MICROBIOLOGIA BALKANICA th BALKAN CONGRESS OF MICROBIOLOGY & 8th CONGRESS OF SERBIAN MICROBIOLOGISTS, oktobar 2011

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2 CIP Katalogizacija u publikaciji Narodna biblioteka Srbije, Beograd Udruženje mikrobiologa Srbije, Beograd. Knjiga radova (Elektronski izvor) Proceedings / MICROBIOLOGIA BALKANICA th BALKAN CONGRESS OF MICROBIOLOGY & 8th CONGRESS OF SERBIAN MICROBIOLOGISTS, oktobar 2011; (organizator) Udruženje mikrobiologa Srbije, Udruženje medicinskih mikrobiolga Srbije; (urednici: Dragojlo Obradović, Lazar Ranin, Špiro Radulović) Beograd 1 elektronski optički disk (CD-ROM); 12cm Sistemski zahtevi: Nisu navedeni. Nasl. sa naslovnog ekrana. Radovi na engleskom jeziku.- Tekst latinica. Tiraž 600. Abstracts. Registar ISBN Udruženje mikrobiologa Srbije, Beograd. KNJIGA RADOVA / PROCEEDINGS MICROBIOLOGIA BALKANICA th BALKAN CONGRESS OF MICROBIOLOGY & 8th CONGRESS OF SERBIAN MICROBIOLOGISTS, oktobar 2011 Izdaje / Published by: Udruženje mikrobiologa Srbije Nemanjina 6, Beograd, Srbija, tel/fax: , dobradovic@agrifaculty.bg.ac.rs Za izdavača / For Publisher: Dragojlo Obradović, predsednik Udruženja Urednici/Editors: Dragojlo Obradović Lazar Ranin Špiro Radulović ISBN Kompjuterska obrada teksta / Computer Layout: Jelena Zovko Belić Tiraž / Circulation 600 primeraka / 600 copy Umnožavanje / Copying Megaphone d.o.o., Vladimira Rolovića 105, Beograd, Srbija

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4 Sensitivity of Real-time PCR method for detection of Erwinia amylovora in plant material Milan Ivanović, Nemanja Kuzmanović, Katarina Gašić, Anđelka Ćalić, Aleksa Obradović University of Belgrade, Faculty of Agriculture Introduction Gram-negative bacterium Erwinia amylovora is the causal agent of fire blight, one of the most destructive diseases of pome fruits. Presumptive diagnosis of the disease can be relatively simple when typical symptoms are evident. However, when the pathogen is present in low concentration no symptoms of diagnostic value are visible. In order to detect latent infection use of modern and sensitive methods is necessary. Nowadays, Real-time PCR is one of the available methods for detection of E. amylovora in symptomless plant material. Aim Sensitivity of Real-time PCR detection of E. amylovora in pear leaves was studied. Material and methods Pear leaves and twigs were crushed in the antioxidant maceration buffer and inoculated with a range of bacterial suspensions from 2, CFU/ml (sample No. 1) to 2, CFU/ml (sample No. 6). Bacterial DNA was extracted according to the procedure of Taylor et al. (2001) and Real-time PCR reaction targeting chromosomal DNA amsc gene according to Pirc et al. (2009). The ams region of the E. amylovora chromosome is involved in synthesis of the capsular polysaccharide amylovoran, which is considered unique to E. amylovora. Specific primers Ams116F, Ams189R and TaqMan Ams141T probe were used in the reaction for 74 base pair fragment amplification. Automatic baseline was used and threshold was set manually crossing exponential phase of control amplification curves. Results In our study, the lowest concentration of bacteria detected by Realtime PCR was 2, CFU/ml. Number of cycles required for reaching the defined fluorescence value varied between the samples: in sample No. 2 the fluorescence level was reached in 36th amplification cycle, whereas in sample 6 the same level was reached in 26th cycle. Fewer amplification cycles are needed when higher initial amount of bacterial DNA is present in the sample. In our laboratory, Real-time PCR has proven to be the most sensitive method for detection of E. amylovora in plant material. Real-time PCR was 10 times more sensitive than Nested PCR and 100 times more sensitive when compared to other conventional PCR procedures. Conclusion High sensitivity of Real-time PCR is the most efficient method for the detection of the pathogen in plant tissue. Low pathogen concentration can be efficiently detected in less than 5 hours. In addition, inhibitors and plant DNA did not reduce the sensitivity of the reaction. In spite of the advantages, high cost of the equipment and reagents stands as a limiting factor for the wider introduction of this method in our laboratories. Keywords Real-time PCR, sensitivity, Erwinia amylovora This study was supported by the Serbian Ministry of Education and Science, through the Project III46008.

5 Sensitivity of Real-time PCR method for detection of Erwinia amylovora in plant material Ivanović Milan, Kuzmanović Nemanja, Gašić Katarina, Ćalić Anđelka, Obradović Aleksa University of Belgrade, Faculty of Agriculture, Department of Plant Pathology, Belgrade, Serbia Introduction Gram-negative bacterium Erwinia amylovora is the causal agent of fire blight, one of the most destructive diseases of pome fruits. Presumptive diagnosis of the disease can be relatively simple when typical symptoms are evident (Picture 1). However, when the pathogen is present in low concentration no symptoms of diagnostic value are visible. In order to detect latent infection use of modern and sensitive methods is necessary. Real-time PCR is one whose application in plant pathology is increasing lately, especially for detection of pathogens in symptomless material. Picture 1. Erwinia amylovora. Twig and fruit necrosis, typical symptoms of fire blight on pear and apple trees. Material and methods In order to artificially inoculate plant material pear leaves and twigs were crushed in the antioxidant maceration buffer and inoculated with a range of bacterial suspensions from 2,1 101 CFU/ml (sample No. 1) to 2,1 106 CFU/ml (sample No. 6). Bacterial DNA was extracted according to the procedure of Taylor et al. (2001) and Real-time PCR reaction targeting chromosomal DNA amsc gene according to Pirc et al. (2009). The ams region of the E. amylovora chromosome is involved in synthesis of the capsular polysaccharide amylovoran, which is considered unique to E. amylovora. Specific primers Ams116F, Ams189R and TaqMan Ams141T probe (Table 1) were used in the reaction for 74 base pair fragment amplification. Reaction volume of 20µl contained, in final concentrations: 0,9µM primers, 0,2µM probe, 1 TaqMan Fast Universal PCR Master Mix and 4µl of sample DNA. The baseline was set automatically, and the fluorescence threshold was set manually to intersect with the linear part of the amplification curves of all Real-time PCR assays, resulting in the final Ct value for each well. Picture Real-Time PCR System (Applied Biosystems, USA) used for detection of E. amylovora Results In our study, the lowest concentration of bacteria detected by Real-time PCR was 2,1 102 CFU/ml (Picture 3). Number of cycles required for reaching the defined fluorescence value varied between the samples: in sample No. 2 the fluorescence level was reached in 36th amplification cycle, whereas in sample 6 the same level was reached in 26th cycle. Fewer amplification cycles are needed when higher initial amount of bacterial DNA is present in the sample. Comparative studies in our laboratory showed that Real-time PCR is 10 times more sensitive than Nested PCR and 100 times more sensitive when compared to the other conventional PCR procedures Sample 6 Table 1. Primers and TaqMan probes used for Real-time PCR detection of E. amylovora Assay Name Ams (amsc gene) Ams116F Ams189R Ams141T Sequence (5-3 ) Amplicon length TCCCACATACTGTGAATCATCCA GGGTATTTGCGCTAATTTTATTCG FAM-CCAGAATCTGGCCCGCGTATACCG-BHQ1 74 bp Table 2. Sensitivity of Real-time PCR compared to conventional PCR for the detection of E. amylovora in spiked samples PCR method Real-time Conventional Concentration of E. amylovora (CFU/ml) Pirc et al. (2009) Llop et al. (2001) Taylor et al. (2001) Stöger et al. (2006) Ekstrakcija Taylor i sar. (2001) Th Picture 3. Real-time PCR with Taqman probe and primers Ams116F/Ams189R for detection and quantitative amplification of E. amylovora DNA. Fluorescence signal is in relation to the amount of template: sample 6-2,1 106 CFU/ml, and four tenfold dilutions, respectively (in curves from left to right); Th - cycle threshold. Table 3. Reaction conditions used in this study for detection of E. amylovora 2 min at 50 C 10 min at 95 C 40 cycles of 15 s at 95 C 1 min at 60 C UNG activation step polymerase activation DNA denaturation annealing and extension Conclusion Real-time PCR is the most efficient method for the detection of the pathogen in plant tissue. Low pathogen concentration can be efficiently detected in less than 5 hours. In addition, inhibitors and plant DNA did not reduce the sensitivity of the reaction. Therefore, it could be recommended for use when reliable detection of a low number of E. amylovora is needed or when diagnosis is difficult due to lack of symptoms and presence of the pathogen under culturable level. However, in spite of the advantages, high cost of the equipment and reagents stands as a limiting factor for the wider introduction of this method in our laboratories. This work was supported by the Serbian Ministry of Education and Science, through the project III46008