Folia Microbiol. 52 (2), 175–182 (2007)
http://www.biomed.cas.cz/mbu/folia/
Comparison of Specificity and Sensitivity of Immunochemical and Molecular Techniques for Reliable Detection of Erwinia amylovora B. KOKOŠKOVÁa, I. MRÁZb, J. HÝBLOVÁc,d aDepartment of Bacteriology, Plant Medicine Division, Research Institute of Crop Production, 161 06 Prague, Czechia
e-mail
[email protected] bDepartment of Plant Virology, Institute of Plant Molecular Biology, Biological Centre of the Academy of Sciences
of the Czech Republic, 370 05 České Budějovice, Czechia
cDepartment of Protein Biochemistry, Institute of Organic Chemistry and Biochemistry, Academy of Sciences
of the Czech Republic, 166 10 Prague, Czechia
dFaculty of Agrobiology, Food and Natural Resources, Czech University of Agriculture Prague, 161 21 Prague, Czechia
Received 27 April 2006 Revised version 23 October 2006
ABSTRACT. Erwinia amylovora [(BURRILL) WINSLOW et al.] (Ea), the causal agent of fire blight, was detected in plant samples and pure bacterial cultures by means of PCR, IFAS and ELISA. Polyclonal antibodies of Neogen Europe Ltd. were used for IFAS and PTA-ELISA and laboratory-generated primers EaF72 and EaR560 for PCR. Using the BIOLOG system and an immature pear fruit assay, identities of all Ea strains were confirmed as the fire blight bacterium. In assays of pure Ea cultures, PTA-ELISA, and both IFAS and PCR were sensitive to concentrations 106–105 and 105–104 CFU/mL, respectively. When saprophytic bacteria associated with Ea in plant samples were tested as potentially cross-reacting bacteria, PTA-ELISA and IFAS gave 20 and 14 % cross-reactions, respectively. In plant samples, the presence of Ea was more reliably detected by IFAS (at a dilution of 1 : 1000) than by PTA-ELISA (to dilution 1 : 100). The capacity to detect Ea might be increased using an optimized PCR, but for PCR prepared from infected plant samples it was necessary to use the bacterial DNA isolated with a DNeasy Plant Mini Kit (Qiagen). In this case the PCR was sensitive to a concentration of 105 CFU/mL. PCR was much more specific than either immunochemical technique, because no false positives were observed when primers EaF72 and EaR560 were used. Abbreviations DAS-ELISA Ab Ea ELISA ELISA-DASI FB FITC IF
double antibody sandwich enzyme-linked immunosorbent assay antibody(ies) Erwinia amylovora enzyme-linked immunosorbent assay ELISA-double antibody sandwich indirect fire blight fluorescein isothiocyanate immunofluorescence
IFAS IgG–AP IP-ELISA PCR PFGE PTA-ELISA SA
indirect fluorescent antibody stain immunoglobulin–alkaline phosphatase immunoprinting-ELISA polymerase chain reaction pulse-field gel electrophoresis plate-trapped antigen-enzyme-linked immunosorbent assay slide agglutination
Erwinia amylovora [(BURRILL) WINSLOW et al. 1920], which causes FB, is included among the quarantine organisms in many countries around the world where strict quarantine measures are used (OEPP/EPPO 1992; EPPO/CABI 1997). FB is probably the most serious disease affecting fruit and ornamental plants in the family Rosaceae. Economically important hosts include Pyrus spp., Malus spp., Cydonia spp., Crataegus spp., Cotoneaster spp., Sorbus spp. and others (Van der Zwet and Beer 1991; Sobiczewski et al. 1997). Management measures include surveys of presumptive plants for disease, laboratory tests, eradication of infected plants, and control and regulation of propagative and cultivated plant materials (Van der Zwet and Beer 1991). The pathogen can survive as an endophyte or epiphyte for variable periods of time depending on environmental factors. Winter survival is mainly on the margins of lesions and cankers of host plants that become active in the spring under favorable weather conditions (Vanneste 2000). An important step in the control of the disease depends on fast and specific detection of the pathogen. At present, some of the most frequently used methods to identify Ea are based on serology, among them the SA test, various types of ELISA, and IF. The success of each serological method depends on the type of specific Ab. The engagement of polyclonal Ab enables the detection of a broader spectrum of pathogen strains; however, cross-reactions can
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occur (Zielke et al. 1993; Sobiczewski et al. 1997). Monoclonal Ab prepared to one determinant are more specific than polyclonal Ab, but they can be overspecific and do not detect all strains in populations of the target bacterium (Lin et al. 1987; Schaad et al. 1990). Usually, a mixture of several monoclonal Ab is necessary to allow recognition of all representative strains of the FB pathogen (Gorris et al. 1996a). SA is an easy, fast, less expensive test that is usually quite reliable (Lelliott 1967). Nevertheless, cross-reactions with diverse bacteria such as Pseudomonas syringae, Pantoea agglomerans and P. dispersa (previously Erwinia herbicola) are sometimes observed. Additional tests are recommended for correct identification (Pánková et al. 1998; Vanneste 2000). The disadvantage of the SA test is its requirement of pure cultures for assay. ELISA and IF tests are very well suited for routine screening of large numbers of samples taken directly from plant tissues. Evaluation is very rapid and results are accurate when highly specific Ab are used (McLaughlin et al. 1989; Gorris et al. 1996a). In contrast, IF results in fewer false positives, because it allows for an evaluation of the shape and size of bacterial cells. Generally, the low sensitivity of indirect ELISA is a very serious limitation in the routine detection of bacterial pathogens, including Ea (Lopez et al. 1987). Therefore, for the monitoring of plants without obvious or unusual symptoms, more precise methods are required. To fulfil this need, an ELISA-DASI assay was designed by Gorris et al. (1996b), based on the use of polyclonal and monoclonal Ab and enrichment of samples with semiselective media before detection of Ea. An interesting technique for the detection of Ea, which also employs monoclonal Ab, is IP-ELISA (Cambra et al. 1996). It relies on using nitrocellulose membranes to immobilize plant material sampled for testing. IP-ELISA may facilitate putative diagnosis on symptomatic plant material even under field conditions but needs further confirmation because of specificity problems. Classical diagnostic methods, based on using of selective or semiselective media (Miller and Schroth 1972; Ishimaru and Klos 1984; Bereswill et al. 1998; Jones and Geider 2001) cannot detect small quantities of Ea, because detection is correlated primarily with obvious disease symptoms. Culturing can fail due to competition or inhibition by saprophytic bacteria. DNA hybridization techniques were developed with DNA from a 29-kb plasmid which is common to Ea strains. The FB pathogen can thus be detected by colony hybridization with the whole-plasmid DNA or with its cloned fragments as probes (Falkenstein et al. 1988). The pathogen is detected with radioactive (32P) or with less sensitive nonradioactive probes (Steinbrenner et al. 1990). The labor and time-consuming steps of colony or DNA hybridization of Ea were circumvented by the use of specific and sensitive PCR analysis (Bereswill et al. 1992). New molecular methods, such as real-time PCR (Salm and Geider 2004) and PFGE (Jock et al. 2002) and others are used for analysis of Ea in detail, but those methods are not yet available for routine testing. Biochemical identification of Ea strains is usually conducted by fatty acid analysis using gas chromatography (Sasser 1990; Van der Zwet and Wells 1993) and/or using the microbial identification system BIOLOG Bacteria (USA) based on an extensive spectrum of carbon utilization reactions (Jones et al. 1993; Krejzar and Kokošková 1999). Test of pathogenicity on immature pear fruits or shoots of various host plants of FB pathogen confirms or disproves laboratory results with definite validity (OEPP/EPPO 1992; EPPO/CABI 1997). Reliability of all diagnostic techniques has been reviewed (Bereswill et al. 1995; Sobiczewski et al. 1997).The FB pathogen is usually isolated easily from symptomatic plant material by dilution plating, followed by laboratory tests that aid in reliable identification. If detection methods are used without isolation of the bacterium from disease samples or from healthy looking plants, more precise methods are required. Our objective was to compare reliability of commonly available polyclonal Ab for ELISA and IF tests, and PCR with primers reported by Bereswill et al. (1995) and those developed in our laboratory for detection and identification of Ea. MATERIAL AND METHODS Detection and determination of the FB bacterium was conducted with SA, PTA-ELISA, IFAS and PCR. Specificity and sensitivity of all methods were compared. Bacterial strains. Isolates of Ea originating from samples of different host plants (hawthorn, pear, apple) with typical and atypical symptoms of FB were analyzed. Strains were recovered from infected tissues, cultivated on meat peptone agar (MPA) and incubated at 25 °C. The identity of all Ea strains tested was confirmed using the BIOLOG GN MicroPlate System (BIOLOG, USA) and the immature pear fruit assay.
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PTA-ELISA and IFAS. Commercial polyclonal Ab (Neogen Europe, UK) were used for diagnosis of the FB bacterium in immunochemical tests according to the manufacturer’s recommendations. In both tests, a rabbit polyclonal Ab raised to whole cells of a typical European Ea strain was employed. This Ab used an anti-rabbit IgG–AP conjugate in PTA-ELISA and an anti-rabbit FITC for IFAS. The diluted Ab were not used because this involves the risk of false-negatives with some Ea strains. Bacterial suspensions for tests were adjusted spectrophotometrically to absorbance A620 ≈ 0.1 which is the corresponding concentration 108 CFU/mL, and diluted to 104 CFU/mL. Positive and/or negative reaction in PTA-ELISA was recorded with an ELISA reader. ELISA threshold level was 0.2 at A405. IF slides were observed under a light microscope fitted for epifluorescence at 1000× magnification using a mercury lamp and a suitable filter system. Preparation of bacterial samples and isolation of DNA for PCR. When pure bacterial cultures were used, bacterial suspensions were adjusted spectrophotometrically to A620 ≈ 0.1 (108 CFU/mL), and diluted to 104 CFU/mL. One μL of each dilution of bacterial suspension was directly added to the PCR reaction mix. When infected plant samples were used, it was necessary to use a DNeasy Plant Mini Kit (Qiagen). DNA isolation was performed according to the manufacturer’s protocol for each dilution of homogenized infected plant sample. PCR and gel electrophoresis. PCR was performed with a Mini Cycler (MJ Research, USA). Primers EaF72 5´-CGT GAC GCT GGA TGA AAT GC-3´ and EaR560 5´-CTC CCG CAT CCA GTC AAG TG-3´ designed in our laboratory for detection and determination of Ea were synthesized by Generi Biotech (Hradec Králové, Czechia). The reaction mix contained 10 μL of 2× PPP Master Mix (75 mmol/L Tris-HCl, pH 8.8; 20 mmol/L (NH4)2SO4; 100 ppm Tween 20; 2.5 mmol/L MgCl2; dNTP 200 μmol/L each; 2.5 U Taq purple DNA polymerase and stabilization additives) obtained from TopBio (Czechia); 0.5 μL of each primer (20 pmol/μL); 1 μL of DNA template and 8 μL ddH2O. The PCR reaction started with the DNA denaturation step (95 °C, 5 min) and continued for 40 cycles (94 °C, 1 min; 68 °C, 40 s) and a final extension (72 °C, 2 min). Amplified PCR products (3 μL) were mixed with 2 μL of loading buffer containing the Sybr Green (Sigma-Aldrich) stain and run on 1 % agarose gel. Optimized and non-optimized PCR protocol. When primers AMSbL and AMSbR designed for specific Ea detection were used (Bereswill et al. 1992, 1995), some optimization steps were necessary. These steps were based on an increase in annealing temperature from 49 to 55 °C and shortening of annealing time from 2 min to 30 s (Kokošková and Mráz 2005). BIOLOG system. Ea strains and bacteria associating with Ea in symptomatic plant samples were identified using the microbial identification system BIOLOG Bacteria. Strains were characterized biochemically using the BIOLOG GN MicroPlate SystemTM following manufacturer’s instructions. Evaluation was performed with naked eye after 4 h and 1 d of incubation. Cultures were identified using the MicroLogTM 2 database for Gram-negative bacteria. RESULTS AND DISCUSSION In our experiments where polyclonal Ab of Neogen Europe were used, PTA-ELISA was sensitive at a value of 105 CFU/mL, whereas IFAS was able to detect Ea at 104 CFU/mL for some strains (Table I). Hutschemackers et al. (1990) reported similar results, with IF showing a detection limit of 104 CFU/mL. Zielke et al. (1993) conducted IF and DAS-ELISA tests for detecting Ea where polyclonal Ab were used. Ea was detected at 105 CFU/mL. The sensitivity of PCR (primers EaF72 and EaR560) was
