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A comparative study of molecular diagnostic methods designed to detect the crayfish plague pathogen, Aphanomyces astaci
Veterinary Microbiology 153 (2011) 343–353
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A comparative study of molecular diagnostic methods designed to detect the crayfish plague pathogen, Aphanomyces astaci Stephen Tuffs a,b, Birgit Oidtmann a,* a
Centre for Environment, Fisheries and Aquaculture Science, Barrack Road, Weymouth, Dorset, United Kingdom Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, Glasgow Biomedical Research Centre, University of Glasgow, 120 University Place, Glasgow G12 8TA, United Kingdom
b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 December 2010 Received in revised form 9 June 2011 Accepted 15 June 2011
Crayfish plague is the most important disease of freshwater crayfish with a significant impact on European species. We compared the analytical test sensitivity and specificity of three published PCR assays for the detection of Aphanomyces astaci, the causative agent of crayfish plague: a conventional PCR assay targeting the ITS region and two TaqMan1 real time assays, targeting either the ITS region or the chitinase gene. We also tested a variation of the conventional assay, by changing one of the primers. Test specificity was assessed using DNA from a range of A. astaci strains and an array of closely related Oomycetes, host tissue and DNA from other organisms that may be present in a diagnostic sample. Sensitivity was assessed using genomic A. astaci DNA from mycelium and zoospores. All assays were found to be of good to excellent sensitivity with levels of detection ranging from 1 (real time assay targeting the ITS region), over 10 (conventional PCR) to 100 zoospores (real time assay targeting the chitinase gene). All three published assays were also specific for A. astaci and did not cross-react with any other test organisms included in this study. The tested variation of the conventional PCR assay with a changed forward primer led to amplification of some non A. astaci DNA. Advantages and disadvantages, including suitable application are discussed for each assay. Crown Copyright ß 2011 Published by Elsevier B.V. All rights reserved.
Keywords: Aphanomyces astaci Real time PCR PCR Crayfish plague Carrier Saprolegniaceae Oomycota
1. Introduction Crayfish plague is the most important disease of freshwater crayfish species; it is caused by the Oomycete pathogen Aphanomyces astaci and has been heavily implicated in the decline of native crayfish species across Europe, as infection in these species is fatal (Edgerton et al., 2004; Edsman et al., 2010; Fu¨reder et al., 2010). North American crayfish may be infected with A. astaci, without normally developing disease (Unestam, 1969, 1972; Unestam and Weiss, 1970; Die´guez-Uribeondo and So¨derha¨ll, 1993; Cerenius et al., 2010). They can therefore act as carriers, and their introduction into Europe has established
* Corresponding author. Tel.: +44 1305 206661; fax: +44 1305 206601. E-mail address: [email protected] (B. Oidtmann).
a potential reservoir for the pathogen which represents a serious threat to native European crayfish species (Holdich et al., 2009). Fast and accurate diagnosis is important if the disease is to be controlled, therefore in recent years molecular diagnostic methods, based on PCR have been developed to detect A. astaci with the aim of being more robust, time and resource effective than culture dependent diagnostic methods. A conventional PCR method was described by Oidtmann et al. (2006) which targets the internal transcribed spacer (ITS) region of the 5.8 s rRNA gene. The method has been widely applied both for the detection of carrier status in North American crayfish and to confirm the pathogen in disease outbreaks in European crayfish species (Schulz et al., 2006; Jones et al., 2009; Kozubı´kova´ et al., 2009; Kozubı´kova´ et al., 2010; Camma` et al., 2010).
0378-1135/$ – see front matter . Crown Copyright ß 2011 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2011.06.012
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Quantitative real time polymerase chain reaction (RTPCR) has also been explored as a potential diagnostic alternative and two quantitative TaqMan1 assays have been developed, one, a TaqMan1 minor groove binder (MGB) RT-PCR targeting the ITS region (Vra˚lstad et al., 2009), the other, using a conventional, TaqMan1 probe and targeting chitinase (chi1, chi2 and chi3) encoding genes in the organism (Hochwimmer et al., 2009).All three of these studies provided appropriate and informative validation data, however in each paper a different approach was taken to validate the PCR method, making direct comparison of these methods difficult. The aim of this study was to use a consistent sensitivity and specificity testing to compare these three methods and consider their suitability for different applications. 2. Materials and methods 2.1. Oomycetes and other organisms tested The various A. astaci strains and other organisms used in this study are listed in Table 1. These included some Saprolegniales that were isolated from a crayfish plague outbreak in the North of the United Kingdom in 2009: a strain of A. astaci designated 197901 (HQ660705) and a Saprolegnia species (HQ660706) with homology to S. ferax, designated 197907. 2.2. DNA extraction DNA from Oomycete species was obtained by extracting DNA from pure mycelium grown in broth cultures using the DNeasy plant kit mini (Qiagen) with an additional tissue lysis step. Approximately 100 mg of mycelium was transferred into a fast prep tube containing lysis matrix A (MP biomedical) and lysis buffer from Qiagen plant kit mini. Tissue disruption was conducted on the Fast prep1 24 (MP biomedical) and DNA was extracted from the lysate using the Qiagen DNA plant kit mini. DNA was eluted into 100 ml of elution buffer (provided with the kit). DNA from bacterial samples was extracted using the heat shock method. This involved dissolving 2 colonies of each bacterial species in 50 ml of molecular grade H2O and heating it to 94 8C for 5 min before placing immediately on ice. Crayfish DNA was obtained from muscle tissue of the various crayfish species and extracted using the EZ1 DNeasy tissue kit and Robot (Qiagen). This kit was also used to extract DNA from Thelohania contejeani. DNA samples were analysed using the Nanodrop ND1000 spectrophotometer to determine the DNA concentration of the sample. The concentration of the DNA was then standardised to 5 ng/ml. 2.3. Confirmation of amplifiable template DNA Each DNA sample used in this study was checked for amplifiable DNA by PCR using primers known to amplify the target DNA. The Oomycete species were tested using primers ITS1 and ITS4 described by White et al. (1990) and bacterial species using universal 16S primers (Weisburg
Table 1 Species and strains from which DNA was extracted and used to assess PCR methods. Species
Aphanomyces astaci Genetic group A B B B B B B B C D undetermined Aphanomyces species Aphanomyces frigidophilus Aphanomyces invadans Aphanomyces invadans Aphanomyces invadans Aphanomyces species Aphanomyces laevis Aphanomyces cladogamus Saprolegnia species Saprolegnia parasitica Saprolegnia parasitica Saprolegnia parasitica Saprolegnia diclina Saprolegnia species (ferax like) Crayfish species Astacus leptodactylus Astacus astacus Austropotamobius pallipes Procambarus clarkii Austropotamobius torrentium Pacifastacus leniusculus Crayfish parasites Thelohania contejeani Other oomycete species Pythium flevoense Bacterial species Citrobacter freundii Aeromonas hydrophila Hafnia alvei Variovirax sp.
Strain
Sv Ti Yx Ho¨ FDL 457 M96/1 M96/2 Sa Kv Pc 197901
NJM 9701 GWR NJM 002 NJM 9510 CBS 107.52 CBS 108.29
ITS Genbank accession numbers
AY683893 AY310500 AY310499
AY683894 AY683896 HQ660705 EU443838 EU422990
AY310497 AY353920
Wild ‘S’ ‘121’ 197907
HQ660706
CBS 232.72
AY598691
197901
et al., 1991). Presence of amplifiable crayfish DNA was confirmed using a method and primers described by Lo et al. (1996) for the detection of decapod DNA and the presence of amplifiable T. contejeani was confirmed using a method and primers described by Feller (2002). 2.4. PCR assay conditions PCR conditions followed the protocols described in the original publications (Oidtmann et al., 2006; Hochwimmer et al., 2009; Vra˚lstad et al., 2009), unless stated otherwise below. The TaqMan1 real-time PCR assays were run on the StepOnePlusTM Real-Time PCR System (Applied Biosystems) and analysed using the Step one software. The volume of template DNA submitted was 5 ml unless otherwise stated in the subsequent sections.
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For the standard PCR assay, the PCR mix contained 1U of Go Taq polymerase (Promega Scientific), 5 PCR buffer, MgCl2 at a final concentration of 2.5 mM, and dNTPs at a total final concentration of 0.25 mM (conditions otherwise as in Oidtmann et al., 2006). A second standard PCR was conducted using the specifications as described above, but where the forward primer was replaced with a modified forward primer, primer 42v2 (50 -GCT TGT GCT G-GG ATG TTC TT-30 ); the gap shows the deletion of an adenosine residue from the original primer. To test the effect of varying the number of amplification cycles, the sensitivity of the standard PCR assays was tested at 50, 40 and 35 cycles for both standard assays. Each PCR reaction was replicated 6 times (except the tests of varying cycle numbers which were repeated 3 times each) and each set of replicates included 1 negative water control. 2.5. Testing assay sensitivity DNA of A. astaci strain FDL 457 was extracted, analysed using the Nanodrop ND-1000 spectrophotometer and adjusted to a standard concentration of 10 ng/ml. A 10 fold serial dilution was established; from each dilution 5 ml of the template solution was submitted for PCR. Strain FDL 457 was chosen as it had been recently passaged and reisolated from a crayfish host and was the most viable defined strain available. Zoospores were obtained from A. astaci mycelium according to the method described by Cerenius et al. (1988). Zoospores were counted in a Neubauer counting chamber to determine the spore concentration in the water. The zoospore suspension was then concentrated by spinning for 5 min at 3000 g, removal of supernatant and re-suspension of the spores in a smaller water volume. This step was repeated several times until the suspension contained an estimated 100,000 spores (in a final volume of 180 ml; spore concentration estimate based on counts of 3 samples taken from zoospore suspension). The zoospore suspension was submitted to DNA extraction with the DNeasy Plant kit mini (Qiagen) and eluted into 180 ml. This solution was diluted 6 times in a ten-fold serial dilution, and 1.8 ml of each dilution step submitted to PCR. The amount of DNA submitted to PCR from this dilution series corresponds to DNA of 1000, 100, 10, 1, 0.1 0.01 and 0.001 spores.
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2.6. Testing for assay specificity To evaluate whether each of the methods assessed can detect various strains of A. astaci, each PCR method was run containing template DNA from a number of different A. astaci strains (see Table 1). These samples were diluted to a standard concentration of 5 ng/ml before 2.5 ml of template was submitted for PCR. The final amount used in each reaction was 12.5 ng of DNA. Table 1 lists DNA templates from the isolates and species that were included to test the specificity of each PCR assay. DNA was extracted as previously described, diluted to a concentration of 5 ng/ml and submitted to PCR as described above with a total of 12.5 ng DNA in each PCR reaction. These samples were chosen either because they are closely related to A. astaci or because they may be present in a diagnostic sample for crayfish plague. Also as an added check of assay specificity, each primer and probe was checked using a BLAST search to look for any potential identity with other species. 3. Results 3.1. Sensitivity assessment with extracted mycelium DNA The results from the sensitivity testing using mycelium DNA are compared in Table 2. The lowest amount of DNA detected from the 10-fold dilution series by both standard PCR assays regardless of cycle number was 500 fg. The results for the variations in PCR cycle numbers tested were replicated consistently (Figs. 1 and 2) with both primer sets used. Results from the analysis of the ITS TaqMan1 PCR suggest the assay can consistently detect down to 50 fg mycelium DNA in a PCR reaction. The assay amplified also lower quantities of DNA, however a positive result was not observed in all six replicates (Table 2) and at 5 fg, the Ct values observed for each replicate increasingly varied resulting in a mean Ct value with a high standard deviation (Table 2, Fig. 3). The lowest quantity of DNA which produced 100% positive results in the 6 replicates in the chitinase assay was 500 fg. In the subsequent dilution step amplification occurred in 66% of the samples (albeit at Cts above 40) and for the final dilution step no positive results were yielded (Fig. 4). At 500 fg the standard deviation of the assay
Table 2 Results of sensitivity testing using DNA from A. astaci mycelium. Quantitya
50 ng 5 ng 500 pg 50 pg 5 pg 500 fg 50 fg 5 fg a b
ITS assay
Chi assay b
Standard ITS PCR
Ct mean
Standard deviation
% detection (n = 6)
Ct mean
Standard deviation
% detection (n = 6)
% detectionb (n = 9) (primer 42)
% detectionb (n = 9) (primer 42 version 2)
12.782 17.485 20.577 25.364 29.500 32.842 36.064 38.802
0.062 0.083 0.035 0.044 0.168 0.140 0.269 0.744
100 100 100 100 100 100 100 83
18.682 22.366 26.407 31.064 35.877 39.580 43.293 Undetermined
0.103 0.119 0.057 0.245 0.232 0.942 3.027 0
100 100 100 100 100 100 66 0
100 100 100 100 100 100 0 0
100 100 100 100 100 100 0
Total genomic A. astaci DNA in PCR reaction. Percentage of qRT-PCR yielding positive results for each standard.
b
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Fig. 1. Sensitivity of standard PCR assay with Primers 42 and 640 at various cycle numbers using different amounts of A. astaci genomic DNA from mycelium (1 = 50 ng, 2 = 5 ng, 3 = 500 pg, 4 = 50 pg 5 = 5 pg, 6 = 500 fg, 7 = 50 fg and 8 = 5 fg). (i) 50 cycles, (ii) 40 cycles, (iii) 35 cycles. M: 100 bp marker, C: negative control.
Fig. 2. Sensitivity of standard PCR assay with Primers 42v2 and 640 at various cycle numbers using different amounts of A. astaci genomic DNA from mycelium (1 = 50 ng, 2 = 5 ng, 3 = 500 pg, 4 = 50 pg, 5 =5 pg, 6 = 500 fg, 7 = 50 fg and 8 = 5 fg). (i) 50 cycles, (ii) 40 cycles, (iii) 35 cycles. M: 100 bp marker, C: negative control.
increased to almost one (Table 2), suggesting that at this amount of template DNA quantification of A. astaci DNA is less reliable. 3.2. Sensitivity assessment with extracted zoospore DNA The results of the zoospore sensitivity assessment are shown in Table 3 and Figs. 5–8 and demonstrate again that the Vra˚lstad et al. (2009) method is the most sensitive. The results suggest this assay can amplify DNA from one zoospore as all six replicates from this point were positive. The standard deviation for this point was relatively high. During the mycelium sensitivity test the chitinase assay and the standard ITS assay were able to detect similar levels of mycelium DNA (500 fg). However the assays differed in sensitivity when using DNA extracted from
zoospores. The standard PCR method could consistently detect 10 zoospores and the chitinase assay 100 zoospores. This suggests that the standard PCR is slightly more sensitive than the chitinase assay. Interestingly, even at high zoospore concentrations the standard deviation among replicates using the chitinase assay was above 0.4 and the results in Fig. 8 show a very poor standard curve. 3.3. Assessment of specificity Each assay was able to produce a product from each A. astaci strain tested. With both RT-PCR assays the Ct values varied greatly however in each case amplification of the template was observed. The standard PCR assay produced a product for each template at the expected product size
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Table 3 Results of sensitivity testing using DNA from A. astaci zoospores. Quantity (spores)a
1000 100 10 1 0.1 0.01 0.001 a b
ITS assay
Chi assay
Ct mean
Standard deviation
% detection (n = 6)
28.729 36.091 39.219 40.455 40.455 39.707 40.229
0.047 0.161 0.460 0.896 0.896 0.572 0.577
100 100 100 100 33 67 33
b
Standard ITS PCR b
Ct mean
Standard deviation
% detection (n = 6)
% detectionb (n = 9) (primer 42)
% detectionb (n = 9) (primer 42 version 2)
36.432 37.022 41.254 41.258 Undetermined Undetermined Undetermined
0.452 0.423 1.290
100 100 67 16
100 100 100 0 0 0 0
100 100 100 0 0 0 0
Estimated number of A. astaci spores from which DNA was used in PCR reaction. Percentage of qRT-PCR yielding positive results for each standard.
Fig. 3. Amplification plot and quantification curve of A. astaci FDL 457 genomic DNA from mycelium using ITS qRT-PCR. Graph (A) presents the amplification plot of 10 fold serial dilutions from 50 ng to 5 fg. The bold line indicates the threshold used to create the standard curve (Ct threshold: 0.078961). Graph (B) presents the standard curve determined from this amplification plot. The outliers at 5 fg have been omitted from the analysis. The cumulative average of the six replicates is shown; it has a slope of 3.918, y-intercept of 43.881 (when x = 1 fg) and a correlation coefficient of 0.996. The quantity unit on the standard curve is ng.
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Fig. 4. Amplification plot and quantification curve of A. astaci FDL 457 genomic DNA from mycelium using Chi3 qRT-PCR. Graph (A) presents the amplification plot of 10 fold serial dilutions from 50 ng to 5 fg. The bold line indicates the threshold used to create the standard curve (Ct threshold: 0.018185). Graph (B) presents the standard curve determined from this amplification plot. The outlier at 50 fg has been omitted from the analysis; samples below 50 fg did not amplify. The cumulative average of the six replicates is shown; it has a slope of 4.277, y-intercept of 51.27 (Ct when x = 1 fg) and a correlation coefficient of 0.995. The quantity unit on the standard curve is ng/ml.
(569 bps). No difference was observed between the two tested primers sets. There was no amplification of non-A. astaci DNA templates using either RT-assays or the published standard PCR single round assay. The standard PCR assay using the modified forward primer cross reacted with two non-A. astaci templates. In one case (Citrobacter freundii) the product was of a clearly different size (200 bp), which could be easily distinguished from the expected PCR product for A. astaci (569 bp). However, when Hafnia alvei was submitted to the assay, a product of similar size (approximately 500 bp) to the product expected from A.
astaci was obtained. This product was sequenced and confirmed to be of bacterial origin. All primers and probes from these assays were submitted to a BLAST search. These BLAST searches found that no primer (and probe) combination showed identical sequence with any organism other than A. astaci. 4. Discussion Existing data for the three assays at the centre of this study were presented differently both with respect to sensitivity and specificity. Each approach gave very useful
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Fig. 5. Sensitivity of standard PCR assay with primers 42 and 640 at various cycle numbers using different numbers of A. astaci spores. (i) 50 cycles, (ii) 40 cycles, (iii) 35 cycles. M: 100 bp marker, C: negative control.
Fig. 6. Sensitivity of standard PCR assay with Primers 42v2 and 640 at various cycle numbers using different numbers of A. astaci spores. (i) 50 cycles, (ii) 40 cycles, (iii) 35 cycles. M: 100 bp marker, C: negative control.
information about each assay, however, the use of different approaches made a comparison of analytical test performance difficult. Vra˚lstad et al. (2009) had reported a limit of detection (95% of replicates amplified) of 160 fg; Hochwimmer et al. (2009) a detection down to 25 copies and Oidtmann et al. (2006) down to 100 fg or 1 spore. Test specificity was tested across different ranges of target DNA in the original studies. Here, we compared three PCR based detection methods of A. astaci, a step not taken in the previous validations of these methods. Our results largely confirmed the results of the original publications. Overall, the qRT-PCR targeted at the ITS region emerged as the most sensitive method, followed by the standard PCR assay and finally the qRT-PCR targeting the chitinase genes. The difference in sensitivity noted between the two ITS targeted PCR methods most likely relates to the technology used; agarose gel electrophoresis requires a high level of DNA before a band can be visualised compared to qRT-PCR which only requires a few copies to generate a signal. The chitinase assay was about 10–100 times less sensitive compared to the qRT-PCR targeted at the ITS region. This can be explained by the number of targets in the A. astaci genome. The chitinase assay targets
three homologues (chi1, chi2 and chi3); therefore the number of targets in the A. astaci genome for this assay will be approximately 3 copies. In contrast, the ITS region represent a multi-copy region (Vra˚lstad et al., 2009; Makkonen et al., 2011). An extrapolation of Oomycete genome size suggests that 100 fg of A. astaci DNA is equivalent to 0.4–4.5 genomic units (Kamoun, 2003; Oidtmann et al., 2006). One zoospore can be considered the equivalent for a genomic unit. The consistent amplification at corresponding dilutions of spore DNA vs. genomic DNA concentrations measured photometrically reported for both ITS targeted assays (500 fg or 10 spores detected by the standard PCR; 50 fg or 1 spore detected by the real-time PCR) are in line with the genome size suggested by Kamoun (2003). Using the chitinase gene assay, A. astaci was consistently detected at 500 fg, and 100 spores. Given that 500 fg could represent up to approximately 20 genomic units, the results observed for the chitinase gene assay are also consistent and the test sensitivity of this assay is likely to lie just above 10 spores. Whereas Oidtmann et al. (2006) had previously only reported the test sensitivity at 50 cycles, we now tested
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Fig. 7. Amplification plot and quantification curve of A. astaci FDL 457 genomic DNA from counted zoospores using ITS qRT-PCR. Graph (A) presents the amplification plot of 10 fold serial dilutions from 1000 to 0.001 zoospores. The bold line indicates the threshold used to create the standard curve (Ct threshold: 0.21725). Graph (B) presents the standard curve determined from this amplification plot. The outliers at 0.1 spores where omitted and reactions containing 0.01 spores or less did not amplify. The cumulative average of the six replicates is shown; it has a slope of 3.569, y-intercept (Ct when x = 1) of 39.654 and a correlation coefficient of 0.993.
lower cycle numbers and observed no difference between 35, 40 or 50 cycles. Furthermore, although the majority of A. astaci strains sequenced to date lack the additional adenosine residue included in primer 42, there was no difference in test sensitivity between primer 42 and the new primer 42v2, designed to adjust for this. The assay consistently detected 500 fg of genomic DNA or DNA extracted from 10 zoospores. It appears that the additional nucleotide present in primer 42 has little effect on the priming efficacy. A limited effect of primer-template mismatches that are located at some distance from the 30 end of the primer have previously been described (Kwok et al., 1990; Yao et al., 2006; Stadhouders et al., 2010). Previously, Oidtmann et al. (2006) had reported detection down to 1 spore using 50 cycles, having used a different PCR reaction mix compared to this study. The difference between the results reported by Oidtmann et al. (2006) and this study are possibly explained by slight pipetting inaccuracies in the preparation of the dilution
series. The difference in master nix used may have also had an impact. Hochwimmer et al. (2009) also described a Sybr green version of their assay. In the Sybr green assay 2 sets of primers pairs (4 primers) are used in a single PCR reaction. We tested the Sybr green version in preliminary experiments during this study and repeatedly observed a melting curve which was difficult to interpret. As a result of these problems and the fact the TaqMan1 assay described by Hochwimmer et al. (2009) allowed for direct comparison with the ITS real time assay, the Sybr green assay was omitted from this study. The specificity testing conducted here confirmed that all assays detect A. astaci DNA across all 4 genogroups that have been previously described (Huang et al., 1994; Dieguez-Uribeondo et al., 1995; Oidtmann et al., 1999), as also shown by Vra˚lstad et al., 2009, for the ITS realtime PCR assay. Furthermore, none of the previously published assays showed any cross-reactivity with any
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Fig. 8. Amplification plot and quantification curve of A. astaci FDL 457 genomic DNA from counted zoospores using Chi3 qRT-PCR. Graph (A) presents the amplification plot of 10 fold serial dilutions from 1000 to 0.001 zoospores. The bold line indicates the threshold used to create the standard curve (Ct threshold: 0.021302). Graph (B) presents the standard curve determined from this amplification plot. 2 replicates of 10 zoospores and 1 replicate amplified with 1 zoospore out of the six, replicates under one zoospore did not amplify. The cumulative average of the six replicates is shown; it has a slope of 1.652, y-intercept (Ct when x = 1) of 41.021 and a correlation coefficient of 0.806.
of the organisms tested in this study. However, some cross reaction of two of the assays tested has previously been suggested. Hochwimmer et al. (2009) reported that their qRT-PCR for the chi genes also amplified A. helicoides and Leptolegnia caudata (which were not included in the specificity testing undertaken here). Furthermore, Kozubı´kova´ et al. (2009) tested North American crayfish populations in the Czech Republic for carrier status and reported that in one case sequencing of a PCR product generated using the single round PCR assay as described by Oidtmann et al. (2006), lead to the discovery of a so far unknown Aphanomyces species closely related to A. astaci (see also Die´guez-Uribeondo et al., 2009). An observation made in this study was that the variation of the forward primer sequence of the standard PCR (primer 42v2) reduced specificity of the assay and its use therefore not recommended.
Sequence differences would have theoretically existed between the reverse primer of the qRT-PCR targeting the ITS region and the target DNA (See Fig. 9). Given the observations with the standard PCR assay, such differences appear not to impact on test sensitivity. It is useful to compare the three methods and analyse their pros and cons. The high sensitivity of the ITS qRT-PCR is a clear advantage over the other assays, especially if the method is applied to detect carrier status in North American crayfish, since these animals are likely to carry A. astaci at a much lower level of pathogen DNA than moribund animals. The higher sensitivity of the assay may lead to the detection of further carriers, where the standard assay may fail to detect due to lower analytical test sensitivity. The method also appeared to be highly specific in all studies undertaken to date. A possible disadvantage of qRT-PCR is that false positives are more difficult to detect. Amplicons generated
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Fig. 9. Alignment of published sequences showing the regions of the ITS and 5.8S rRNA gene of A. astaci targeted by primers and probe from both the Vra˚lstad et al. (2009) (framed) and Oidtmann et al. (2006) (shaded grey) PCR methods. Genbank accession numbers for each of the sequences are shown in brackets.
by qRT-PCR are not usually confirmed by sequencing and even if they were, the shortness of the amplicon may limit its information value. Several new Aphanomyces species have been detected in North American crayfish over the last few years (Die´guez-Uribeondo et al., 2009), the relevance of which remains to be further studied. None of the new species identified so far would lead to crossreaction with the ITS qRT-PCR assay. However, researchers using PCR as a diagnostic tool need to remain aware of the possible presence of this far undetected Aphanomyces species that may cross-react. When high Ct values are observed using the ITS qRTPCR, Vra˚lstad et al. (2009) recommend the use of confirmatory analysis to determine if such results represent false positives or trace amounts of target DNA and suggest collection of additional material. However, if no additional material is available, the diagnosis may be unresolved. The above also largely summarises the advantages and disadvantages of the conventional PCR assay. An advantage of the conventional PCR is the ability to confirm positives by sequencing, however, this is at the cost of a slightly reduced sensitivity. Sequencing of the PCR product may also provide additional information that may be used for molecular epidemiological studies and also allows discrimination of new emerging strains or closely related species. Following the points highlighted above, at present the qRT-PCR targeting the chi genes does not appear to offer any further advantage over the ITS assays. Given the crossreactions observed by Hochwimmer et al. (2009), and the fact that far fewer sequence data are available for the
Chitinase genes, some further work might be necessary to demonstrate the benefits of using this assay. Given the results of this study, we would consider the ITS qRT-PCR to be the preferred method for screening populations of North American crayfish for carrier status. Occasional confirmation of results using the conventional assay followed by sequencing would be advisable. Both the standard PCR and the ITS qRT-PCR are suitable for diagnosing disease outbreaks in the highly susceptible species. Follow up confirmation by sequencing is recommended. In the case of the ITS qRT-PCR, a different PCR amplicon would need to be generated to provide sufficient sequence information (e.g. by using the standard PCR assay). The continued detection of new Aphanomyces species in aquatic animals highlight the challenges faced when designing a diagnostic PCR assay and underline that it remains important to isolate Oomycetes from freshwater crayfish, to sequence PCR products and compare PCR results to histology results where possible. It also underlines that combining the application of multiple PCR assays can be beneficial and provide additional information. Acknowledgements We would like to warmly acknowledge Richard Paley, Tim Bean and David Stone for useful discussions and advice and Patricia Brown for looking after the Oomycete cultures. This work was funded by Defra project F1172. The authors would also like to extend special thanks to the reviewers for valuable comments.
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Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
A comparative study of molecular diagnostic methods designed to detect the crayfish plague pathogen, Aphanomyces astaci Stephen Tuffs a,b, Birgit Oidtmann a,* a
Centre for Environment, Fisheries and Aquaculture Science, Barrack Road, Weymouth, Dorset, United Kingdom Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, Glasgow Biomedical Research Centre, University of Glasgow, 120 University Place, Glasgow G12 8TA, United Kingdom
b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 December 2010 Received in revised form 9 June 2011 Accepted 15 June 2011
Crayfish plague is the most important disease of freshwater crayfish with a significant impact on European species. We compared the analytical test sensitivity and specificity of three published PCR assays for the detection of Aphanomyces astaci, the causative agent of crayfish plague: a conventional PCR assay targeting the ITS region and two TaqMan1 real time assays, targeting either the ITS region or the chitinase gene. We also tested a variation of the conventional assay, by changing one of the primers. Test specificity was assessed using DNA from a range of A. astaci strains and an array of closely related Oomycetes, host tissue and DNA from other organisms that may be present in a diagnostic sample. Sensitivity was assessed using genomic A. astaci DNA from mycelium and zoospores. All assays were found to be of good to excellent sensitivity with levels of detection ranging from 1 (real time assay targeting the ITS region), over 10 (conventional PCR) to 100 zoospores (real time assay targeting the chitinase gene). All three published assays were also specific for A. astaci and did not cross-react with any other test organisms included in this study. The tested variation of the conventional PCR assay with a changed forward primer led to amplification of some non A. astaci DNA. Advantages and disadvantages, including suitable application are discussed for each assay. Crown Copyright ß 2011 Published by Elsevier B.V. All rights reserved.
Keywords: Aphanomyces astaci Real time PCR PCR Crayfish plague Carrier Saprolegniaceae Oomycota
1. Introduction Crayfish plague is the most important disease of freshwater crayfish species; it is caused by the Oomycete pathogen Aphanomyces astaci and has been heavily implicated in the decline of native crayfish species across Europe, as infection in these species is fatal (Edgerton et al., 2004; Edsman et al., 2010; Fu¨reder et al., 2010). North American crayfish may be infected with A. astaci, without normally developing disease (Unestam, 1969, 1972; Unestam and Weiss, 1970; Die´guez-Uribeondo and So¨derha¨ll, 1993; Cerenius et al., 2010). They can therefore act as carriers, and their introduction into Europe has established
* Corresponding author. Tel.: +44 1305 206661; fax: +44 1305 206601. E-mail address: [email protected] (B. Oidtmann).
a potential reservoir for the pathogen which represents a serious threat to native European crayfish species (Holdich et al., 2009). Fast and accurate diagnosis is important if the disease is to be controlled, therefore in recent years molecular diagnostic methods, based on PCR have been developed to detect A. astaci with the aim of being more robust, time and resource effective than culture dependent diagnostic methods. A conventional PCR method was described by Oidtmann et al. (2006) which targets the internal transcribed spacer (ITS) region of the 5.8 s rRNA gene. The method has been widely applied both for the detection of carrier status in North American crayfish and to confirm the pathogen in disease outbreaks in European crayfish species (Schulz et al., 2006; Jones et al., 2009; Kozubı´kova´ et al., 2009; Kozubı´kova´ et al., 2010; Camma` et al., 2010).
0378-1135/$ – see front matter . Crown Copyright ß 2011 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2011.06.012
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Quantitative real time polymerase chain reaction (RTPCR) has also been explored as a potential diagnostic alternative and two quantitative TaqMan1 assays have been developed, one, a TaqMan1 minor groove binder (MGB) RT-PCR targeting the ITS region (Vra˚lstad et al., 2009), the other, using a conventional, TaqMan1 probe and targeting chitinase (chi1, chi2 and chi3) encoding genes in the organism (Hochwimmer et al., 2009).All three of these studies provided appropriate and informative validation data, however in each paper a different approach was taken to validate the PCR method, making direct comparison of these methods difficult. The aim of this study was to use a consistent sensitivity and specificity testing to compare these three methods and consider their suitability for different applications. 2. Materials and methods 2.1. Oomycetes and other organisms tested The various A. astaci strains and other organisms used in this study are listed in Table 1. These included some Saprolegniales that were isolated from a crayfish plague outbreak in the North of the United Kingdom in 2009: a strain of A. astaci designated 197901 (HQ660705) and a Saprolegnia species (HQ660706) with homology to S. ferax, designated 197907. 2.2. DNA extraction DNA from Oomycete species was obtained by extracting DNA from pure mycelium grown in broth cultures using the DNeasy plant kit mini (Qiagen) with an additional tissue lysis step. Approximately 100 mg of mycelium was transferred into a fast prep tube containing lysis matrix A (MP biomedical) and lysis buffer from Qiagen plant kit mini. Tissue disruption was conducted on the Fast prep1 24 (MP biomedical) and DNA was extracted from the lysate using the Qiagen DNA plant kit mini. DNA was eluted into 100 ml of elution buffer (provided with the kit). DNA from bacterial samples was extracted using the heat shock method. This involved dissolving 2 colonies of each bacterial species in 50 ml of molecular grade H2O and heating it to 94 8C for 5 min before placing immediately on ice. Crayfish DNA was obtained from muscle tissue of the various crayfish species and extracted using the EZ1 DNeasy tissue kit and Robot (Qiagen). This kit was also used to extract DNA from Thelohania contejeani. DNA samples were analysed using the Nanodrop ND1000 spectrophotometer to determine the DNA concentration of the sample. The concentration of the DNA was then standardised to 5 ng/ml. 2.3. Confirmation of amplifiable template DNA Each DNA sample used in this study was checked for amplifiable DNA by PCR using primers known to amplify the target DNA. The Oomycete species were tested using primers ITS1 and ITS4 described by White et al. (1990) and bacterial species using universal 16S primers (Weisburg
Table 1 Species and strains from which DNA was extracted and used to assess PCR methods. Species
Aphanomyces astaci Genetic group A B B B B B B B C D undetermined Aphanomyces species Aphanomyces frigidophilus Aphanomyces invadans Aphanomyces invadans Aphanomyces invadans Aphanomyces species Aphanomyces laevis Aphanomyces cladogamus Saprolegnia species Saprolegnia parasitica Saprolegnia parasitica Saprolegnia parasitica Saprolegnia diclina Saprolegnia species (ferax like) Crayfish species Astacus leptodactylus Astacus astacus Austropotamobius pallipes Procambarus clarkii Austropotamobius torrentium Pacifastacus leniusculus Crayfish parasites Thelohania contejeani Other oomycete species Pythium flevoense Bacterial species Citrobacter freundii Aeromonas hydrophila Hafnia alvei Variovirax sp.
Strain
Sv Ti Yx Ho¨ FDL 457 M96/1 M96/2 Sa Kv Pc 197901
NJM 9701 GWR NJM 002 NJM 9510 CBS 107.52 CBS 108.29
ITS Genbank accession numbers
AY683893 AY310500 AY310499
AY683894 AY683896 HQ660705 EU443838 EU422990
AY310497 AY353920
Wild ‘S’ ‘121’ 197907
HQ660706
CBS 232.72
AY598691
197901
et al., 1991). Presence of amplifiable crayfish DNA was confirmed using a method and primers described by Lo et al. (1996) for the detection of decapod DNA and the presence of amplifiable T. contejeani was confirmed using a method and primers described by Feller (2002). 2.4. PCR assay conditions PCR conditions followed the protocols described in the original publications (Oidtmann et al., 2006; Hochwimmer et al., 2009; Vra˚lstad et al., 2009), unless stated otherwise below. The TaqMan1 real-time PCR assays were run on the StepOnePlusTM Real-Time PCR System (Applied Biosystems) and analysed using the Step one software. The volume of template DNA submitted was 5 ml unless otherwise stated in the subsequent sections.
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For the standard PCR assay, the PCR mix contained 1U of Go Taq polymerase (Promega Scientific), 5 PCR buffer, MgCl2 at a final concentration of 2.5 mM, and dNTPs at a total final concentration of 0.25 mM (conditions otherwise as in Oidtmann et al., 2006). A second standard PCR was conducted using the specifications as described above, but where the forward primer was replaced with a modified forward primer, primer 42v2 (50 -GCT TGT GCT G-GG ATG TTC TT-30 ); the gap shows the deletion of an adenosine residue from the original primer. To test the effect of varying the number of amplification cycles, the sensitivity of the standard PCR assays was tested at 50, 40 and 35 cycles for both standard assays. Each PCR reaction was replicated 6 times (except the tests of varying cycle numbers which were repeated 3 times each) and each set of replicates included 1 negative water control. 2.5. Testing assay sensitivity DNA of A. astaci strain FDL 457 was extracted, analysed using the Nanodrop ND-1000 spectrophotometer and adjusted to a standard concentration of 10 ng/ml. A 10 fold serial dilution was established; from each dilution 5 ml of the template solution was submitted for PCR. Strain FDL 457 was chosen as it had been recently passaged and reisolated from a crayfish host and was the most viable defined strain available. Zoospores were obtained from A. astaci mycelium according to the method described by Cerenius et al. (1988). Zoospores were counted in a Neubauer counting chamber to determine the spore concentration in the water. The zoospore suspension was then concentrated by spinning for 5 min at 3000 g, removal of supernatant and re-suspension of the spores in a smaller water volume. This step was repeated several times until the suspension contained an estimated 100,000 spores (in a final volume of 180 ml; spore concentration estimate based on counts of 3 samples taken from zoospore suspension). The zoospore suspension was submitted to DNA extraction with the DNeasy Plant kit mini (Qiagen) and eluted into 180 ml. This solution was diluted 6 times in a ten-fold serial dilution, and 1.8 ml of each dilution step submitted to PCR. The amount of DNA submitted to PCR from this dilution series corresponds to DNA of 1000, 100, 10, 1, 0.1 0.01 and 0.001 spores.
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2.6. Testing for assay specificity To evaluate whether each of the methods assessed can detect various strains of A. astaci, each PCR method was run containing template DNA from a number of different A. astaci strains (see Table 1). These samples were diluted to a standard concentration of 5 ng/ml before 2.5 ml of template was submitted for PCR. The final amount used in each reaction was 12.5 ng of DNA. Table 1 lists DNA templates from the isolates and species that were included to test the specificity of each PCR assay. DNA was extracted as previously described, diluted to a concentration of 5 ng/ml and submitted to PCR as described above with a total of 12.5 ng DNA in each PCR reaction. These samples were chosen either because they are closely related to A. astaci or because they may be present in a diagnostic sample for crayfish plague. Also as an added check of assay specificity, each primer and probe was checked using a BLAST search to look for any potential identity with other species. 3. Results 3.1. Sensitivity assessment with extracted mycelium DNA The results from the sensitivity testing using mycelium DNA are compared in Table 2. The lowest amount of DNA detected from the 10-fold dilution series by both standard PCR assays regardless of cycle number was 500 fg. The results for the variations in PCR cycle numbers tested were replicated consistently (Figs. 1 and 2) with both primer sets used. Results from the analysis of the ITS TaqMan1 PCR suggest the assay can consistently detect down to 50 fg mycelium DNA in a PCR reaction. The assay amplified also lower quantities of DNA, however a positive result was not observed in all six replicates (Table 2) and at 5 fg, the Ct values observed for each replicate increasingly varied resulting in a mean Ct value with a high standard deviation (Table 2, Fig. 3). The lowest quantity of DNA which produced 100% positive results in the 6 replicates in the chitinase assay was 500 fg. In the subsequent dilution step amplification occurred in 66% of the samples (albeit at Cts above 40) and for the final dilution step no positive results were yielded (Fig. 4). At 500 fg the standard deviation of the assay
Table 2 Results of sensitivity testing using DNA from A. astaci mycelium. Quantitya
50 ng 5 ng 500 pg 50 pg 5 pg 500 fg 50 fg 5 fg a b
ITS assay
Chi assay b
Standard ITS PCR
Ct mean
Standard deviation
% detection (n = 6)
Ct mean
Standard deviation
% detection (n = 6)
% detectionb (n = 9) (primer 42)
% detectionb (n = 9) (primer 42 version 2)
12.782 17.485 20.577 25.364 29.500 32.842 36.064 38.802
0.062 0.083 0.035 0.044 0.168 0.140 0.269 0.744
100 100 100 100 100 100 100 83
18.682 22.366 26.407 31.064 35.877 39.580 43.293 Undetermined
0.103 0.119 0.057 0.245 0.232 0.942 3.027 0
100 100 100 100 100 100 66 0
100 100 100 100 100 100 0 0
100 100 100 100 100 100 0
Total genomic A. astaci DNA in PCR reaction. Percentage of qRT-PCR yielding positive results for each standard.
b
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Fig. 1. Sensitivity of standard PCR assay with Primers 42 and 640 at various cycle numbers using different amounts of A. astaci genomic DNA from mycelium (1 = 50 ng, 2 = 5 ng, 3 = 500 pg, 4 = 50 pg 5 = 5 pg, 6 = 500 fg, 7 = 50 fg and 8 = 5 fg). (i) 50 cycles, (ii) 40 cycles, (iii) 35 cycles. M: 100 bp marker, C: negative control.
Fig. 2. Sensitivity of standard PCR assay with Primers 42v2 and 640 at various cycle numbers using different amounts of A. astaci genomic DNA from mycelium (1 = 50 ng, 2 = 5 ng, 3 = 500 pg, 4 = 50 pg, 5 =5 pg, 6 = 500 fg, 7 = 50 fg and 8 = 5 fg). (i) 50 cycles, (ii) 40 cycles, (iii) 35 cycles. M: 100 bp marker, C: negative control.
increased to almost one (Table 2), suggesting that at this amount of template DNA quantification of A. astaci DNA is less reliable. 3.2. Sensitivity assessment with extracted zoospore DNA The results of the zoospore sensitivity assessment are shown in Table 3 and Figs. 5–8 and demonstrate again that the Vra˚lstad et al. (2009) method is the most sensitive. The results suggest this assay can amplify DNA from one zoospore as all six replicates from this point were positive. The standard deviation for this point was relatively high. During the mycelium sensitivity test the chitinase assay and the standard ITS assay were able to detect similar levels of mycelium DNA (500 fg). However the assays differed in sensitivity when using DNA extracted from
zoospores. The standard PCR method could consistently detect 10 zoospores and the chitinase assay 100 zoospores. This suggests that the standard PCR is slightly more sensitive than the chitinase assay. Interestingly, even at high zoospore concentrations the standard deviation among replicates using the chitinase assay was above 0.4 and the results in Fig. 8 show a very poor standard curve. 3.3. Assessment of specificity Each assay was able to produce a product from each A. astaci strain tested. With both RT-PCR assays the Ct values varied greatly however in each case amplification of the template was observed. The standard PCR assay produced a product for each template at the expected product size
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Table 3 Results of sensitivity testing using DNA from A. astaci zoospores. Quantity (spores)a
1000 100 10 1 0.1 0.01 0.001 a b
ITS assay
Chi assay
Ct mean
Standard deviation
% detection (n = 6)
28.729 36.091 39.219 40.455 40.455 39.707 40.229
0.047 0.161 0.460 0.896 0.896 0.572 0.577
100 100 100 100 33 67 33
b
Standard ITS PCR b
Ct mean
Standard deviation
% detection (n = 6)
% detectionb (n = 9) (primer 42)
% detectionb (n = 9) (primer 42 version 2)
36.432 37.022 41.254 41.258 Undetermined Undetermined Undetermined
0.452 0.423 1.290
100 100 67 16
100 100 100 0 0 0 0
100 100 100 0 0 0 0
Estimated number of A. astaci spores from which DNA was used in PCR reaction. Percentage of qRT-PCR yielding positive results for each standard.
Fig. 3. Amplification plot and quantification curve of A. astaci FDL 457 genomic DNA from mycelium using ITS qRT-PCR. Graph (A) presents the amplification plot of 10 fold serial dilutions from 50 ng to 5 fg. The bold line indicates the threshold used to create the standard curve (Ct threshold: 0.078961). Graph (B) presents the standard curve determined from this amplification plot. The outliers at 5 fg have been omitted from the analysis. The cumulative average of the six replicates is shown; it has a slope of 3.918, y-intercept of 43.881 (when x = 1 fg) and a correlation coefficient of 0.996. The quantity unit on the standard curve is ng.
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Fig. 4. Amplification plot and quantification curve of A. astaci FDL 457 genomic DNA from mycelium using Chi3 qRT-PCR. Graph (A) presents the amplification plot of 10 fold serial dilutions from 50 ng to 5 fg. The bold line indicates the threshold used to create the standard curve (Ct threshold: 0.018185). Graph (B) presents the standard curve determined from this amplification plot. The outlier at 50 fg has been omitted from the analysis; samples below 50 fg did not amplify. The cumulative average of the six replicates is shown; it has a slope of 4.277, y-intercept of 51.27 (Ct when x = 1 fg) and a correlation coefficient of 0.995. The quantity unit on the standard curve is ng/ml.
(569 bps). No difference was observed between the two tested primers sets. There was no amplification of non-A. astaci DNA templates using either RT-assays or the published standard PCR single round assay. The standard PCR assay using the modified forward primer cross reacted with two non-A. astaci templates. In one case (Citrobacter freundii) the product was of a clearly different size (200 bp), which could be easily distinguished from the expected PCR product for A. astaci (569 bp). However, when Hafnia alvei was submitted to the assay, a product of similar size (approximately 500 bp) to the product expected from A.
astaci was obtained. This product was sequenced and confirmed to be of bacterial origin. All primers and probes from these assays were submitted to a BLAST search. These BLAST searches found that no primer (and probe) combination showed identical sequence with any organism other than A. astaci. 4. Discussion Existing data for the three assays at the centre of this study were presented differently both with respect to sensitivity and specificity. Each approach gave very useful
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Fig. 5. Sensitivity of standard PCR assay with primers 42 and 640 at various cycle numbers using different numbers of A. astaci spores. (i) 50 cycles, (ii) 40 cycles, (iii) 35 cycles. M: 100 bp marker, C: negative control.
Fig. 6. Sensitivity of standard PCR assay with Primers 42v2 and 640 at various cycle numbers using different numbers of A. astaci spores. (i) 50 cycles, (ii) 40 cycles, (iii) 35 cycles. M: 100 bp marker, C: negative control.
information about each assay, however, the use of different approaches made a comparison of analytical test performance difficult. Vra˚lstad et al. (2009) had reported a limit of detection (95% of replicates amplified) of 160 fg; Hochwimmer et al. (2009) a detection down to 25 copies and Oidtmann et al. (2006) down to 100 fg or 1 spore. Test specificity was tested across different ranges of target DNA in the original studies. Here, we compared three PCR based detection methods of A. astaci, a step not taken in the previous validations of these methods. Our results largely confirmed the results of the original publications. Overall, the qRT-PCR targeted at the ITS region emerged as the most sensitive method, followed by the standard PCR assay and finally the qRT-PCR targeting the chitinase genes. The difference in sensitivity noted between the two ITS targeted PCR methods most likely relates to the technology used; agarose gel electrophoresis requires a high level of DNA before a band can be visualised compared to qRT-PCR which only requires a few copies to generate a signal. The chitinase assay was about 10–100 times less sensitive compared to the qRT-PCR targeted at the ITS region. This can be explained by the number of targets in the A. astaci genome. The chitinase assay targets
three homologues (chi1, chi2 and chi3); therefore the number of targets in the A. astaci genome for this assay will be approximately 3 copies. In contrast, the ITS region represent a multi-copy region (Vra˚lstad et al., 2009; Makkonen et al., 2011). An extrapolation of Oomycete genome size suggests that 100 fg of A. astaci DNA is equivalent to 0.4–4.5 genomic units (Kamoun, 2003; Oidtmann et al., 2006). One zoospore can be considered the equivalent for a genomic unit. The consistent amplification at corresponding dilutions of spore DNA vs. genomic DNA concentrations measured photometrically reported for both ITS targeted assays (500 fg or 10 spores detected by the standard PCR; 50 fg or 1 spore detected by the real-time PCR) are in line with the genome size suggested by Kamoun (2003). Using the chitinase gene assay, A. astaci was consistently detected at 500 fg, and 100 spores. Given that 500 fg could represent up to approximately 20 genomic units, the results observed for the chitinase gene assay are also consistent and the test sensitivity of this assay is likely to lie just above 10 spores. Whereas Oidtmann et al. (2006) had previously only reported the test sensitivity at 50 cycles, we now tested
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Fig. 7. Amplification plot and quantification curve of A. astaci FDL 457 genomic DNA from counted zoospores using ITS qRT-PCR. Graph (A) presents the amplification plot of 10 fold serial dilutions from 1000 to 0.001 zoospores. The bold line indicates the threshold used to create the standard curve (Ct threshold: 0.21725). Graph (B) presents the standard curve determined from this amplification plot. The outliers at 0.1 spores where omitted and reactions containing 0.01 spores or less did not amplify. The cumulative average of the six replicates is shown; it has a slope of 3.569, y-intercept (Ct when x = 1) of 39.654 and a correlation coefficient of 0.993.
lower cycle numbers and observed no difference between 35, 40 or 50 cycles. Furthermore, although the majority of A. astaci strains sequenced to date lack the additional adenosine residue included in primer 42, there was no difference in test sensitivity between primer 42 and the new primer 42v2, designed to adjust for this. The assay consistently detected 500 fg of genomic DNA or DNA extracted from 10 zoospores. It appears that the additional nucleotide present in primer 42 has little effect on the priming efficacy. A limited effect of primer-template mismatches that are located at some distance from the 30 end of the primer have previously been described (Kwok et al., 1990; Yao et al., 2006; Stadhouders et al., 2010). Previously, Oidtmann et al. (2006) had reported detection down to 1 spore using 50 cycles, having used a different PCR reaction mix compared to this study. The difference between the results reported by Oidtmann et al. (2006) and this study are possibly explained by slight pipetting inaccuracies in the preparation of the dilution
series. The difference in master nix used may have also had an impact. Hochwimmer et al. (2009) also described a Sybr green version of their assay. In the Sybr green assay 2 sets of primers pairs (4 primers) are used in a single PCR reaction. We tested the Sybr green version in preliminary experiments during this study and repeatedly observed a melting curve which was difficult to interpret. As a result of these problems and the fact the TaqMan1 assay described by Hochwimmer et al. (2009) allowed for direct comparison with the ITS real time assay, the Sybr green assay was omitted from this study. The specificity testing conducted here confirmed that all assays detect A. astaci DNA across all 4 genogroups that have been previously described (Huang et al., 1994; Dieguez-Uribeondo et al., 1995; Oidtmann et al., 1999), as also shown by Vra˚lstad et al., 2009, for the ITS realtime PCR assay. Furthermore, none of the previously published assays showed any cross-reactivity with any
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Fig. 8. Amplification plot and quantification curve of A. astaci FDL 457 genomic DNA from counted zoospores using Chi3 qRT-PCR. Graph (A) presents the amplification plot of 10 fold serial dilutions from 1000 to 0.001 zoospores. The bold line indicates the threshold used to create the standard curve (Ct threshold: 0.021302). Graph (B) presents the standard curve determined from this amplification plot. 2 replicates of 10 zoospores and 1 replicate amplified with 1 zoospore out of the six, replicates under one zoospore did not amplify. The cumulative average of the six replicates is shown; it has a slope of 1.652, y-intercept (Ct when x = 1) of 41.021 and a correlation coefficient of 0.806.
of the organisms tested in this study. However, some cross reaction of two of the assays tested has previously been suggested. Hochwimmer et al. (2009) reported that their qRT-PCR for the chi genes also amplified A. helicoides and Leptolegnia caudata (which were not included in the specificity testing undertaken here). Furthermore, Kozubı´kova´ et al. (2009) tested North American crayfish populations in the Czech Republic for carrier status and reported that in one case sequencing of a PCR product generated using the single round PCR assay as described by Oidtmann et al. (2006), lead to the discovery of a so far unknown Aphanomyces species closely related to A. astaci (see also Die´guez-Uribeondo et al., 2009). An observation made in this study was that the variation of the forward primer sequence of the standard PCR (primer 42v2) reduced specificity of the assay and its use therefore not recommended.
Sequence differences would have theoretically existed between the reverse primer of the qRT-PCR targeting the ITS region and the target DNA (See Fig. 9). Given the observations with the standard PCR assay, such differences appear not to impact on test sensitivity. It is useful to compare the three methods and analyse their pros and cons. The high sensitivity of the ITS qRT-PCR is a clear advantage over the other assays, especially if the method is applied to detect carrier status in North American crayfish, since these animals are likely to carry A. astaci at a much lower level of pathogen DNA than moribund animals. The higher sensitivity of the assay may lead to the detection of further carriers, where the standard assay may fail to detect due to lower analytical test sensitivity. The method also appeared to be highly specific in all studies undertaken to date. A possible disadvantage of qRT-PCR is that false positives are more difficult to detect. Amplicons generated
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Fig. 9. Alignment of published sequences showing the regions of the ITS and 5.8S rRNA gene of A. astaci targeted by primers and probe from both the Vra˚lstad et al. (2009) (framed) and Oidtmann et al. (2006) (shaded grey) PCR methods. Genbank accession numbers for each of the sequences are shown in brackets.
by qRT-PCR are not usually confirmed by sequencing and even if they were, the shortness of the amplicon may limit its information value. Several new Aphanomyces species have been detected in North American crayfish over the last few years (Die´guez-Uribeondo et al., 2009), the relevance of which remains to be further studied. None of the new species identified so far would lead to crossreaction with the ITS qRT-PCR assay. However, researchers using PCR as a diagnostic tool need to remain aware of the possible presence of this far undetected Aphanomyces species that may cross-react. When high Ct values are observed using the ITS qRTPCR, Vra˚lstad et al. (2009) recommend the use of confirmatory analysis to determine if such results represent false positives or trace amounts of target DNA and suggest collection of additional material. However, if no additional material is available, the diagnosis may be unresolved. The above also largely summarises the advantages and disadvantages of the conventional PCR assay. An advantage of the conventional PCR is the ability to confirm positives by sequencing, however, this is at the cost of a slightly reduced sensitivity. Sequencing of the PCR product may also provide additional information that may be used for molecular epidemiological studies and also allows discrimination of new emerging strains or closely related species. Following the points highlighted above, at present the qRT-PCR targeting the chi genes does not appear to offer any further advantage over the ITS assays. Given the crossreactions observed by Hochwimmer et al. (2009), and the fact that far fewer sequence data are available for the
Chitinase genes, some further work might be necessary to demonstrate the benefits of using this assay. Given the results of this study, we would consider the ITS qRT-PCR to be the preferred method for screening populations of North American crayfish for carrier status. Occasional confirmation of results using the conventional assay followed by sequencing would be advisable. Both the standard PCR and the ITS qRT-PCR are suitable for diagnosing disease outbreaks in the highly susceptible species. Follow up confirmation by sequencing is recommended. In the case of the ITS qRT-PCR, a different PCR amplicon would need to be generated to provide sufficient sequence information (e.g. by using the standard PCR assay). The continued detection of new Aphanomyces species in aquatic animals highlight the challenges faced when designing a diagnostic PCR assay and underline that it remains important to isolate Oomycetes from freshwater crayfish, to sequence PCR products and compare PCR results to histology results where possible. It also underlines that combining the application of multiple PCR assays can be beneficial and provide additional information. Acknowledgements We would like to warmly acknowledge Richard Paley, Tim Bean and David Stone for useful discussions and advice and Patricia Brown for looking after the Oomycete cultures. This work was funded by Defra project F1172. The authors would also like to extend special thanks to the reviewers for valuable comments.
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