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Laboratory of Fish Pathology, Faculty of Veterinary Sciences, University of Zaragoza, 50013 Zaragoza, Spain
Correspondence
José Luis Balcázar
balcazar{at}unizar.es
Received 27 March 2006
Accepted 6 November 2006
Abbreviations: Ct, cycle threshold; FAM, 6-carboxyfluorescein; LUX, Light Upon eXtension.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of the A. salmonicida strains determined in this study are AM296501–AM296510.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONREFERENCES |
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Methods currently used to identify A. salmonicida within infected fish can be time-consuming, labour-intensive and hard to implement because of difficulties in distinguishing the bacterium from other bacterial species given the wide variety of existing biochemical profiles, the absence of an efficient selective medium and the poor plating efficiency of the bacterium in mixed cultures. For this reason, attention has focused on the application of different serological techniques (Adams & Thompson, 1990; Gilroy & Smith, 2003), and DNA sequence-based methods employing PCR have been developed (Gustafson et al., 1992; Hiney et al., 1992; Miyata et al., 1996).
Real-time PCR technology may be able to provide an innovative method of detecting and enumerating several organisms in different environments (Sharkey et al., 2004). With this method, PCR product accumulation is continuously monitored during cycle progression by means of fluorescent detection. In addition, this technique is characterized by a large dynamic range of quantification, sensitivity and throughput capacity and requires no post-amplification manipulation, thus avoiding possible carry-over contamination.
A fluorescence-based PCR technique that uses a fluorogenic primer labelled with a single fluorophore was recently developed as a cost-effective alternative to other fluorescence-based PCR techniques (Nazarenko et al., 2002a, b). Light Upon eXtension (LUX) primers (Invitrogen) are designed with the fluorophore attached near the 3' end and a short complementary sequence at the 5' end that creates a hairpin structure. This structure effectively quenches the fluorophore so that a separate quenching moiety is not required. When the primer is incorporated into a double-stranded PCR product and extended by DNA polymerase, the fluorophore is dequenched, resulting in a significant increase in fluorescent signal (Lowe et al., 2003).
The most frequent target of species-specific A. salmonicida DNA probes and PCR assays is a 6.4 kb cryptic plasmid known to occur in 90 % of A. salmonicida subsp. salmonicida (Hiney et al., 1992; Sørum et al., 1993; O'Brien et al., 1994; Mooney et al., 1995; Byers et al., 2002). This locus is also found in all A. salmonicida subsp. achromogenes strains tested and some A. salmonicida subsp. masoucida strains. No reaction has been found with over 60 related aeromonads and aquatic bacteria tested to date (Mooney et al., 1995).
The present study employed this locus to develop a quantitative real-time PCR assay using fluorogenic primers. Assay sensitivity and specificity were examined in pure or mixed cultures, and enumeration of A. salmonicida by real-time PCR in tissue samples from rainbow trout (Oncorhynchus mykiss) was carried out.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Strains were obtained from the following sources: ATCC (American Type Culture Collection, Manassas, VA, USA), NCIMB (National Collections of Industrial and Marine Bacteria, Aberdeen, UK) and LMG (Laboratory for Microbiology, Ghent University, Belgium), as indicated in Table 1; strains CLFP 501–505, isolated from rainbow trout during a furunculosis outbreak at fish farms, from the Autonomous Community of Aragon, Spain; strains 2320, 793 and 4814 from Dr D. Colquhoun (Department for Fish Health, National Veterinary Institute, Oslo, Norway); strains 265/87, MT-004, F-661-2/89, Fin 3, 870626-1/1C, S-228/91, S-123-91-1, Vib 293, Vib 305, Vib 281 and M 147/92 from Dr B. K. Gudmundsdóttir (Institute for Experimental Pathology, University of Iceland, Reykjavík, Iceland); strains 146 and 147 from Dr José Guijarro (Department of Functional Biology, University of Oviedo, Spain); strains CECT 4342, CECT 4224, CECT 4245, CECT 4227, CECT 4257, CECT 4199, CECT 5761 and CECT 4237 from Dr María José Figueras (Biology and Microbiology Research Unit, Rovira and Virgili University, Spain); and strains Rs 146 and Rs 147 from Dr Eva Jansson (Department of Wild Life, Fish and Environment, National Veterinary Institute, Sweden). All strains were grown in brain heart infusion broth (Scharlau Chemie) overnight at 22 °C, with the exception of Flavobacterium psychrophilum, which was grown in Anackel–Ordal broth (0.5 % tryptone, 0.05 % yeast extract, 0.02 % sodium acetate, 0.02 % beef extract, adjusted to pH 7.4) for 2 days at 15 °C with agitation, and Renibacterium salmoninarun, which was grown in modified KDM2 broth (1 % bactopeptone, 0.05 % yeast extract, 0.05 % L-cysteine, adjusted to pH 6.5) for 6 days at 15 °C with agitation.
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DNA extractions. Bacterial cultures (1.0 ml) were homogenized in 200 µl TE buffer [10 mM Tris/HCl (pH 8.0), 1 mM EDTA], centrifuged at 12 000 g for 1 min and pellets were extracted using InstaGene Matrix (Bio-Rad) following the manufacturer's instructions. DNA yield and purity were determined spectrophotometrically by measuring A260 : A280 ratios (Gene Quant pro RNA–DNA calculator; Amersham Pharmacia Biotech). Concentrated DNA was stored at –20 °C.
Fluorogenic real-time PCR. Oligonucleotide sequences were derived from a specific DNA probe for A. salmonicida (GenBank accession no. X64214). The probe is specific for a region of the DNA fragment that has been used previously as a species-specific genetic probe (Hiney et al., 1992). LUX designer software (www.invitrogen.com/lux) was used to design the LUX fluorogenic primers [5'-CGGAACGTAATCTGAATTGTTCTTTTC(FAM)G-3'] and (5'-ATTGCTTATCGAGGCAGCCAAC-3').
For this assay, each 50 µl PCR contained 25 µl Platinum Quantitative PCR SuperMix-UDG (uracil DNA glycosylase) [60 U Platinum Taq DNA polymerase ml–1, 40 mM Tris/HCl (pH 8.4), 100 mM KCl, 6 mM MgCl2, 400 µM each dGTP, dATP and dCTP, 800 µM dUTP, 40 U UDG ml–1 and stabilizers], FAM-labelled LUX primer and corresponding unlabelled primer (200 nM each final concentration), 1 µl ROX reference dye (Invitrogen) and 10 µl template. An ABI PRISM 7000 was programmed as follows: 50 °C for 2 min and denaturation at 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s, 55 °C for 30 s and 72 °C for 30 s. Data were analysed using ABI PRISM 7000 sequence detection software, version 1.2.3 (Applied Biosystems). The cycle threshold (Ct) was calculated as the cycle number at which the reaction became exponential. The Ct of each sample was then compared with a standard curve and the result was expressed as absolute amounts of genomic DNA. Standard curves for quantification were plotted from triplicate samples by using Ct values of tenfold dilutions of template extracted from 8x106 c.f.u. A. salmonicida NCIMB 1102 ml–1. To confirm the results of the real-time PCR, 10 µl aliquots of PCR products were analysed by electrophoresis on ethidium bromide-stained 2 % agarose gels (Bio-Rad).
Sensitivity and specificity of the real-time PCR assay. The specificity of the real-time PCR was determined by comparing PCR products derived from A. salmonicida (n=16) and non-A. salmonicida strain (n=26) DNA templates. For sensitivity assays, DNA was extracted from pure cultures of A. salmonicida NCIMB 1102 (8x106 c.f.u. ml–1) to generate a standard curve used for enumeration of unknown samples.
Detection of seed A. salmonicida in fish tissue homogenate. Tissue samples (250 mg kidney, liver, spleen or intestine) from rainbow trout were homogenized in a 1 ml suspension of early stationary phase A. salmonicida NCIMB 1102 cells diluted in deionized water to a final concentration of 32, 320 and 3200 c.f.u. (g tissue)–1. Homogenates were centrifuged at 12 000 g for 1 min and DNA was extracted using InstaGene matrix following the manufacturer's instructions. Enumeration of A. salmonicida by real-time PCR was compared with plate count values. Statistical analysis was based on Student's t-test using paired and equal variance of log c.f.u. g–1. All statistics were performed using SPSS for Windows version 11.5 (SPSS).
Detection of A. salmonicida in naturally infected fish. Tissue samples (250 mg) of diseased fish associated with outbreaks on different fish farms were also tested. Samples were homogenized in 1 ml deionized water. Homogenates were centrifuged at 12 000 g for 1 min and DNA was extracted as described above.
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Primer design
LUX fluorogenic primers were designed from a specific DNA probe for A. salmonicida (Hiney et al., 1992), as 16S rRNA gene sequence analysis did not provide sufficient information for the delineation and identification of most species of the genera Aeromonas. It has been reported that A. salmonicida and Aeromonas bestiarum type strains differ by only two nucleotides, at positions 1011 and 1018 (Martínez-Murcia et al., 2005).
A computer search using tBLASTX (Altschul et al., 1997) for the matching sequences in GenBank including updates against the DNA probe for A. salmonicida (GenBank accession no. X64214) as a query sequence revealed more than 97 % similarity with plasmids from A. salmonicida subspecies listed in the NCBI genome database. The tBLASTX search also found that the DNA probe for A. salmonicida shared 47 % identity with the YopP gene from Yersinia enterocolitica (GenBank accession no. AF023202), which induces apoptosis in mouse macrophages (Mills et al., 1997). Because of the similarity between the DNA probe for A. salmonicida and the YopP gene from Y. enterocolitica, the extreme 5'-end region of the sequence was targeted, as this is where the most sequence variation occurs between the two.
Specificity of detection
To improve the diagnosis capabilities for this bacterial pathogen, a real-time PCR assay was developed. Species specificity was confirmed by positive amplification of all 16 isolates of A. salmonicida and lack of a product from ten other strains of Aeromonas species (Aeromonas encheleia, Aeromonas eucrenophila, Aeromonas sobria, A. bestiarum, Aeromonas veronii, Aeromonas allosaccharophila, Aeromonas culicicola, Aeromonas media and Aeromonas hydrophila) and 16 strains of common bacterial fish pathogens (Flavobacterium psychrophilum, Moritella viscosa, Photobacterium phosphoreum, Renibacterium salmoninarum, Vibrio tubiashii, Vibrio fluvialis, Vibrio splendidus, Vibrio furnissii, Vibrio pelagius, Vibrio aestuarianus, Vibrio anguillarum and Yersinia ruckeri) (Table 1
). The size of the amplified PCR product (131 bp) was verified by gel electrophoresis (not shown).
Byers et al. (2002) showed that primers PAAS1 and PAAS2, previously designed from the same sequence used in the present study (Hiney et al., 1992), did not produce any false-positive reactions with 27 non-target bacterial DNA extracts and these primers were considered to be specific for A. salmonicida. However, the PAAS primer set correctly identified only 285/308 isolates as A. salmonicida and was therefore determined to have an in vitro sensitivity of 93 %. The failure of the PAAS primer set to identify 100 % of the A. salmonicida isolates appeared to be related to the primer target site, which had previously been shown to occur on a 6.4 kb cryptic plasmid (Sørum et al., 1993) and to be present in approximately 90 % of A. salmonicida isolates (Mooney et al., 1995). It is therefore reasonable to speculate that the specificity of the reaction could be related to the virulence of the strains, as all A. salmonicida strains used in the present study were isolated from disease outbreaks in O. mykiss, Salmo salar, Salvelinus alpinus and Zoarces viviparus. In addition, the probe sequence homology with the YopP gene from Y. enterocolitica could confirm this hypothesis.
Sensitivity of detection
The detection sensitivity of the real-time PCR assay was determined by testing triplicate sets of genomic DNA prepared from serial dilutions (8x101 to 8x106 c.f.u. ml–1) of A. salmonicida NCIMB 1102. The results were reported as Ct values versus log starting quantities of DNA. Positive signals were found in all dilutions except those where the DNA concentration was below 5x10–4 ng µl–1 (80 c.f.u. ml–1). The Ct values were also plotted against c.f.u. ml–1 (8x101 to 8x106 c.f.u. ml–1) in tenfold serial dilutions of the A. salmonicida NCIMB 1102 culture used for extracting genomic DNA. Based on this approach, a correlation was observed between Ct and c.f.u. ml–1 of the starting quantity of A. salmonicida NCIMB 1102 DNA (Fig. 1).
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Rn signal and indicated that the assay is useful for quantitative measurements of A. salmonicida. The real-time PCR detection limit for A. salmonicida DNA derived from pure culture was 0.5 pg PCR mixture µl–1, or the equivalent of 80 c.f.u. (ml culture)–1, which would extrapolate to 16 c.f.u. per PCR assay, assuming 100 % extraction efficiency.
Detection of A. salmonicida in fish tissues
The Ct values obtained from the analysis of tissue samples were extrapolated to the corresponding previously calculated standard curve. The presence of fish tissue did not affect real-time PCR detection of A. salmonicida NCIMB 1102, and the results of plate counts yielded a good correlation (r=0.85) between the two assays (Table 2). On the other hand, amplification products were not detected when tissues were inoculated independently with 32 c.f.u. (g culture)–1.
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). These preliminary results showed that bacterial concentrations were in the normal range of values.
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The relative quantification of DNA using fluorogenic primers is comparable in sensitivity and dynamic range to other published methods of quantification, such as TaqMan probes (Best et al., 2005), molecular beacons (Xi et al., 2003) and DNA-binding dyes (SYBR Green) (De Medici et al., 2003). However, direct comparisons among methods for quantification are difficult as these methods are functionally different and may be affected differently by various factors.
In conclusion, the fluorogenic primer method is rapid, uses fewer toxic chemicals and is a useful tool for the sensitive and accurate detection of A. salmonicida in fish tissue. However, further studies should be performed in order to establish diagnostic agreement with serological methods at different stages post-infection.
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ACKNOWLEDGEMENTS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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