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1 CSIRO Livestock Industries, F. D. McMaster Laboratory Chiswick, Armidale, New South Wales 2350, Australia
2 School of Veterinary Science, The University of Melbourne, Werribee, Victoria 3030, Australia
Correspondence
Amir H. Noormohammadi
amirh{at}unimelb.edu.au
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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400 bp were generated from 35 different M. synoviae strains/isolates from chickens and subjected to mutation scanning analysis. Analysis of the amplicons by single-strand conformation polymorphism (SSCP) revealed 10 distinct profiles (A–J). Sequencing of the amplicons representing these profiles revealed that each profile related to a unique sequence, some differing from each other by only one base-pair substitution. Comparative high-resolution melting (HRM) curve analysis of the amplicons using SYTO 9 green fluorescent dye also displayed profiles which were concordant with the same 10 SSCP profiles (A–J) and their sequences. For both mutation detection methods, the Australian M. synoviae strains represented one of the A, B, C or D profiles, while the USA strains represented one of the E, F, G, H, I or J profiles. The results presented in this study show that the PCR-based SSCP or HRM curve analyses of vlhA provide high-resolution mutation detection tools for the detection and identification of M. synoviae strains. In particular, the HRM curve analysis is a rapid and effective technique which can be performed in a single test tube in less than 2 h.
A table of raw data from HRM curve analysis is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Provisional diagnosis and monitoring of M. synoviae infection is usually made using serological assays, while definitive diagnosis is made by isolation and identification of the organism. However, the latter is generally expensive and time consuming (requiring 1–2 weeks to complete). Recently, PCR has been employed for identification of suspected cultures or for rapid detection of M. synoviae directly from clinical samples (Garcia et al., 1995; Kiss et al., 1997; Lauerman et al., 1993; Silveira et al., 1996; Wang et al., 1997). Further identification of the M. synoviae isolates (i.e. defining the strain involved) is very useful for epidemiological tracing and is of critical importance in countries in which poultry flocks are vaccinated with the live attenuated M. synoviae strain MS-H (Vaxsafe MS, Bioproperties Australia). RFLP has been described elsewhere (Morrow et al., 1990b) for classification of M. synoviae strains and is currently in use in our laboratory for classification of new field M. synoviae strains. However, RFLP is time consuming, since it requires isolation and culture of the organism and extraction of genomic DNA, and can be unreliable, particularly because of genomic rearrangements that may occur in progenies of a single M. synoviae isolate (Noormohammadi et al., 2000). Also, sequence variation in an individual isolate may go undetected, because only a relatively small number of restriction enzymes scans a subset of a putatively variable site. An arbitrarily primed PCR has also been described (Fan et al., 1995) for interspecies classification of M. synoviae, but the reproducibility of this technique as a routine procedure has not been evaluated. More recently, sequence analysis of the single-copy conserved region of the M. synoviae vlhA gene (Noormohammadi et al., 2000) has been used for classification of M. synoviae strains and epidemiological investigations (Bencina et al., 2001; Hong et al., 2004). These studies have suggested that strain identification based on the vlhA PCR should be regarded as a preliminary typing method for M. synoviae. However, this procedure is considered time consuming (requiring sequencing of the PCR product and subsequent analysis) and is relatively expensive as a routine diagnostic tool.
PCR-based mutation detection techniques, such single-strand conformation polymorphism (SSCP) and melting-curve analysis, provide useful and cost-effective alternatives for the direct analysis of genetic variation, particularly when large numbers of samples are to be analysed. Such techniques usually rely on the physical properties or the modification of DNA fragments for the separation of molecules of the same or very similar size (i.e. differing by only one or a few nucleotides). SSCP has been described extensively in the literature as a relatively rapid technique for the discrimination of variants of a given bacterium (Charvalos et al., 1996; Guttman et al., 1996; Speldooren et al., 1998; Telenti et al., 1993; Tokue et al., 1994), but so far has not been assessed for discrimination of M. synoviae strains. Also, high-resolution melting (HRM) curve analysis without using a fluorescence hybridization probe has been introduced recently as a rapid technique for genotyping and mutation scanning (Gundry et al., 2003). The introduction of new fluorescent dyes, such as LCGreen I (Wittwer et al., 2003) and SYTO 9 green (Krypuy et al., 2006; Monis et al., 2005), has brought further improvement to HRM curve analysis for clinical (Reed & Wittwer, 2004; Wittwer et al., 2003; Zhou et al., 2005) and/or epidemiological (Cheng et al., 2006; Odell et al., 2005; Robinson et al., 2006) investigations.
The main purpose of the present study was to compare SSCP and SYTO 9 green HRM curve analyses for the detection of inter-strain nucleotide variation in the M. synoviae vlhA gene, as alternatives to sequencing, and to assess their usefulness for routine diagnostic and epidemiological applications.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). All cultures were grown in mycoplasma broth (MB) as modified (Morrow et al., 1998) from the original formulation of Frey et al. (1968).
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), with some modifications. Briefly, 0.5 ml of mycoplasma cells was harvested at late-exponential phase by centrifugation at 20 000 g, the pellet was washed two times in PBS, solubilized in 300 µl RLT lysis buffer (Qiagen) and incubated at 4 °C overnight. A 15 µl volume of Qiaex II matrix (Qiagen) and 300 µl 70 % ethanol were added to the suspension, and the matrix was resuspended by vortexing. This suspension was loaded into a multispin MSK-100 column (Axygen). Columns were centrifuged for 30 s at 10 000 g in a microcentrifuge and the flow-through discarded. The matrix was washed once with 600 µl buffer RW1 (Qiagen) and twice with 500 µl buffer RPE (Qiagen). For each wash, columns were centrifuged for 30 s at 10 000 g. The columns were centrifuged for 90 s at 18 000 g to dry the matrix. The matrix was overlaid with 30 µl distilled water. After incubation at room temperature for 1 min, columns were centrifuged for 1 min at 10 000 g. The genomic DNA samples were used immediately or stored at –20 °C prior to use in the PCR.
PCR amplification.
On the basis of the vlhA1 gene sequence (the first characterised M. synoviae vlhA gene variant) (GenBank database accession no. AF035624), two oligonucleotide primers Link (5'-TACTATTAGCAGCTAGTGC-3') and MSCons-R (5'-AGTAACCGATCCGCTTAAT-3') were designed and used to amplify 350–400 bp of the single-copy conserved 5' end of the vlhA genes (Noormohammadi et al., 2000) from different M. synoviae strains. PCR was performed using Platinum Taq DNA Polymerase High Fidelity (Invitrogen) with some modifications. A 50 µl reaction mixture contained 200 µM each of dATP, dCTP, dGTP and dTTP, 2 mM MgSO4, 250 µM each primer, 1 U Platinum Taq DNA Polymerase High Fidelity, 5 µl 10x Platinum Taq DNA polymerase buffer and 1 µl extracted M. synoviae genomic DNA. The reaction mixture was incubated at 94 °C for 2 min, then subjected to 35 cycles of 96 °C for 15 s, 54 °C for 15 s and 68 °C for 20 s.
In each set of reactions, chicken and M. gallisepticum DNA and distilled H2O were included as negative controls. All PCR products were purified using the QIAquick PCR purification kit (Qiagen) prior to nucleotide sequencing or SSCP analysis.
Agarose gel electrophoresis and SSCP analysis.
After PCR, individual amplicons were mixed with an equal volume of loading buffer (10 mM NaOH, 95 % formamide, 0.05 % of both bromophenol blue and xylene cyanole) and the intensity of selected samples was verified on ethidium bromide-stained 2 % agarose gels using TBE (65 mM Tris/HCl, 27 mM boric acid, 1 mM EDTA, pH 9; Bio-Rad) as the buffer and 
X174-HaeIII (Promega) as a size marker. After denaturation at 94 °C for 15 min and snap cooling on a freeze block (–20 °C), the samples were subjected to SSCP analysis, as recently described (Gasser et al., 2004). In brief, samples (12 µl each) were loaded into the wells of precast GMA S-12x13 gels (96x261 mm; product no. 3548, Elchrom Scientific) and subjected to electrophoresis for 16.5 h at 72 V and 7.2 °C (constant) in a horizontal SEA2000 apparatus (Elchrom Scientific) connected to a MultiTemp III (Pharmacia) cooling system. After electrophoresis, gels were stained for 15 min with ethidium bromide (0.5 µg ml–1), destained in water for the same time and then photographed (using 667 film, Polaroid) upon UV transillumination. SSCP profiles were demonstrated to be reproducible on different days using amplicons produced on different days (data not shown).
HRM curve analysis.
Amplification of target sequences was carried out on a RotorGene thermal cycler (RG6000, Corbett Research). Each 20 µl reaction consisted of 200 µM each of dATP, dCTP, dGTP and dTTP, 1 mM MgCl2, 250 µM each primer (Link and MSCons-R), 5 µM SYTO 9 green fluorescent nucleic acid stain (Invitrogen), 0.5 U Taq DNA polymerase (Promega), 2 µl 10x Taq DNA polymerase buffer and 1 µl extracted M. synoviae genomic DNA. The reaction mixture was incubated at 96 °C for 2 min and then subjected to 40 cycles of 96 °C for 15 s, 54 °C for 15 s and 72 °C for 20 s. Optical measurements in the green channel (excitation at 470 nm and detection at 510 nm) were recorded during the extension step. After completion of 40 PCR cycles, melting-curve data were generated by increasing the temperature from 70 to 99 °C at 0.2 °C s–1 and recording fluorescence. HRM curve analysis was performed using the software Rotor-Gene 1.7.27 and the HRM algorithm provided. Normalization regions of 79.88–80.00 and 83.08–84.23 and a confidence threshold of 90 % were applied, and profiles giving an identity of less than 95 % to any of the existing profiles were considered as distinct profiles.
Sequencing and nucleotide sequence analyses.
Amplicons were purified using the QIAquick PCR purification kit (Qiagen), eluted in 30 µl H2O and then subjected to automated sequencing (BigDye chemistry, Applied Biosystems) in both directions, using the same primers as for PCR. The nucleotide sequences were compared with each other and with those previously available in GenBank (accession nos AF464936, AF035624 and AF314230). Computer analysis of the nucleotide sequences was performed using the programs provided by the Australian National Genomic Information Service (http://www.angis.org.au). The computer program CLUSTALW-fast (Thompson et al., 1994) was used to create multiple sequence alignments, and OldDistances was used to calculate pairwise similarities between sequences.
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RESULTS |
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) from genomic DNA extracted from 35 M. synoviae strains/isolates (Table 1
). PCR products of the expected size range were generated from all M. synoviae strains/isolates, but not from M. gallisepticum or chicken DNA (Fig. 1). SSCP analysis of the PCR products resulted in 10 distinct profiles named A–J. An example of each profile is presented in Fig. 2. The number of bands per profile varied from two to four, considering both single-stranded and double-stranded DNA. Most Australian strains/isolates, including the vaccine strain MS-H and its parent strain 86079/7NS, produced profile A (see Table 1). Some other Australian strains/isolates, however, produced profiles B, C or D. All USA strains, but none of the Australian strains, produced one of the profiles E, F, G, H, I or J. Repeated examination of all isolates/strains by SSCP did not alter the profile produced by each strain/isolate.
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). All M. synoviae strains/isolates with identical SSCP profiles (MS-H, 94011/V-18d, 94042/6a, 94046/W1B-17a and 94029/1a; 93148/23-22b and 94041/12a; WVU-1853-C+ and YA) had identical nucleotide sequences. In total, 10 different genotypes of M. synoviae were defined by sequencing. There was a complete concordance between SSCP profiles and the defined nucleotide sequence types. Differences between nucleotide sequences ranged from 0.3 % (between 94027/10a and T2/3X) to 14.5 % (between K1968 and K1938). Hence, the lowest level of nucleotide variation detectable by SSCP was 1 nt between strains 94027/10a and T2/3X (see Fig. 3 and Table 2).
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and Table 3). Analyses of the normalized curves (Fig. 4b) revealed that M. synoviae strains with distinct SSCP profiles also represented distinct HRM genotypes (Table 3). M. synoviae strains with identical SSCP profiles (and an identical nucleotide sequence of the vlhA PCR product) generated identical genotypes (see Supplementary Table S1). For example, M. synoviae isolates 86079/7NS, MS-H and 94011/V-18d produced the same HRM curve profile (A). Similarly, isolates 94027/19a and 94027/6a, and 93148/23-22b and 94041/12a, also produced the same profiles, B and C, respectively. Several HRM runs (performed on different days) using at least one representative from each profile resulted in only minor variations in melting temperatures of the peaks, but following normalization of data, the profiles were consistent with those described above (Table 4). Further details of HRM runs are available in Supplementary Table S1.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), in which, in contrast to SYBR Green I, SYTO 9 has been found to produce highly reproducible DNA melting and to be less selective in incorporation into particular amplicons.
All HRM curve profiles generated from M. synoviae strains in this study were found to have a major peak and a shorter shoulder peak. However, only one single DNA band could be detected when PCR products were subjected to agarose gel electrophoresis (see Fig. 1). It is unlikely that the shoulder peaks resulted from primer dimers, since such a peak was not detected in a no-template control that contained both primers. In addition, the melting temperature of the shoulder peak varied from one strain to another. It is also very unlikely that PCR products consisted of two variant sequences, since all M. synoviae cultures used in this study were grown from single colonies and all produced only one (and not more than one) shoulder peak. More importantly, within a single strain of M. synoviae, the vlhA single-copy gene region targeted for PCR in this study is known to be conserved (Bencina et al., 2001; Hong et al., 2004; Noormohammadi et al., 2000, 2002). Examination of the nucleotide sequence of the targeted vlhA gene region revealed an uneven distribution of G/C nucleotides through the length of the region. In the type strain WVU-1853-C+, the 5' end (bp 1–240) of this region has a G/C content of 46 %, while the 3' end (bp 241–398) is AT rich and has a G/C content of only 36 %. Thus it is likely that the presence of shoulder peaks is a result of uneven G/C distribution throughout the targeted DNA. However, irrespective of the cause, it seemed that the presence of the shoulder peak and its combination with the major peak contributed to the profiles generated (see normalized graphs in Fig. 4b), increasing the power of the HRM curve technique by adding further variation to the normalized melting curves.
This study describes, for what is believed to be the first time, the use of real-time PCR followed by melting-curve and SSCP analysis for detection and strain classification of M. synoviae isolates. An arbitrary-primed PCR has previously been described for detection of M. synoviae strain variations (Fan et al., 1995), but the reproducibility of the method is still under question and the results are difficult to interpret. In addition, the approach does not determine whether the profile variation detected relates to genomic rearrangements that commonly occur within single isolates (Noormohammadi et al., 2000). PCR followed by sequencing of the amplified product has also been described (Hong et al., 2004) for detection of M. synoviae strain variations; however, this approach is considerably more time consuming and the results often require interpretation.
Due to identical sequences over the vlhA gene region examined in this study, the PCR HRM curve analysis was unable to discriminate between the vaccine strain MS-H (or its parent strain 86079/7NS or its reisolates after passage in vivo) and the Australian isolates 93220/C-27a and 93107/5-5b. The last two have been shown to be distinct from MS-H by RFLP of genomic DNA (Markham et al., 1998). Recent studies in our laboratory have revealed that progenies of a single M. synoviae strain isolated after infection in vivo may exhibit different RFLP patterns (unpublished data). However, it is not known whether the isolates 93220/C-27a and 93107/5-5b are closely related to the MS-H vaccine (or its parent strain 76079/7NS), but have undergone genomic rearrangement, or whether they are indeed distinct strains but have an identical vlhA single-copy conserved 5' region. Hence, when an unknown Australian isolate exhibits SSCP or HRM curve patterns similar to those of the MS-H vaccine, confirmation of the results by conventional RFLP of genomic DNA may be useful.
PCR HRM curve analysis was able to discriminate between all the Australian vaccine/field isolates and the overseas (USA) strains examined in this study. This feature is particularly useful for discrimination of the vaccine strain from local field strains if the MS-H vaccine were to be introduced into countries such as the USA in which HRM curve profile A (MS-H type) is not found.
In summary, the combination of PCR and HRM curve analysis is a rapid and specific technique for the characterization of M. synoviae isolates. The entire process including extraction of DNA, PCR and HRM curve analysis can be completed within 2 h. Research in our laboratory on other avian pathogens has shown that a separately performed PCR step using conventional thermocyclers with SYTO 9 may be added immediately before HRM curve analysis. Studies are currently under way in our laboratory to adopt this assay for direct identification of M. synoviae in clinical specimens collected from diseased birds.
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ACKNOWLEDGEMENTS |
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Edited by: G. Firrao
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 13 December 2006;
revised 7 March 2007;
accepted 7 March 2007.
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