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Rapid and efficient screening of a Representational Difference Analysis library using reverse Southern hybridisation: Identification of genetic differences between Haemophilus parasuis isolates
Journal of Microbiological Methods 68 (2007) 326 – 330 www.elsevier.com/locate/jmicmeth
Rapid and efficient screening of a Representational Difference Analysis library using reverse Southern hybridisation: Identification of genetic differences between Haemophilus parasuis isolates John F. Lancashire a , C. Turni b , P.J. Blackall b , Michael P. Jennings a,⁎ a b
School of Molecular and Microbial Sciences, The University of Queensland, Brisbane, Queensland 4072, Australia Department of Primary Industries and Fisheries, Animal Research Institute, Yeerongpilly 4105, Queensland, Australia. Received 23 June 2006; accepted 12 September 2006 Available online 3 November 2006
Abstract Representational Difference Analysis (RDA) is an established technique used for isolation of specific genetic differences between or within bacterial species. This method was used to investigate the genetic basis of serovar-specificity and the relationship between serovar and virulence in Haemophilus parasuis. An RDA clone library of 96 isolates was constructed using H. parasuis strains H425(P) (serovar 12) and HS1967 (serovar 4). To screen such a large clone library to determine which clones are strain-specific would typically involved separately labelling each clone for use in Southern hybridisation against genomic DNA from each of the strains. In this study, a novel application of reverse Southern hybridisation was used to screen the RDA library: genomic DNA from each strain was labelled and used to probe the library to identify strain-specific clones. This novel approach represents a significant improvement in methodology that is rapid and efficient. © 2006 Elsevier B.V. All rights reserved. Keywords: Representational Difference Analysis; Southern hybridisation; Haemophilus parasuis
1. Introduction The application of Representational Difference Analysis (RDA) to identify genetic differences between two complex genomes by PCR-based subtractive hybridisation (Lisitsyn et al., 1993) and subsequent adaptation to analysis of bacterial genomes (Tinsley and Nassif, 1996), has been widely used to identify strain-specific and species-specific virulence determinants. This is a particularly useful tool in genetic studies of veterinary pathogens where few complete genome sequences and microarrays are currently available. Following subtractive hybridisation and specific PCR enrichment, the RDA products are usually cloned into a library and screened by Southern hybridisation analysis to determine which clones are strain-specific. The latter can be quite expensive and time-consuming when an extensive library is analysed.
⁎ Corresponding author. Tel.: +61 7 3365 4879; fax: +61 7 3365 4620. E-mail address: [email protected] (M.P. Jennings). 0167-7012/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2006.09.011
Haemophilus parasuis is the causative agent of Glässer's disease, porcine polyserositis and arthritis, and is of significance in the pork industry worldwide (Oliveira et al., 2003). There is considerable antigenic heterogeneity in H. parasuis. The Kielstein–Rapp-Gabrielson (KRG) scheme (Kielstein and Rapp-Gabrielson, 1992), originally based on heat stable antigens tested by agar gel precipitation, is the internationally recognized standard scheme and consists of 15 serovars (Del Rio et al., 2003). The scheme is technically demanding with limited serotyping resources available worldwide. The fact that non-typable isolates are commonly encountered (Kielstein and Rapp-Gabrielson, 1992; Rafiee and Blackall, 2000) suggests that additional serovars may exist. The use of an indirect haemagglutination technology in the KRG scheme has reduced the number of non-typable isolates in some studies (Del Rio et al., 2003; Tadjine et al., 2004) but not in all studies (Turni and Blackall, 2005). As well as antigenic variation, there is a well-recognized variation in the virulence of isolates of H. parasuis. This variation in virulence seems to have some correlation with the KRG serotyping scheme. Of the 15 known serovars, serovars 1,
J.F. Lancashire et al. / Journal of Microbiological Methods 68 (2007) 326–330
5, 10, 12, 13 and 14 have been shown to be highly virulent causing death or moribundity within four days, serovars 2, 4 and 15 were moderately virulent causing polyserositis, but not death, serovar 8 was considered to be of mild virulence resulting in mild symptoms and lesions, while serovars 3, 6, 7, 9 and 11 were avirulent causing no clinical symptoms or lesions (Kielstein and Rapp-Gabrielson, 1992). However, attempts to link outer membrane proteins and DNA profiles with virulence have been inconclusive (Hill et al., 2003; Ruiz et al., 2001). In this study, a novel application of reverse Southern hybridisation was developed and validated for rapid screening of the RDA clone library to determine the genetic basis of serovar-specificity and the relationship between serovars and virulence.
327
2. Materials and methods 2.1. Bacterial strains, culture media, and plasmids Escherichia coli strains were cultured in Luria-Bertani medium (LB) with appropriate antibiotics. Strain JM109 (Promega) was used to construct the RDA library using the pGEM®T-Easy vector system (Promega). All H. parasuis strains used in this study were obtained from the culture collection at the Animal Research Institute, Brisbane, Australia. H. parasuis strains used in RDA were H425(P), the international serovar 12 reference strain H425 which had been re-isolated after passage through swine, and Australian isolate HS1967 (serovar 4). Additional serovar 4 strains HS84 and HS1911, and serovar 12
`
Fig. 1. Diagram of experimental outline for RDA and reverse Southern hybridisation procedures. Enriched tester-specific RDA products were Sau3A1-digested and ligated to the second round adaptors (1). The enriched tester-specific products from second round RDA were cloned in a library (2). Reverse Southern hybridisation of the RDA clone library using a DIG-labelled tester genomic DNA probe (A), and DIG-labelled driver genomic DNA probe (B).
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J.F. Lancashire et al. / Journal of Microbiological Methods 68 (2007) 326–330
Fig. 2. Visualisation of RDA PCR products by gel electrophoresis. Lanes contain the products from PCR enrichment of (1) undiluted subtractive hybridisation mixture and (2) 1/10 dilution of the hybridisation mixture as reaction template. DNA size is indicated in kb.
strains HS1750 and HS1754 (all sourced from Australian pigs) were used. The H. parasuis strains were grown on TM/SN prepared as described previously (Reid and Blackall, 1987). Briefly, TM/SN is prepared from a basal medium known as TM, which contains 1% Biosate Peptone, 1% NaCl, 0.1% starch, 0.05% glucose and 1.5% agar. Immediately before pouring, TM/SN is supplemented with the following sterile additives: 0.0025% NADH, 0.005% thiamine, 1% heat inactivated horse serum and 5% oleic acid bovine serum albumin (O-A) complex. The OA complex consists of 4.75% bovine serum albumin (Fraction V) (JRH Biosciences, U.S.A.) in normal saline (with the normal saline containing 0.06% oleic acid and 5% 0.05N NaOH). When required, a liquid medium version of TM/SN, termed TMB, was prepared by omitting the agar from TM/SN. 2.2. DNA techniques Restriction and DNA modification enzymes were purchased from New England Biolabs. Taq polymerase for amplifying probes by PCR was purchased from Fisher Biotech. Molecular biology methods used were as previously described (Ausubel, 1994).
igenin (DIG)-High Prime (Roche). The 96 clones in the library were digested with EcoRI, and then the RDA product inserts were separated from the plasmid backbone by electrophoresis on 0.7% agarose gels then transferred to GeneScreen® Hybridisation Transfer membrane (NEN™ Life Science Products) by capillary action (Ausubel, 1994). Duplicate blots were separately hybridised with H425(P) or HS1967 genomic DNA probes for 16 h at 68 °C. Washes and detection were carried out (using the DIG DNA Labelling and Detection Kit, Roche) as recommended by the manufacturer. Conventional Southern hybridisation analysis was performed by transferring MfeI-digested H. parasuis chromosomal DNA to a nitrocellulose membrane as described above. Probes were made by PCR amplification of the cloned RDA products using the universal primers M13 forward and M13 reverse, then labelling using DIG High Prime. 2.5. DNA sequencing Cloned RDA products were PCR amplified in triplicate using the universal primers M13 forward and M13 reverse. ABI Prism BigDye Terminator Version 3.1 (PE Applied Biosystems) was used for DNA sequencing. Following EDTA/ethanol precipitation, samples were sent to the Australian Genome Research Facility (AGRF) for automated sequencing using an ABI 3730 × l 96-capillary automatic sequencer (PE Applied Biosystems). Sequence data were aligned and annotated using MacVector version 7.2 (Accelrys), and gene identities assigned using searches against the nucleotide and protein databases at NCBI using the tBLAST-n algorithm (Altschul et al., 1997). 3. Results and discussion
2.3. Construction of an RDA library
3.1. Construction of an H. parasuis RDA library
An outline of the experimental procedure is shown in Fig. 1. Briefly, the RDA procedure was performed as previously described (Tinsley and Nassif, 1996). H. parasuis strain HS1967 (serovar 4) was used as the tester, and strain H425(P) (serovar 12) was used as the driver. Two rounds of RDA were completed using adapters RBam and JBam (Tinsley and Nassif, 1996). In the second round of kinetic enrichment by PCR, a 1/ 10 dilution of the hybridisation mixture was used. These PCR products were column purified (Gel Extraction Kit, Qiagen), and then T-A cloned into pGEM®T-Easy and transformed into E. coli JM109 competent cells by heat-shock transformation according to the manufacturer's instructions (pGEM®T-Easy vector system, Promega).
The genetic differences between H. parasuis strains HS1967 (serovar 4) and H425(P) (serovar 12) were isolated by RDA. Two rounds of subtractive hybridisation and kinetic enrichment were performed and the resultant products (Fig. 2) were labelled and used to probe chromosomal DNA from HS1967 and H425(P), confirming enrichment of HS1967specific DNA (result not shown). The PCR products were then ligated into pGEM®T-Easy and transformed into E. coli
2.4. Reverse Southern hybridisation analysis The reverse Southern hybridisation procedure was used in a novel application for rapid screening of the RDA library for strain-specific cloned DNA fragments. The probes were prepared using 6 μg of MfeI digested H. parasuis strain H425 (P) and HS1967 genomic DNA, isolated as previously described (Ausubel, 1994), which was separately labelled using digox-
Fig. 3. Validation of reverse Southern hybridisation technique. (A) 0.7% agarose gel of EcoRI digested pGEM®-T-Easy containing H. parasuis genomic DNA inserts sized 1.4 kb and 0.8 kb. (B) DIG-labelled MfeI digested HS1967 genomic DNA. Gel loadings are 1 kb DNA ladder then EcoRI-digested pGEM®-T-Easy clones.
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product, and were separated on duplicate 0.7% agarose gels by electrophoresis then transferred to separate membranes for Southern analysis using an H425(P) or HS1967 genomic DNA probe. A comparison between separately probed duplicate blots (Fig. 4) rapidly identifies whether an insert band is present in one blot and not the other (tester-specific) or both blots (nontester-specific). From a total of 96 library clones, there were 18 isolates identified as HS1967-specific. These were then sequenced, duplicate clones eliminated, revealing 13 HS1967specific genes.
Fig. 4. Example of reverse Southern hybridisation rapid screening of an RDA library. Cloned RDA products were released from the vector by EcoRI digest, then transferred to a nitrocellulose membrane in dulplicate for reverse Southern hybridisation screening. (A) HS1967 genomic DNA probe. (B) H425(P) genomic DNA probe.
JM109 competent cells. Vectors containing an RDA product insert were selected by blue–white screening and small-scale plasmid preparations were made.
3.2.3. Application of reverse Southern hybridisation analysis for rapid screening of large clone libraries The conventional Southern hybridisation procedure is based upon using a high copy number labelled PCR product probe against a less abundant genomic target DNA sequence. In this study, reverse Southern hybridisation was applied and validated for use in screening large RDA libraries. This method provides the advantage of rapid single step screening and uses fewer materials and reagents to deliver more cost-effective screening of large RDA libraries compared to conventional Southern hybridisation.
3.2. Isolation of tester-specific clones from the RDA library Acknowledgements 3.2.1. Application and validation of reverse Southern hybridisation It is usual practice to confirm that the cloned RDA products are tester-specific by labelling each individual clone to use as a probe for Southern blots of tester and driver chromosomal DNA. This is an appropriate approach for RDA that yields several distinct products. However, in this study there were considerable genetic differences identified between the two H. parasuis strains. In these circumstances, the expense of separately labelling RDA products and the time taken to complete separate Southern blots was inappropriate. These factors were overcome by the novel use of reverse Southern hybridisation (Buhariwalla et al., 1995). Rather than screen each clone individually, a scheme was devised such that two probes consisting of separately labeled genomic DNA from the tester and driver strains could be used against blots consisting of a panel of RDA library clones (reverse Southern hybridisation; see Fig. 1). To determine whether labelled chromosomal DNA would provide a sufficient signal in Southern hybridisation against the cloned fragments, a previously cloned H. parasuis genomic DNA fragment was digested with EcoRI and separated from the vector by gel electrophoresis, then transferred to a membrane for reverse Southern hybridisation using the HS1967 genomic DNA probe. The result (Fig. 3) indicates that the genomic probe specifically binds to the H. parasuis genomic DNA and not to the vector backbone, indicating adequate specificity and sensitivity of the genomic probe to provide sufficient signal intensity for determination of tester-specificity. 3.2.2. Screening the RDA library by reverse Southern hybridisation 96 small-scale plasmid preparations made from the RDA library were digested with EcoRI to release the cloned RDA
Work in the Jennings laboratory and John Lancashire's PhD scholarship was supported by an ARC Linkage Grant LP0218929 and Bioproperties (Australia) Pty Ltd. References Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. Ausubel, F., 1994. Current Protocols in Molecular Biology. Wiley, New York. Buhariwalla, H., Greaves, S., Magrath, R., Mithen, R., 1995. Development of specific Pcr primers for the amplification of polymorphic DNA from the obligate root pathogen Plasmodiophora–Brassicae. Physiol. Mol. Plant Pathol. 47, 83–94. Del Rio, M.L., Gutierrez, C.B., Rodriguez Ferri, E.F., 2003. Value of indirect hemagglutination and coagglutination tests for serotyping Haemophilus parasuis. J. Clin. Microbiol. 41, 880–882. Hill, C.E., Metcalf, D.S., MacInnes, J.I., 2003. A search for virulence genes of Haemophilus parasuis using differential display RT-PCR. Vet. Microbiol. 96, 189–202. Kielstein, P., Rapp-Gabrielson, V.J., 1992. Designation of 15 serovars of Haemophilus parasuis on the basis of immunodiffusion using heat-stable antigen extracts. J. Clin. Microbiol. 30, 862–865. Lisitsyn, N., Lisitsyn, N., Wigler, M., 1993. Cloning the differences between two complex genomes. Science 259, 946–951. Oliveira, S., Blackall, P.J., Pijoan, C., 2003. Characterization of the diversity of Haemophilus parasuis field isolates by use of serotyping and genotyping. Am. J. Vet. Res. 64, 435–442. Rafiee, M., Blackall, P.J., 2000. Establishment, validation and use of the Kielstein–Rapp-Gabrielson serotyping scheme for Haemophilus parasuis. Aust. Vet. J. 78, 172–174. Reid, G.G., Blackall, P.J., 1987. Comparison of adjuvants for an inactivated infectious coryza vaccine. Avian Dis. 31, 59–63. Ruiz, A., Oliveira, S., Torremorell, M., Pijoan, C., 2001. Outer membrane proteins and DNA profiles in strains of Haemophilus parasuis recovered from systemic and respiratory sites. J. Clin. Microbiol. 39, 1757–1762.
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Tadjine, M., Mittal, K.R., Bourdon, S., Gottschalk, M., 2004. Development of a new serological test for serotyping Haemophilus parasuis isolates and determination of their prevalence in North America. J. Clin. Microbiol. 42, 839–840. Tinsley, C.R., Nassif, X., 1996. Analysis of the genetic differences between Neisseria meningitidis and Neisseria gonorrhoeae: two closely related
bacteria expressing two different pathogenicities. Proc. Natl. Acad. Sci. U. S. A. 93, 11109–11114. Turni, C., Blackall, P.J., 2005. Comparison of the indirect haemagglutination and gel diffusion test for serotyping Haemophilus parasuis. Vet. Microbiol. 106, 145–151.
Rapid and efficient screening of a Representational Difference Analysis library using reverse Southern hybridisation: Identification of genetic differences between Haemophilus parasuis isolates John F. Lancashire a , C. Turni b , P.J. Blackall b , Michael P. Jennings a,⁎ a b
School of Molecular and Microbial Sciences, The University of Queensland, Brisbane, Queensland 4072, Australia Department of Primary Industries and Fisheries, Animal Research Institute, Yeerongpilly 4105, Queensland, Australia. Received 23 June 2006; accepted 12 September 2006 Available online 3 November 2006
Abstract Representational Difference Analysis (RDA) is an established technique used for isolation of specific genetic differences between or within bacterial species. This method was used to investigate the genetic basis of serovar-specificity and the relationship between serovar and virulence in Haemophilus parasuis. An RDA clone library of 96 isolates was constructed using H. parasuis strains H425(P) (serovar 12) and HS1967 (serovar 4). To screen such a large clone library to determine which clones are strain-specific would typically involved separately labelling each clone for use in Southern hybridisation against genomic DNA from each of the strains. In this study, a novel application of reverse Southern hybridisation was used to screen the RDA library: genomic DNA from each strain was labelled and used to probe the library to identify strain-specific clones. This novel approach represents a significant improvement in methodology that is rapid and efficient. © 2006 Elsevier B.V. All rights reserved. Keywords: Representational Difference Analysis; Southern hybridisation; Haemophilus parasuis
1. Introduction The application of Representational Difference Analysis (RDA) to identify genetic differences between two complex genomes by PCR-based subtractive hybridisation (Lisitsyn et al., 1993) and subsequent adaptation to analysis of bacterial genomes (Tinsley and Nassif, 1996), has been widely used to identify strain-specific and species-specific virulence determinants. This is a particularly useful tool in genetic studies of veterinary pathogens where few complete genome sequences and microarrays are currently available. Following subtractive hybridisation and specific PCR enrichment, the RDA products are usually cloned into a library and screened by Southern hybridisation analysis to determine which clones are strain-specific. The latter can be quite expensive and time-consuming when an extensive library is analysed.
⁎ Corresponding author. Tel.: +61 7 3365 4879; fax: +61 7 3365 4620. E-mail address: [email protected] (M.P. Jennings). 0167-7012/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2006.09.011
Haemophilus parasuis is the causative agent of Glässer's disease, porcine polyserositis and arthritis, and is of significance in the pork industry worldwide (Oliveira et al., 2003). There is considerable antigenic heterogeneity in H. parasuis. The Kielstein–Rapp-Gabrielson (KRG) scheme (Kielstein and Rapp-Gabrielson, 1992), originally based on heat stable antigens tested by agar gel precipitation, is the internationally recognized standard scheme and consists of 15 serovars (Del Rio et al., 2003). The scheme is technically demanding with limited serotyping resources available worldwide. The fact that non-typable isolates are commonly encountered (Kielstein and Rapp-Gabrielson, 1992; Rafiee and Blackall, 2000) suggests that additional serovars may exist. The use of an indirect haemagglutination technology in the KRG scheme has reduced the number of non-typable isolates in some studies (Del Rio et al., 2003; Tadjine et al., 2004) but not in all studies (Turni and Blackall, 2005). As well as antigenic variation, there is a well-recognized variation in the virulence of isolates of H. parasuis. This variation in virulence seems to have some correlation with the KRG serotyping scheme. Of the 15 known serovars, serovars 1,
J.F. Lancashire et al. / Journal of Microbiological Methods 68 (2007) 326–330
5, 10, 12, 13 and 14 have been shown to be highly virulent causing death or moribundity within four days, serovars 2, 4 and 15 were moderately virulent causing polyserositis, but not death, serovar 8 was considered to be of mild virulence resulting in mild symptoms and lesions, while serovars 3, 6, 7, 9 and 11 were avirulent causing no clinical symptoms or lesions (Kielstein and Rapp-Gabrielson, 1992). However, attempts to link outer membrane proteins and DNA profiles with virulence have been inconclusive (Hill et al., 2003; Ruiz et al., 2001). In this study, a novel application of reverse Southern hybridisation was developed and validated for rapid screening of the RDA clone library to determine the genetic basis of serovar-specificity and the relationship between serovars and virulence.
327
2. Materials and methods 2.1. Bacterial strains, culture media, and plasmids Escherichia coli strains were cultured in Luria-Bertani medium (LB) with appropriate antibiotics. Strain JM109 (Promega) was used to construct the RDA library using the pGEM®T-Easy vector system (Promega). All H. parasuis strains used in this study were obtained from the culture collection at the Animal Research Institute, Brisbane, Australia. H. parasuis strains used in RDA were H425(P), the international serovar 12 reference strain H425 which had been re-isolated after passage through swine, and Australian isolate HS1967 (serovar 4). Additional serovar 4 strains HS84 and HS1911, and serovar 12
`
Fig. 1. Diagram of experimental outline for RDA and reverse Southern hybridisation procedures. Enriched tester-specific RDA products were Sau3A1-digested and ligated to the second round adaptors (1). The enriched tester-specific products from second round RDA were cloned in a library (2). Reverse Southern hybridisation of the RDA clone library using a DIG-labelled tester genomic DNA probe (A), and DIG-labelled driver genomic DNA probe (B).
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J.F. Lancashire et al. / Journal of Microbiological Methods 68 (2007) 326–330
Fig. 2. Visualisation of RDA PCR products by gel electrophoresis. Lanes contain the products from PCR enrichment of (1) undiluted subtractive hybridisation mixture and (2) 1/10 dilution of the hybridisation mixture as reaction template. DNA size is indicated in kb.
strains HS1750 and HS1754 (all sourced from Australian pigs) were used. The H. parasuis strains were grown on TM/SN prepared as described previously (Reid and Blackall, 1987). Briefly, TM/SN is prepared from a basal medium known as TM, which contains 1% Biosate Peptone, 1% NaCl, 0.1% starch, 0.05% glucose and 1.5% agar. Immediately before pouring, TM/SN is supplemented with the following sterile additives: 0.0025% NADH, 0.005% thiamine, 1% heat inactivated horse serum and 5% oleic acid bovine serum albumin (O-A) complex. The OA complex consists of 4.75% bovine serum albumin (Fraction V) (JRH Biosciences, U.S.A.) in normal saline (with the normal saline containing 0.06% oleic acid and 5% 0.05N NaOH). When required, a liquid medium version of TM/SN, termed TMB, was prepared by omitting the agar from TM/SN. 2.2. DNA techniques Restriction and DNA modification enzymes were purchased from New England Biolabs. Taq polymerase for amplifying probes by PCR was purchased from Fisher Biotech. Molecular biology methods used were as previously described (Ausubel, 1994).
igenin (DIG)-High Prime (Roche). The 96 clones in the library were digested with EcoRI, and then the RDA product inserts were separated from the plasmid backbone by electrophoresis on 0.7% agarose gels then transferred to GeneScreen® Hybridisation Transfer membrane (NEN™ Life Science Products) by capillary action (Ausubel, 1994). Duplicate blots were separately hybridised with H425(P) or HS1967 genomic DNA probes for 16 h at 68 °C. Washes and detection were carried out (using the DIG DNA Labelling and Detection Kit, Roche) as recommended by the manufacturer. Conventional Southern hybridisation analysis was performed by transferring MfeI-digested H. parasuis chromosomal DNA to a nitrocellulose membrane as described above. Probes were made by PCR amplification of the cloned RDA products using the universal primers M13 forward and M13 reverse, then labelling using DIG High Prime. 2.5. DNA sequencing Cloned RDA products were PCR amplified in triplicate using the universal primers M13 forward and M13 reverse. ABI Prism BigDye Terminator Version 3.1 (PE Applied Biosystems) was used for DNA sequencing. Following EDTA/ethanol precipitation, samples were sent to the Australian Genome Research Facility (AGRF) for automated sequencing using an ABI 3730 × l 96-capillary automatic sequencer (PE Applied Biosystems). Sequence data were aligned and annotated using MacVector version 7.2 (Accelrys), and gene identities assigned using searches against the nucleotide and protein databases at NCBI using the tBLAST-n algorithm (Altschul et al., 1997). 3. Results and discussion
2.3. Construction of an RDA library
3.1. Construction of an H. parasuis RDA library
An outline of the experimental procedure is shown in Fig. 1. Briefly, the RDA procedure was performed as previously described (Tinsley and Nassif, 1996). H. parasuis strain HS1967 (serovar 4) was used as the tester, and strain H425(P) (serovar 12) was used as the driver. Two rounds of RDA were completed using adapters RBam and JBam (Tinsley and Nassif, 1996). In the second round of kinetic enrichment by PCR, a 1/ 10 dilution of the hybridisation mixture was used. These PCR products were column purified (Gel Extraction Kit, Qiagen), and then T-A cloned into pGEM®T-Easy and transformed into E. coli JM109 competent cells by heat-shock transformation according to the manufacturer's instructions (pGEM®T-Easy vector system, Promega).
The genetic differences between H. parasuis strains HS1967 (serovar 4) and H425(P) (serovar 12) were isolated by RDA. Two rounds of subtractive hybridisation and kinetic enrichment were performed and the resultant products (Fig. 2) were labelled and used to probe chromosomal DNA from HS1967 and H425(P), confirming enrichment of HS1967specific DNA (result not shown). The PCR products were then ligated into pGEM®T-Easy and transformed into E. coli
2.4. Reverse Southern hybridisation analysis The reverse Southern hybridisation procedure was used in a novel application for rapid screening of the RDA library for strain-specific cloned DNA fragments. The probes were prepared using 6 μg of MfeI digested H. parasuis strain H425 (P) and HS1967 genomic DNA, isolated as previously described (Ausubel, 1994), which was separately labelled using digox-
Fig. 3. Validation of reverse Southern hybridisation technique. (A) 0.7% agarose gel of EcoRI digested pGEM®-T-Easy containing H. parasuis genomic DNA inserts sized 1.4 kb and 0.8 kb. (B) DIG-labelled MfeI digested HS1967 genomic DNA. Gel loadings are 1 kb DNA ladder then EcoRI-digested pGEM®-T-Easy clones.
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product, and were separated on duplicate 0.7% agarose gels by electrophoresis then transferred to separate membranes for Southern analysis using an H425(P) or HS1967 genomic DNA probe. A comparison between separately probed duplicate blots (Fig. 4) rapidly identifies whether an insert band is present in one blot and not the other (tester-specific) or both blots (nontester-specific). From a total of 96 library clones, there were 18 isolates identified as HS1967-specific. These were then sequenced, duplicate clones eliminated, revealing 13 HS1967specific genes.
Fig. 4. Example of reverse Southern hybridisation rapid screening of an RDA library. Cloned RDA products were released from the vector by EcoRI digest, then transferred to a nitrocellulose membrane in dulplicate for reverse Southern hybridisation screening. (A) HS1967 genomic DNA probe. (B) H425(P) genomic DNA probe.
JM109 competent cells. Vectors containing an RDA product insert were selected by blue–white screening and small-scale plasmid preparations were made.
3.2.3. Application of reverse Southern hybridisation analysis for rapid screening of large clone libraries The conventional Southern hybridisation procedure is based upon using a high copy number labelled PCR product probe against a less abundant genomic target DNA sequence. In this study, reverse Southern hybridisation was applied and validated for use in screening large RDA libraries. This method provides the advantage of rapid single step screening and uses fewer materials and reagents to deliver more cost-effective screening of large RDA libraries compared to conventional Southern hybridisation.
3.2. Isolation of tester-specific clones from the RDA library Acknowledgements 3.2.1. Application and validation of reverse Southern hybridisation It is usual practice to confirm that the cloned RDA products are tester-specific by labelling each individual clone to use as a probe for Southern blots of tester and driver chromosomal DNA. This is an appropriate approach for RDA that yields several distinct products. However, in this study there were considerable genetic differences identified between the two H. parasuis strains. In these circumstances, the expense of separately labelling RDA products and the time taken to complete separate Southern blots was inappropriate. These factors were overcome by the novel use of reverse Southern hybridisation (Buhariwalla et al., 1995). Rather than screen each clone individually, a scheme was devised such that two probes consisting of separately labeled genomic DNA from the tester and driver strains could be used against blots consisting of a panel of RDA library clones (reverse Southern hybridisation; see Fig. 1). To determine whether labelled chromosomal DNA would provide a sufficient signal in Southern hybridisation against the cloned fragments, a previously cloned H. parasuis genomic DNA fragment was digested with EcoRI and separated from the vector by gel electrophoresis, then transferred to a membrane for reverse Southern hybridisation using the HS1967 genomic DNA probe. The result (Fig. 3) indicates that the genomic probe specifically binds to the H. parasuis genomic DNA and not to the vector backbone, indicating adequate specificity and sensitivity of the genomic probe to provide sufficient signal intensity for determination of tester-specificity. 3.2.2. Screening the RDA library by reverse Southern hybridisation 96 small-scale plasmid preparations made from the RDA library were digested with EcoRI to release the cloned RDA
Work in the Jennings laboratory and John Lancashire's PhD scholarship was supported by an ARC Linkage Grant LP0218929 and Bioproperties (Australia) Pty Ltd. References Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. Ausubel, F., 1994. Current Protocols in Molecular Biology. Wiley, New York. Buhariwalla, H., Greaves, S., Magrath, R., Mithen, R., 1995. Development of specific Pcr primers for the amplification of polymorphic DNA from the obligate root pathogen Plasmodiophora–Brassicae. Physiol. Mol. Plant Pathol. 47, 83–94. Del Rio, M.L., Gutierrez, C.B., Rodriguez Ferri, E.F., 2003. Value of indirect hemagglutination and coagglutination tests for serotyping Haemophilus parasuis. J. Clin. Microbiol. 41, 880–882. Hill, C.E., Metcalf, D.S., MacInnes, J.I., 2003. A search for virulence genes of Haemophilus parasuis using differential display RT-PCR. Vet. Microbiol. 96, 189–202. Kielstein, P., Rapp-Gabrielson, V.J., 1992. Designation of 15 serovars of Haemophilus parasuis on the basis of immunodiffusion using heat-stable antigen extracts. J. Clin. Microbiol. 30, 862–865. Lisitsyn, N., Lisitsyn, N., Wigler, M., 1993. Cloning the differences between two complex genomes. Science 259, 946–951. Oliveira, S., Blackall, P.J., Pijoan, C., 2003. Characterization of the diversity of Haemophilus parasuis field isolates by use of serotyping and genotyping. Am. J. Vet. Res. 64, 435–442. Rafiee, M., Blackall, P.J., 2000. Establishment, validation and use of the Kielstein–Rapp-Gabrielson serotyping scheme for Haemophilus parasuis. Aust. Vet. J. 78, 172–174. Reid, G.G., Blackall, P.J., 1987. Comparison of adjuvants for an inactivated infectious coryza vaccine. Avian Dis. 31, 59–63. Ruiz, A., Oliveira, S., Torremorell, M., Pijoan, C., 2001. Outer membrane proteins and DNA profiles in strains of Haemophilus parasuis recovered from systemic and respiratory sites. J. Clin. Microbiol. 39, 1757–1762.
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