Abstract Book of EAVLD2012 - eavld congress 2012

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S4 - P - 06 SEPRION-COATED MICROCANTILEVER SENSORS FOR PRP SC DETECTION Daniela Meloni¹, Danilo Pitardi¹, Maria Mazza¹, Riccardo Castagna 2 , Ivan Ferrante 2 , Carlo Ricciardi 2 , Elena Bozzetta¹. ¹Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, Istopatologia e Test Rapidi, Turin, Italy; 2 Politecnico di Torino, Scienza dei Materiali e Ingegneria Chimica, Turin, Italy. Cantilever, Seprion, TSE, PrP sc detection Introduction The current state of technology to detect animal TSEs relies upon the post-mortem detection of prions, considering the EU trend in the age limit for testing [1] and in order to improve surveillance and food safety, new technologies should be developed to allow the ante mortem tests for PrP sc proteins. The new tests should be body fluids-based and performed on presymptomatic animals, but to be effective they would need to detect small amounts of PrP sc and to differentiate PrP c from PrP sc . Mechanical biosensors appear to be one of the most promising techniques for molecular diagnostics [2]. Dynamic-mode mechanical biosensors are oscillating devices with a resonance frequency that changes when molecules land on the cantilever. Thanks to the monitoring of different resonance modes over arrays of several cantilever-based microbalances, the selective identification and quantification of adsorbed mass of the order of few picograms with very high precision was recently demonstrated [3]. Here we propose the development of a biosensing platform for PrP sc detection based on microcantilever arrays coated with a selective polymer (Seprion from IDEXX Lab. Inc.). Materials & methods Microcantilever arrays were fabricated in a clean room making use of standard microtechnologies such as KOH bulk etching, optical lithography and Reactive Ion Etching; they were dipped into coating solution for few minutes inside a laminar flow bench, washed with PBS and MilliQ water, and finally dried on a plate dryer. An optical lever setup is used to monitor cantilever resonance curve while vibrating in vacuum environment; a piezoactuator is employed for the excitation. We calculated the arithmetic mean and uncertainty of relative frequency deviation Δf/f over the modes for each cantilever, and then we used the “weighted average”method to have the best estimation of the true value Δf/f of the array [3]. PrP sc was extracted from scrapie positive samples, brainstems were homogenised in lysis buffer and centrifuged at 22.000 g for 20 minutes, the supernatant was collected and incubated with proteinase K (20 μg/ml) for 1 hour at 37° C. After centrifugation at 215.000 g for 1 hour, the pellet was dissolved in distilled water. The coated arrays were incubated for 2h in a water-based solution containing pathologic PrP sc or simply water as negative control experiment (“blank”). Samples were then rinsed in DI water and finally dried in a steam of nitrogen. Fig. 2. Micro-cantilever array Fig3. PrP sc capture Results Microcantilever arrays were successfully treated with both 0.1% and 0.2% coating solution: the calculated mass surface densities seemed to be rather similar over the arrays and dilution, showing a good homogeneity of the functionalization procedure. Preliminary data exhibited a relevant negative relative frequency deviation Δf/f up to nearly 10 times higher respect to blank signals, showing that the polymer remained active in immobilizing the target molecule and thus an increment of mass occurred on cantilever surfaces when exposed to PrP sc solutions. Discussion & conclusions Cantilever-based microbalances were successfully coated with Seprion polymer to selectively immobilize pathogenic PrP sc . Preliminary data showed that the technique looks promising, but more focused experiments are needed to exploit its potentiality. References 1. European Commission, Brussels, 16.7.2010, COM (2010) 384 final. Communication from the Commission to the European Parliament and the Council. The TSE Road map 2, a Strategy paper on Transmissible Spongiform Encephalopathies for 2010-2015. 2. Arlett, J.L., Myers, E.B., Roukes, M.L. (2011). Comparative advantages of mechanical biosensors. Nat. Nanotechnol. 6, 4:203-15. 3. Ricciardi, C., Fiorilli, S., Bianco, S., Canavese, G., Castagna, R., Ferrante, I., Digregorio, G., Marasso, S.L., Napione, L., Bussolino, F. (2010). Development of microcantilever based biosensor array to detect Angiopoietin-1, a marker of tumor angiogenesis. Biosens. Bioelect., 25:1193-1198 Figure 1: Flow chart explaining the new technique
S4 - P - 07 EVALUATION OF DIFFERENT EXTRACTION METHODS FOR THE DETECTION OF MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS IN BOVINE FAECES Jean-Louis Moyen 1 , Laure Brugère 1 , Julie Charrot 2 , Eric Sellal 2 , Line Kirsty 3 , Adrian McGoldrick 3 1 Laboratoire départemental d’Analyse et de Recherche de la Dordogne,24660 Coulounieix-Chamiers France 2 Laboratoire Service International, Lisieux, France 3 Animal Health and Veterinary Laboratories Agency Starcross, Exeter, United Kingdom M; avium susp. paratuberculosis, MAP, Introduction Johne’s disease or bovine paratuberculosis occurs throughout the world. It is a chronic, untreatable, intestinal disease of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP) 1 . The profile and interest in the disease has risen in recent years, and consequently, it will be beneficial to have accurate information about prevalence in order to formulate policy and appropriate control strategies. Central to gaining accurate prevalence data in any country, is the availability of good diagnostic methods. In order to control the disease, MAP has to be diagnosed ante-mortem by either detecting the causative agent itself or by the detection of specific antibodies. The need of a rapid, low cost, and sensitive diagnostic test would be beneficial in the detection and control of the organism. The methods currently available include solid and liquid culture, serological methodologies and more recently, polymerase chain reaction (PCR) based tests. Culture (liquid and solid) is sensitive but the time to detection ranges from 6 to 24 weeks. Serological methods tend to be used in screening programmes since they are quick and cheap, however the sensitivity is low. Available PCR methods allow fast results (2 days) but reported sensitivity also remains low. Sensitivity and the time taken to reach diagnosis are critical points in MAP management control programmes. Indeed, these disease control strategies can often fail because new clinical cases are found even after culling of positive diagnosed animals. In this study, with specific reference to PCR, we hypothesise that the optimization of sample preparation and nucleic acids extraction can improve the sensitivity making PCR a viable option in the control of the disease. Materials & methods Faecal material from cows belonging to certified MAP free herds were used as negative controls. Positive samples originated from naturally infected animals as well as from spiking negative faeces with MAP homogenates. Naturally infected samples, as well as those spiked, were used to prepare a dilution series of samples. Each set of samples were prepared in duplicate. All samples, both naturally infected and those spiked, were thoroughly mixed to ensure homogeneity (as much as practically possible). For a field trial (epidemiological study), sampling was made from undiluted samples. The heterogeneity between samples is increased but the aliquots are more representative using real faeces. Sample preparation conditions were comparable (size, dilution, shaking, bead beating, pK incubation and spinning) In this study we compared 3 PCR methods. The first was an IS900 real time PCR test from LSI (Taqman) 2,3 using a Qiagen spin column extraction method with 1g faeces and 4 ml H2O. The alternative methods were: An ambion magnetic beads method on Magmax KF96 developped by AHVLA 4 and an LSI magnetic beads (MV384) method on Magmax KF96 optimized by the LDAR24. Results Spiked and diluted faeces: All negative samples were negative Detection of higher dilutions depends on the methods Tab.1. spiked samples – CT values for serial dilutions for each method 10 -2 1/2 10 -2 1/4 10 -2 1/8 10 -2 1/16 10 -2 1/32 10 -2 1/64 Qiagen Spin columns 33.52 34.44 34.73 undet 37.81 38.75 35.54 35.03 35.57 35.53 34.59 undet 33.26 33.97 36.09 37.85 undet 37.39 33.58 38.09 36.97 38.13 undet undet mean 33.98 35.38 35.84 37.17 36.20 38.07 s 1.05 1.86 0.94 1.43 2.28 0.96 Soil kit 24.96 26.39 27.73 28.26 29.33 31.24 25.36 26.14 27.2 28.01 29.77 31.06 25.3 25.81 27.16 28.17 29.4 33.1 26.02 26.49 27.31 28.09 29.64 34.42 mean 25.41 26.21 27.35 28.13 29.54 32.46 s 0.44 0.30 0.26 0.11 0.21 1.60 Ambion AHVLA 28.94 28.08 30.09 30.46 31.3 33 27.21 27.11 31.17 31.89 31.54 32.96 26.9 29.23 30.04 31.31 31.2 32.72 26.96 27.63 31.05 33.06 33.44 33.08 mean 27.50 28.01 30.59 31.68 31.87 32.94 s 0.97 0.90 0.61 1.09 1.06 0.15 Discussion & conclusions The heterogeneity of samples for field trial is important. The best method sensitivity is obtained with AHVLA method. This method is requires more time and needs a larger centrifuge. The LSI magnetic beads method gives a very good sensitivity too. The classic spin column method gives the worst sensitivity The epidemiological sensitivity is much better with the optimized methods and allows better eradication program. The choice between these methods depends on the customer’s requirement. (cost versus sensitivity) References 1. N. Beth Harris, R. G. Barletta Clin. Microbiol. Rev. 2001 14(3): 489-512 Mycobacterium avium ssp paratuberculosis in veterinary medicine 2. E.P. Green, M.L.V. Tizard, M.T. Moss, J. Thompson, D.J. Winterhouse, J.J. McFadden and J. Hermon-Taylor: 1989, Sequence and characteristics of IS900, an insertion element identiefied in a human Crohn’s disease isolate of Mycobacterium paratuberculosis. Nucleic acid research volume 17 number 22 9063-9073 3.. LSI Kit TaqVetM. paratuberculosis advanced V003 4. A. McGoldrich, K. Line 2012 (personnal communication) Sample preparation (sample size, dilution, shaking, bead beating and spinning conditions) is very important Soil kit (Fast Dna MPBIO) gives the best sensitivity but is time consuming and is not usable for field diagnosis
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2 nd CONGRESSOFTHEEUROPEAN ASSOCIAT
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Welcome Dear colleagues, Welcome to
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worked with many diagnostic compani
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Products and services for scientist
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16:4518.15 20:0023:00 [S1.]Oralpres
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Day4 Wednesday,4July2012 4 th sessi
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Oral presentations “General Sessi
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antigens or attenuated vaccines can
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conditions, e.g. Staphylococcus aur
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S1 - O - 1 ORAL FLUIDS - SAMPLE MAT
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S1 - O - 03 INTRODUCTION RATE OF A
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S1 - O -05 VALIDATION OF A COMPETIT
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S1 - O - 07 SUBTYPING OF SWINE INFL
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S1 - O - 09 VIRAL DIAGNOSIS USING T
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S1 - O - 11 USING ORAL FLUID FOR TH
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S1 - O - 13 THE FIRST YEAR OF OBLIG
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S1 - O - 15 SEROLOGICAL ANALYSIS AN
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S1 - O - 17 RING TEST EVALUATION FO
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Oral presentations “Diagnostics a
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S2 - O - 01 DEVELOPMENT OF A RAPID
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S2 - O - 03 EVALUATION OF RAPID HRS
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Oral presentations “Emerging, re-
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advances, the fundamental tools of
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S3 - O - 02 25 YEARS OF PASSIVE SUR
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S3 - O - 04 EQUINE NOCARDIOFORM PLA
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S3 - O - 06 DETECTION OF SCHMALLENB
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S3 - O - 08 SCHMALLENBERGVIRUS: SER
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S3 - O - 10 DIAGNOSTIC ASPECTS OF S
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Oral presentations “New technique
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acid and organic solvent. Then a dr
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S4 - O - 02 BIOLOG GENERATION III,
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S4 - O - 04 MATRIX-ASSISTED LASER D
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S4 - O - 06 15 MINUTE ELISA USING A
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S1 - P - 01 LAWSONIA INTRACELLULARI
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S1 - P - 03 DETECTION OF PRRSV ANTI
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S1 - P - 07 ANALYTICAL EVALUATION O
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S1 - P - 09 PTS FOR BVDV ANTIGEN DE
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S1 - P - 13 IDENTIFICATION OF GLOBI
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S1 - P - 19 CHARACTERIZATION OF EXT
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S1- P - 21 ANALYSIS OF THE EFFICIEN
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S1 - P - 23 RELIABLE AND EARLY DETE
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S1 - P - 25 GENOTYPIC VARIABILITY D
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