. nodosus, CCUG the PCR protocol published by Moore et al. [8] was 27824T (Culture Collection, University of Göteborg used at our institutes, but there were problems with [CCUG], Sweden) was used as a positive control in the non-specific amplicons and faint bands of the correct D. nodosus-specific real-time PCR and was included in product size which made interpretation difficult. More- each run. Every PCR run also included a non-template over, conventional PCR requires agarose gel electro- control in the form of DNase- and RNase-free sterile phoresis for identification of the PCR products, making water (Sigma-Aldrich). Fluorescence signals were ana- it inconvenient for the analysis of a large number of lysed using an automatic setting of the threshold line in samples. A faster and more easily interpreted method, the 7500 software (v.2.0.4) and a manually set threshold such as real-time PCR, was desirable. of 0.01 in the softwares of the Stratagene and Rotor- The aim of this study was to develop a TaqMan-based Gene real-time PCR instruments. real-time PCR assay for detection of D. nodosus and to compare its performance with culturing and with con- Sensitivity of the developed real-time PCR assay and ventional PCR. Another goal was to compare the sensi- comparison of its sensitivity by three different tivity of the developed real-time PCR assay between the laboratories three laboratories participating in this study since it is The detection limit and the amplification efficiency of advantageous to be able to use the same detection the D. nodosus-specific real-time PCR assay were deter- method. The real-time PCR assay was developed in col- mined using 10-fold serial dilutions of chromosomal laboration between the National Veterinary Institute DNA (393 ng to 3.9 ag corresponding to approximately (SVA), the Norwegian Veterinary Institute (NVI) and 3×108 to 3 × 10-3 copies of the D. nodosus genome per the National Veterinary Institute in Denmark (DTU- reaction) obtained from D. nodosus CCUG 27824T VET). Its specificity (inclusivity/exclusivity) was tested at (CCUG), and performing real-time PCR as described SVA and its sensitivity was tested and compared at all above. Each DNA dilution was run in triplicate in the three laboratories. The SVA compared the real-time real-time PCR analysis. The DNA from the D. nodosus PCR assay with culturing for 126 Swedish sheep and the type strain was prepared at the SVA and aliquots of the NVI compared it with conventional PCR for 224 Nor- DNA preparation were sent by regular mail to the NVI wegian sheep. and to the DTU-VET for the comparison of assay sensitivity. Methods Development of a D. nodosus-specific real-time PCR assay Inclusivity and exclusivity testing D. nodosus-specific PCR primers and a TaqMan-probe Fifty-five bacterial and two fungal strains were used to targeting the 16S rRNA gene were designed using pri- test the inclusivity and the exclusivity of the developed mer3 [9], producing an 84-bp fragment. The specificity D. nodosus-specific 16S real-time PCR assay (Table 1). of the assay was checked against GenBank sequences The D. nodosus strains (except for strain CCUG with the BLAST program package http://blast.ncbi.nlm. 27824T) were obtained from the SVA strain collection nih.gov/Blast.cgi[10]. The designed primers and Taq- whereas the strains of Actinobacillus pleuropneumoniae, Man-probe were ordered from Thermo Fisher Scientific Histophilus somni and the two Fusobacterium-strains with the following sequences 5’-CGGGGTTATG- were obtained from CCUG. The Haemophilus influen- TAGCTTGCTATG-3’ (16Sf), 5’-TACGTTGTCCCC- zae strain ATCC 49247 was obtained from ATCC CACCATAA-3’ (16Sr) and 5’-TGGCGGACGGGTG (American Type Culture Collection). The remaining AGTAATATATAGGAATC-3’ (16Sprobe TET-labeled). strains (n = 42), some of which originated from CCUG Frosth et al. Acta Veterinaria Scandinavica 2012, 54:6 Page 3 of 7 http://www.actavetscand.com/content/54/1/6 Table 1 Bacterial and fungal strains used for inclusivity Table 1 Bacterial and fungal strains used for inclusivity and exclusivity testing of the developed real-time PCR and exclusivity testing of the developed real-time
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