Journal of Microbiological Methods 95 (2013) 368–372
Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Note
Whole genome amplification of the obligate intracellular pathogen Coxiella burnetii using multiple displacement amplification Sanjay Kumar a, Shefali Raj Gangoliya a, Mustapha Berri b, Annie Rodolakis b, Syed Imteyaz Alam a,⁎ a b
Biotechnology Division, Defence Research & Development Establishment, Gwalior 474002, India INRA, UMR1282 ISP, Nouzilly, France
a r t i c l e
i n f o
Article history: Received 27 August 2013 Received in revised form 14 October 2013 Accepted 16 October 2013 Available online 25 October 2013
a b s t r a c t This study demonstrates that whole genome multiple displacement amplification (MDA) is a promising technique for downstream genomic analysis of fastidious obligate intracellular pathogens such as Coxiella burnetii. The MDA technology can help in obtaining sufficient genetic material from highly infectious agent and thus minimizing repeated culturing and associated biohazard. © 2013 Elsevier B.V. All rights reserved.
Keywords: Coxiella burnetii Whole genome amplification Multiple displacement amplification
Coxiella burnetii is the etiologic agent of an acute, disabling influenzalike illness in humans termed Q fever. In rare instances chronic disease can occur, usually presenting as hepatitis or endocarditis. C. burnetii infects a wide range of vertebrate and invertebrate hosts and is found worldwide with the exception of New Zealand. Most human Q fever cases are acquired through inhalation of contaminated aerosols originating from products shed from chronically infected domestic livestock (Angelakis and Raoult, 2010; Maurin and Raoult, 1999; Rodolakis, 2006). C. burnetii has an obligate requirement for replication in a eukaryotic host cell vacuole with lysosomal characteristics (Heinzen et al., 1996). Resistance to this harsh intracellular environment correlates with remarkable environmental stability, a trait that distinguishes C. burnetii from other obligate intracellular bacteria. Environmental stability, along with aerosol transmission and an infectious dose approaching one organism, has resulted in classification of C. burnetii as a CDC category B biothreat agent. Unfortunately, research on this pathogen has been hampered by its extremely low infectious dose and is confounded by fastidious nature of the organism with respect to in vitro intracellular proliferation and the need of BSL3 laboratory, which is technically demanding and time consuming. Obtaining sufficient quantity of DNA material of Coxiella for any genetic characterization analysis is a challenge. It is also a potential biohazard for the investigators to repeatedly proliferate C. burnetii under laboratory conditions in quantities sufficient for proteomic or genomic studies. Identification and analysis of pathogen DNA (e.g. PCR of specific targets) is a powerful tool for confirmation of the agent in both environmental and clinical samples. Immunocapture of organisms using antibodies, binding to specific
⁎ Corresponding author. Tel.: +91 751 2340132x276; fax: +91 751 2341148. E-mail address: [email protected] (S.I. Alam). 0167-7012/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mimet.2013.10.008
surface marker is a method of choice for environmental detection of threat agents. These studies rely on availability of sufficient DNA sample for downstream applications. The objective of this study was, thus, to develop an efficient strategy for the amplification of total DNA of suitable quality from small amounts of initial template for genetic analysis and development of countermeasures for this pathogen of medical and military importance. The use of whole genome amplification (WGA) as a tool for the study of infectious bacteria is of growing clinical interest (Hosono et al., 2003). The multiple displacement amplification (MDA) has become the preferred choice due to its high processivity and low error rate. In this study, we used whole genome amplification by multipledisplacement amplification (MDA) (Dean et al., 2002) and produced a large amount of C. burnetii strain RSA 493 DNA. The DNA recovered has been found to contain most of the C. burnetii loci targeted, and thus provides avenues for genomic studies of this fastidious and hazardous bacterium. Attempts were made to construct a bacterial artificial chromosome (BAC) library using the amplified C. burnetii genome with an objective to maintain the pathogen's gene pool for any downstream molecular application and to carry out genome sequence analysis. Total DNA of C. burnetii strain RSA 493 was obtained from INRA, France and was used for multiple displacement amplification using REPLI-g Mini Kit (Qiagen, Germany) as per manufacturer's instructions. The REPLI-g Mini Kit provides highly uniform amplification across the entire genome by bacteriophage Phi29 DNA polymerase, with negligible sequence bias (Hosono et al., 2003). The method is based on MDA technology originally proposed by Dean et al. (2002), which carries out isothermal genome amplification utilizing a uniquely processive DNA polymerase capable of replicating up to 100 kb without dissociating from the genomic DNA template. The DNA polymerase has a 3′–5′ exonuclease proofreading activity to maintain high fidelity
S. Kumar et al. / Journal of Microbiological Methods 95 (2013) 368–372
during replication and is used in the presence of exonuclease resistant primers to achieve high yields of DNA product. Briefly, 2.5 μl (0.22 ng) and 5.0 μl (0.45 ng) of template DNA were mixed with sample buffer and denatured. Twenty nine microliter of reaction buffer and 1 μl of REPLI-g Mini DNA Polymerase were added to the template and the reaction volume was adjusted to 40 μl using nuclease free water. Reaction was carried out for 16 h at 30 °C and at the end of the reaction REPLI-g Mini DNA Polymerase was inactivated by heating the sample for 3 min at 65 °C. Amplified and un-amplified genomic DNA from C. burnetii strain RSA 493 was tested for uniform amplification by PCR amplification of 20 target loci. The details of the loci, the primer sequences used for amplification, the optimized annealing temperatures and their product sizes are summarized in Table 1. BAC library construction was performed as described by Osoegawa et al. (2001) using the CopyRight BAC Cloning Kit (Lucigen Corporation, Middleton, WI, USA). Briefly amplified genomic DNA was subjected to partial digestion with BamHI, size selected on agarose gels, and ligated to the pSMART BAC vector (Lucigen Corporation, Middleton, WI, USA). Ligation products were transformed by electroporation into the BAC-optimized electrocompetent cells (Lucigen Corporation). The positive BAC DNAs were purified using the Qiagen Large-Construct Kit (Qiagen, Germany). Sequencing was carried out on ABI3730 equipment with “Dye Terminator” process using ABI kit version 3.1.
369
To increase the amount of C. burnetii strain RSA 493 DNA, we used a whole genome amplification strategy based on MDA technique, as described by Dean et al. (2001, 2002). This isothermal amplification method employs the MDA property of the bacteriophage Phi29 DNA polymerase to amplify a long (10 to 70 kb) stretch of circular or linear DNA (Blanco et al., 1989), yielding high amount of DNA (approximately 10 μg per 50 μl reaction) from a relatively limited quantity of template (~10 ng) with minimum sequence bias (Dean et al., 2002). In addition, Phi29-based MDA was shown to cover most of the loci present in the original DNA template, including those of an entire phage or plasmid as well as a complete genome from one single cell (Dean et al., 2001, 2002; Hellani et al., 2004; Paez et al., 2004). In spite of the success of the MDA methodology with eukaryotes, the applications for prokaryotic organisms have been limited. Recently, MDA has been used to improve the detection of bacteria in mites (Jeyaprakash and Hoy, 2004) or to amplify genomic DNA from Salmonella enterica serovar Enteritidis cells (Kwon and Cox, 2004), but the genetic content was evaluated for only a few loci. In this study, MDA was applied for the first time in order to produce considerable quantities of C. burnetii DNA by using REPLI-g Mini Kit (Qiagen, Germany). Successful and unbiased amplification was evaluated for representative loci on the genome by PCR analysis of amplified product as described below. Template DNA (0.22 or 0.45 ng) was denatured and added to the reaction mixture. A total yield of approximately 200 and 500 ng was observed at the end of
Table 1 Primers and PCR-amplified genes from C. burnetii genome. ORF no.
Protein
Primers sequence (5′–3′)
Gene locus
Annealing temperature (°C)
CBU_0307
54
696
This study
83,817–84,404
53
588
-do-
902,551–903,234
53
684
-do-
CBU_0090
TolB protein
82,485–83,807
58
1323
-do-
CBU_2079
Hypothetical protein
1980,877–1,981,281
59
405
-do-
CBU_0891
Hypothetical exported membrane-associated protein GtrA family protein
F: ATGTCGGTAAAAAAACTAGTAACA R: TTACATTAAGAATTTGTAGCCAAG F: ATGAGCCGTAAATTTACAGACAA R:TTAATAAGCCTCATAAATTAAATTAAC F: ATGAAAAAACTAACCGTAACTTTC R: CTATTTATTTAAAAAAGTCGCCAC F: ATGAACTCCATGCAAAAACGACA R: CTACACGCCTAAATAAGGAGAC F: ATGAGCTATATTAAACGAGATCAT R: TTAGCCGCAGCCATGACCTC F: GTGAAACGGTATCTTTTTTTTACTAT R: TCAAGGCAGGCTCTTTAAAAGC F:ATGATTAAAAAACTTATTTTATTCGTTTA R: TCAAATCGCTTGATGGGAATT F: ATGAAAAAACGCACAAAAATTATTAT R: TTAATGAACCACTTTTACCTTTGCT F: ATGTCAAAAATCAAGTGGTTTTTAG R: TCAGATAGTATTTTGCGGACTTAAT F: ATGCATAAATTTCGCCTCTCGGG R: TCATTGAACGGACGTCCTCATAA F: ATGCGCTTACAAAGAGTCTTTTCA R: TTAGGAAGGAATTTCCTGGAAATA F: ATGCCGAAAGAAACTAAAACCGA R:TCATTTTCTTCGAGCGATCTTGC F: ATGAGTAGACGTGAGACTCCCA R: TCACCGAGGACTAGACAGACAA F: ATGAGAAGCTTTATTTATATTTTAATT R: TCATTGCATTCGTCGATACAATT F: ATGTGGAGAATGTTGATGTACCC R: TTAAGACTGAGCTTCCTCAGATG F: ATGTCAGCAATTTTACCAACAGA R: TTAACCGCCACTTGCAATGGG F: ATGGTTGATGACGAAAAACGTGA R: TCACCGACGAGTGGGCTTAAC F: TTGATGTTTGAACTGTTCAAGGAGA R: CTACGGATTAACCACTAAGCGAT F: ATGAGTATCGAAAATCAGAAAAC R: TCAACGATCATTCGCTCCATAA F: TATGTATCCACCGTAGCCAGTC R: CCCAACAACACCTCCTTATTC
273,668–274,363
CBU_0952
OmpA-like transmembrane domain Peptidoglycan-associated lipoprotein Hypothetical protein
842,764–843,702
56
939
-do-
1781,325–1,781,774
55
450
-do-
696,836–697,882
54
1047
-do-
1797,132–1,797,785
54
654
-do-
1882,741–1,883,970
55
1230
-do-
1125,521–1,126,249
58
729
-do-
665,529–665,813
52
285
-do-
722,881–723,897
53
1017
-do-
849,530–849,862
51
333
-do-
1793,750–1,794,292
52
543
-do-
602,388–602,819
57
432
-do-
1684,801–1,685,397
50
597
-do-
477,073–477,444
52
372
-do-
1564,167–1,564,901
59
735
-do-
Present as 20 copies in the genome
61
687
Willems et al. (1994)
CBU_0091
CBU_1853 CBU_0754 CBU_1869 CBU_1967 CBU_1184 CBU_0718 CBU_0781 CBU_0898
Efflux transporter, RND family, MFP subunit Hypothetical exported protein Drug resistance transporter, Bcr/CflA family Acyltransferase family protein Hypothetical membrane-associated protein Ankyrin repeat protein ankG
CBU_0658
Thyroglobulin type 1 repeat domain protein Hypothetical membrane-associated protein Hypothetical protein
CBU_1754
Hypothetical protein
CBU_0532
comE operon protein 1
CBU_1626
IcmG protein; putative
CBU_0006
Transposable like element
CBU_1865
Product size (bp)
Reference
370
S. Kumar et al. / Journal of Microbiological Methods 95 (2013) 368–372
MDA amplification for the two initial template amounts, respectively. Diagnostic PCRs with C. burnetii-specific primer for the locus trans (CBU_0006) (Willems et. al., 1994) as well as the eubacterial 16S rRNA gene (Lipski et al., 2001) were all positive. Whole genome amplification by MDA has been shown to representatively amplify nearly complete and unbiased eukaryotic genomes (Dean et al., 2002; Hosono et al., 2003; Paez et al., 2004). To investigate the representative content of the C. burnetii genome amplification by MDA, 20 target loci were selected for analysis (Fig. 1, Table 1). Different sets of primers (Table 1) targeting genes positioned at distinct locations around the C. burnetii chromosomal map (Fig. 1) were used for PCR amplification employing MDA amplified genomic DNA as the template in comparison to un-amplified DNA template (Fig. 2). The selected loci exhibited random distribution on the C. burnetii strain RSA 493 chromosome. The genes were selected as putative surface markers and/ or vaccine candidates based on bioinformatic analysis and reported literature pertaining to expression, surface localization, abundance and immunogenicity of different proteins (data not shown). PCR conditions consisted of initial denaturation at 94 °C for 4 min followed by 30cycles of amplification with denaturation at 94°C for 1min, annealing at 51–61°C (as shown in table 1) for 40s, extension at 72°C for 30 s, and final extension of the incompletely synthesized DNA at 72 °C for 5 min. All 20 primer sets tested gave PCR products of the expected sizes (Fig. 3). Templates from un-amplified and amplified DNA were 2-fold diluted to determine the limiting dilution of PCR amplification of target genes (Table 2). The results indicate fold-amplification in the range of 3 × 102 to 192 × 102 for the selected loci (Table 2). A genomic library
was constructed in BAC cloning system using the amplified genomic DNA (data not shown). The choice of the BAC cloning system allows us to cover the entire genome with a relatively small number of clones, reduce the potential for recombination between DNA fragments, and to avoid lethal overexpression of cloned bacterial genes (Amemiya et al., 1999). Sequencing of ten randomly selected clones using vector specific primers indicated random distribution of cloned fragments from the genome (data not shown), further supporting the unbiased nature of amplification and utility and compatibility of the technique for such applications. In summary, genomic studies require large amounts of high quality DNA, which is often difficult to obtain from fastidious obligate intracellular pathogens. MDA is promising techniques that significantly amplify the genome while simultaneously avoids repeated handling of the infectious agent. The technique is especially valuable for intracellular pathogens and/or for agents with low infectious dose by substantially reducing the associated biohazard and overcoming the bottleneck of obtaining large quantity of DNA for downstream processing.
Acknowledgments We thank Prof. (Dr.) M. P. Kaushik, Director, DRDE, Gwalior, for providing all facilities and support required for this study. The work has been funded by the Defence Research and Development Organization, Government of India.
Fig. 1. Schematic representation of C. burnetii chromosome with locations of gene loci amplified (indicated by arrows) by multiple displacement amplification (MDA). The chromosome atlas is adapted from Genome Atlas Database at www.cbs.dtu.dk.
S. Kumar et al. / Journal of Microbiological Methods 95 (2013) 368–372
371
Fig. 2. Multiple displacement amplification of genomic DNA from C. burnetii using REPLI-g Mini Kit. Genomic DNA template from C. burnetii RSA 493 was used at different concentrations (2.5 μl per reaction) for MDA amplification. Lane 1: Un-amplified genomic DNA 2.5 μl (90 ng ml−1); lanes 2–4: REPLI-g amplification using 2.5 μl amplified genomic DNA at dilutions 1:100; 1:200; and 1:400.
References Amemiya, C.T., Zhong, T.P., Silverman, G.A., Fishman, M.C., 1999. The zebrafish, genomics and genetics. In: Detrich, W., Westerfield, M., Zon, L. (Eds.), Methods in Cell Biology. Academic Press, New York, pp. 235–258. Angelakis, E., Raoult, D., 2010. Q fever. Vet. Microbiol. 140, 297–309. Blanco, L., Bernard, A., Lazaro, J.M., Martin, G., Garmendia, C., Salas, M., 1989. Highly efficient DNA synthesis by the phage phi29 DNA polymerase. Symmetrical mode of DNA replication. J. Biol. Chem. 264, 8935–8940. Dean, F.B., Nelson, J.R., Giesler, T.L., Lasken, R.S., 2001. Rapid amplification of plasmid and phage DNA using phi29 DNA polymerase and multiply-primed rolling circle amplification. Genome Res. 11, 1095–1099. Dean, F.B., Hosono, S., Fang, L., Wu, Z., Faruqi, A.F., Bray-Ward, P., Sun, Z., Zong, Q., Du, Y., Du, J., Driscoll, M., Song, W., Kingsmore, S.F., Egholm, M., Lasken, R.S., 2002. Comprehensive human genome amplification using multiple displacement amplification. Proc. Natl. Acad. Sci. U. S. A. 99, 5261–5266. Heinzen, R.A., Scidmore, M.A., Rockey, D.D., Hackstadt, T., 1996. Differential interaction with endocytic and exocytic pathways distinguish parasitophorous vacuoles of Coxiella burnetii and Chlamydia trachomatis. Infect. Immun. 64, 796–809. Hellani, A., Coskun, S., Benkhalifa, M., Tbakhi, A., Sakati, N., Al-Odaib, A., Ozand, P., 2004. Multiple displacement amplification on a single cell and possible PGD applications. Mol. Hum. Reprod. 10, 847–852. Hosono, S., Faruqi, A.F., Dean, F.B., Du, Y., Sun, Z., Wu, X., Du, J., Kingsmore, S.F., Egholm, M., Larken, R.S., 2003. Unbiased whole-genome amplification directly from clinical samples. Genome Res. 13, 954–964. Jeyaprakash, A., Hoy, M.A., 2004. Multiple displacement amplification in combination with high-fidelity PCR improves detection of bacteria from single females or eggs of Metaseiulus occidentalis (Nesbitt) (Acari: Phytoseiidae). J. Invertebr. Pathol. 86, 111–116. Kwon, Y.M., Cox, M.M., 2004. Improved efficacy of whole genome amplification from bacterial cells. Biotechniques 37, 1–3. Lipski, A., Friedrich, U., Altendorf, K., 2001. Application of rRNA targeted oligonucleotide probes in biotechnology. Appl. Microbiol. Biotechnol. 56, 40–57. Maurin, M., Raoult, D., 1999. Q fever. Clin. Microbiol. Rev. 12, 518–553.
Fig. 3. Comparison of PCR amplification of 20 target genes using specific primers and unamplified (left) and MDA amplified template genomic DNA from C. burnetii. A representative image of PCR product for one dilution (from a range of two-fold serially diluted templates) of un-amplified and MDA-amplified template has been shown for comparison. Dilutions of the two templates were made to determine the limiting dilution for PCR amplification of target genes as reflected in Table 2. Osoegawa, K., Mammoser, A.G., Wu, C., Frengen, E., Zeng, C., Catanese, J.J., de Jong, P.J., 2001. A bacterial artificial chromosome library for sequencing the complete human genome. Genome Res. 11, 483–496. Paez, J.G., Lin, M., Beroukhim, R., Lee, J.C., Zhao, X., Richter, D.J., Gabriel, S., Herman, P., Sasaki, H., Altshuler, D., Li, C., Meyerson, M., Sellers, W.R., 2004. Genome coverage and sequence fidelity of phi29 polymerase based multiple strand displacement whole genome amplification. Nucleic Acids Res. 32, 71. Rodolakis, A., 2006. Q fever, state of art: epidemiology, diagnosis and prophylaxis. Small Rumin. Res. 62, 121–124. Willems, H., Thiele, D., Frohlich-Ritter, R., Krauss, H., 1994. Detection of Coxiella burnetii in cow’s milk using the polymerase chain reaction (PCR). J. Vet. Med. Ser. B 41, 580–587.
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Table 2 Limiting dilution for amplification of target genes using un-amplified and amplified genome of C. burnetii. ORF no.
CBU_0307 CBU_0091 CBU_0952 CBU_0090 CBU_2079 CBU_0891 CBU_1853 CBU_0754 CBU_1869 CBU_1967 CBU_1184 CBU_0718 CBU_0781 CBU_0898 CBU_1865 CBU_0658 CBU_1754 CBU_0532 CBU_1626 CBU_0006 a b
Dilution of un-amplified genomic template (×10−3)a
Dilution of MDA-amplified genomic template (×10−5)
Fold amplification
2
8
32
128
512
2048
8192
1.5
6
24
96
384
1536
6144
+ −b + + + + + + + −b + −b + + + −b + + + +
− − − − − − − − − − − − + − + − + + − −
− − − − − − − − − − − − − − − − − + − −
− − − − − − − − − − − − − − − − − + − −
− − − − − − − − − − − − − − − − − − − −
− − − − − − − − − − − − − − − − − − − −
− − − − − − − − − − − − − − − − − − − −
+ + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + +
+ − + − + + + + − − + + + + + + + + + +
− − − + − + + − − + + + + + − + + + +
− − − + − − − − − − − + + + − + + + −
− − − − − − − − − − − − − + − + − − −
− − − − − − − − − − − − − − − − − − −
The starting dilution 2 × 10−3–9 pg/μl of template DNA. Amplification was observed only at a lower dilution using un-amplified genomic DNA as template.
12 × 102 b3 × 102 12 × 102 3 × 102 192 × 102 12 × 102 48 × 102 48 × 102 3 × 102 b3 × 102 48 × 102 b48 × 102 48 × 102 192 × 102 192 × 102 b12 × 102 192 × 102 3 × 102 192 × 102 48 × 102
Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Note
Whole genome amplification of the obligate intracellular pathogen Coxiella burnetii using multiple displacement amplification Sanjay Kumar a, Shefali Raj Gangoliya a, Mustapha Berri b, Annie Rodolakis b, Syed Imteyaz Alam a,⁎ a b
Biotechnology Division, Defence Research & Development Establishment, Gwalior 474002, India INRA, UMR1282 ISP, Nouzilly, France
a r t i c l e
i n f o
Article history: Received 27 August 2013 Received in revised form 14 October 2013 Accepted 16 October 2013 Available online 25 October 2013
a b s t r a c t This study demonstrates that whole genome multiple displacement amplification (MDA) is a promising technique for downstream genomic analysis of fastidious obligate intracellular pathogens such as Coxiella burnetii. The MDA technology can help in obtaining sufficient genetic material from highly infectious agent and thus minimizing repeated culturing and associated biohazard. © 2013 Elsevier B.V. All rights reserved.
Keywords: Coxiella burnetii Whole genome amplification Multiple displacement amplification
Coxiella burnetii is the etiologic agent of an acute, disabling influenzalike illness in humans termed Q fever. In rare instances chronic disease can occur, usually presenting as hepatitis or endocarditis. C. burnetii infects a wide range of vertebrate and invertebrate hosts and is found worldwide with the exception of New Zealand. Most human Q fever cases are acquired through inhalation of contaminated aerosols originating from products shed from chronically infected domestic livestock (Angelakis and Raoult, 2010; Maurin and Raoult, 1999; Rodolakis, 2006). C. burnetii has an obligate requirement for replication in a eukaryotic host cell vacuole with lysosomal characteristics (Heinzen et al., 1996). Resistance to this harsh intracellular environment correlates with remarkable environmental stability, a trait that distinguishes C. burnetii from other obligate intracellular bacteria. Environmental stability, along with aerosol transmission and an infectious dose approaching one organism, has resulted in classification of C. burnetii as a CDC category B biothreat agent. Unfortunately, research on this pathogen has been hampered by its extremely low infectious dose and is confounded by fastidious nature of the organism with respect to in vitro intracellular proliferation and the need of BSL3 laboratory, which is technically demanding and time consuming. Obtaining sufficient quantity of DNA material of Coxiella for any genetic characterization analysis is a challenge. It is also a potential biohazard for the investigators to repeatedly proliferate C. burnetii under laboratory conditions in quantities sufficient for proteomic or genomic studies. Identification and analysis of pathogen DNA (e.g. PCR of specific targets) is a powerful tool for confirmation of the agent in both environmental and clinical samples. Immunocapture of organisms using antibodies, binding to specific
⁎ Corresponding author. Tel.: +91 751 2340132x276; fax: +91 751 2341148. E-mail address: [email protected] (S.I. Alam). 0167-7012/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mimet.2013.10.008
surface marker is a method of choice for environmental detection of threat agents. These studies rely on availability of sufficient DNA sample for downstream applications. The objective of this study was, thus, to develop an efficient strategy for the amplification of total DNA of suitable quality from small amounts of initial template for genetic analysis and development of countermeasures for this pathogen of medical and military importance. The use of whole genome amplification (WGA) as a tool for the study of infectious bacteria is of growing clinical interest (Hosono et al., 2003). The multiple displacement amplification (MDA) has become the preferred choice due to its high processivity and low error rate. In this study, we used whole genome amplification by multipledisplacement amplification (MDA) (Dean et al., 2002) and produced a large amount of C. burnetii strain RSA 493 DNA. The DNA recovered has been found to contain most of the C. burnetii loci targeted, and thus provides avenues for genomic studies of this fastidious and hazardous bacterium. Attempts were made to construct a bacterial artificial chromosome (BAC) library using the amplified C. burnetii genome with an objective to maintain the pathogen's gene pool for any downstream molecular application and to carry out genome sequence analysis. Total DNA of C. burnetii strain RSA 493 was obtained from INRA, France and was used for multiple displacement amplification using REPLI-g Mini Kit (Qiagen, Germany) as per manufacturer's instructions. The REPLI-g Mini Kit provides highly uniform amplification across the entire genome by bacteriophage Phi29 DNA polymerase, with negligible sequence bias (Hosono et al., 2003). The method is based on MDA technology originally proposed by Dean et al. (2002), which carries out isothermal genome amplification utilizing a uniquely processive DNA polymerase capable of replicating up to 100 kb without dissociating from the genomic DNA template. The DNA polymerase has a 3′–5′ exonuclease proofreading activity to maintain high fidelity
S. Kumar et al. / Journal of Microbiological Methods 95 (2013) 368–372
during replication and is used in the presence of exonuclease resistant primers to achieve high yields of DNA product. Briefly, 2.5 μl (0.22 ng) and 5.0 μl (0.45 ng) of template DNA were mixed with sample buffer and denatured. Twenty nine microliter of reaction buffer and 1 μl of REPLI-g Mini DNA Polymerase were added to the template and the reaction volume was adjusted to 40 μl using nuclease free water. Reaction was carried out for 16 h at 30 °C and at the end of the reaction REPLI-g Mini DNA Polymerase was inactivated by heating the sample for 3 min at 65 °C. Amplified and un-amplified genomic DNA from C. burnetii strain RSA 493 was tested for uniform amplification by PCR amplification of 20 target loci. The details of the loci, the primer sequences used for amplification, the optimized annealing temperatures and their product sizes are summarized in Table 1. BAC library construction was performed as described by Osoegawa et al. (2001) using the CopyRight BAC Cloning Kit (Lucigen Corporation, Middleton, WI, USA). Briefly amplified genomic DNA was subjected to partial digestion with BamHI, size selected on agarose gels, and ligated to the pSMART BAC vector (Lucigen Corporation, Middleton, WI, USA). Ligation products were transformed by electroporation into the BAC-optimized electrocompetent cells (Lucigen Corporation). The positive BAC DNAs were purified using the Qiagen Large-Construct Kit (Qiagen, Germany). Sequencing was carried out on ABI3730 equipment with “Dye Terminator” process using ABI kit version 3.1.
369
To increase the amount of C. burnetii strain RSA 493 DNA, we used a whole genome amplification strategy based on MDA technique, as described by Dean et al. (2001, 2002). This isothermal amplification method employs the MDA property of the bacteriophage Phi29 DNA polymerase to amplify a long (10 to 70 kb) stretch of circular or linear DNA (Blanco et al., 1989), yielding high amount of DNA (approximately 10 μg per 50 μl reaction) from a relatively limited quantity of template (~10 ng) with minimum sequence bias (Dean et al., 2002). In addition, Phi29-based MDA was shown to cover most of the loci present in the original DNA template, including those of an entire phage or plasmid as well as a complete genome from one single cell (Dean et al., 2001, 2002; Hellani et al., 2004; Paez et al., 2004). In spite of the success of the MDA methodology with eukaryotes, the applications for prokaryotic organisms have been limited. Recently, MDA has been used to improve the detection of bacteria in mites (Jeyaprakash and Hoy, 2004) or to amplify genomic DNA from Salmonella enterica serovar Enteritidis cells (Kwon and Cox, 2004), but the genetic content was evaluated for only a few loci. In this study, MDA was applied for the first time in order to produce considerable quantities of C. burnetii DNA by using REPLI-g Mini Kit (Qiagen, Germany). Successful and unbiased amplification was evaluated for representative loci on the genome by PCR analysis of amplified product as described below. Template DNA (0.22 or 0.45 ng) was denatured and added to the reaction mixture. A total yield of approximately 200 and 500 ng was observed at the end of
Table 1 Primers and PCR-amplified genes from C. burnetii genome. ORF no.
Protein
Primers sequence (5′–3′)
Gene locus
Annealing temperature (°C)
CBU_0307
54
696
This study
83,817–84,404
53
588
-do-
902,551–903,234
53
684
-do-
CBU_0090
TolB protein
82,485–83,807
58
1323
-do-
CBU_2079
Hypothetical protein
1980,877–1,981,281
59
405
-do-
CBU_0891
Hypothetical exported membrane-associated protein GtrA family protein
F: ATGTCGGTAAAAAAACTAGTAACA R: TTACATTAAGAATTTGTAGCCAAG F: ATGAGCCGTAAATTTACAGACAA R:TTAATAAGCCTCATAAATTAAATTAAC F: ATGAAAAAACTAACCGTAACTTTC R: CTATTTATTTAAAAAAGTCGCCAC F: ATGAACTCCATGCAAAAACGACA R: CTACACGCCTAAATAAGGAGAC F: ATGAGCTATATTAAACGAGATCAT R: TTAGCCGCAGCCATGACCTC F: GTGAAACGGTATCTTTTTTTTACTAT R: TCAAGGCAGGCTCTTTAAAAGC F:ATGATTAAAAAACTTATTTTATTCGTTTA R: TCAAATCGCTTGATGGGAATT F: ATGAAAAAACGCACAAAAATTATTAT R: TTAATGAACCACTTTTACCTTTGCT F: ATGTCAAAAATCAAGTGGTTTTTAG R: TCAGATAGTATTTTGCGGACTTAAT F: ATGCATAAATTTCGCCTCTCGGG R: TCATTGAACGGACGTCCTCATAA F: ATGCGCTTACAAAGAGTCTTTTCA R: TTAGGAAGGAATTTCCTGGAAATA F: ATGCCGAAAGAAACTAAAACCGA R:TCATTTTCTTCGAGCGATCTTGC F: ATGAGTAGACGTGAGACTCCCA R: TCACCGAGGACTAGACAGACAA F: ATGAGAAGCTTTATTTATATTTTAATT R: TCATTGCATTCGTCGATACAATT F: ATGTGGAGAATGTTGATGTACCC R: TTAAGACTGAGCTTCCTCAGATG F: ATGTCAGCAATTTTACCAACAGA R: TTAACCGCCACTTGCAATGGG F: ATGGTTGATGACGAAAAACGTGA R: TCACCGACGAGTGGGCTTAAC F: TTGATGTTTGAACTGTTCAAGGAGA R: CTACGGATTAACCACTAAGCGAT F: ATGAGTATCGAAAATCAGAAAAC R: TCAACGATCATTCGCTCCATAA F: TATGTATCCACCGTAGCCAGTC R: CCCAACAACACCTCCTTATTC
273,668–274,363
CBU_0952
OmpA-like transmembrane domain Peptidoglycan-associated lipoprotein Hypothetical protein
842,764–843,702
56
939
-do-
1781,325–1,781,774
55
450
-do-
696,836–697,882
54
1047
-do-
1797,132–1,797,785
54
654
-do-
1882,741–1,883,970
55
1230
-do-
1125,521–1,126,249
58
729
-do-
665,529–665,813
52
285
-do-
722,881–723,897
53
1017
-do-
849,530–849,862
51
333
-do-
1793,750–1,794,292
52
543
-do-
602,388–602,819
57
432
-do-
1684,801–1,685,397
50
597
-do-
477,073–477,444
52
372
-do-
1564,167–1,564,901
59
735
-do-
Present as 20 copies in the genome
61
687
Willems et al. (1994)
CBU_0091
CBU_1853 CBU_0754 CBU_1869 CBU_1967 CBU_1184 CBU_0718 CBU_0781 CBU_0898
Efflux transporter, RND family, MFP subunit Hypothetical exported protein Drug resistance transporter, Bcr/CflA family Acyltransferase family protein Hypothetical membrane-associated protein Ankyrin repeat protein ankG
CBU_0658
Thyroglobulin type 1 repeat domain protein Hypothetical membrane-associated protein Hypothetical protein
CBU_1754
Hypothetical protein
CBU_0532
comE operon protein 1
CBU_1626
IcmG protein; putative
CBU_0006
Transposable like element
CBU_1865
Product size (bp)
Reference
370
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MDA amplification for the two initial template amounts, respectively. Diagnostic PCRs with C. burnetii-specific primer for the locus trans (CBU_0006) (Willems et. al., 1994) as well as the eubacterial 16S rRNA gene (Lipski et al., 2001) were all positive. Whole genome amplification by MDA has been shown to representatively amplify nearly complete and unbiased eukaryotic genomes (Dean et al., 2002; Hosono et al., 2003; Paez et al., 2004). To investigate the representative content of the C. burnetii genome amplification by MDA, 20 target loci were selected for analysis (Fig. 1, Table 1). Different sets of primers (Table 1) targeting genes positioned at distinct locations around the C. burnetii chromosomal map (Fig. 1) were used for PCR amplification employing MDA amplified genomic DNA as the template in comparison to un-amplified DNA template (Fig. 2). The selected loci exhibited random distribution on the C. burnetii strain RSA 493 chromosome. The genes were selected as putative surface markers and/ or vaccine candidates based on bioinformatic analysis and reported literature pertaining to expression, surface localization, abundance and immunogenicity of different proteins (data not shown). PCR conditions consisted of initial denaturation at 94 °C for 4 min followed by 30cycles of amplification with denaturation at 94°C for 1min, annealing at 51–61°C (as shown in table 1) for 40s, extension at 72°C for 30 s, and final extension of the incompletely synthesized DNA at 72 °C for 5 min. All 20 primer sets tested gave PCR products of the expected sizes (Fig. 3). Templates from un-amplified and amplified DNA were 2-fold diluted to determine the limiting dilution of PCR amplification of target genes (Table 2). The results indicate fold-amplification in the range of 3 × 102 to 192 × 102 for the selected loci (Table 2). A genomic library
was constructed in BAC cloning system using the amplified genomic DNA (data not shown). The choice of the BAC cloning system allows us to cover the entire genome with a relatively small number of clones, reduce the potential for recombination between DNA fragments, and to avoid lethal overexpression of cloned bacterial genes (Amemiya et al., 1999). Sequencing of ten randomly selected clones using vector specific primers indicated random distribution of cloned fragments from the genome (data not shown), further supporting the unbiased nature of amplification and utility and compatibility of the technique for such applications. In summary, genomic studies require large amounts of high quality DNA, which is often difficult to obtain from fastidious obligate intracellular pathogens. MDA is promising techniques that significantly amplify the genome while simultaneously avoids repeated handling of the infectious agent. The technique is especially valuable for intracellular pathogens and/or for agents with low infectious dose by substantially reducing the associated biohazard and overcoming the bottleneck of obtaining large quantity of DNA for downstream processing.
Acknowledgments We thank Prof. (Dr.) M. P. Kaushik, Director, DRDE, Gwalior, for providing all facilities and support required for this study. The work has been funded by the Defence Research and Development Organization, Government of India.
Fig. 1. Schematic representation of C. burnetii chromosome with locations of gene loci amplified (indicated by arrows) by multiple displacement amplification (MDA). The chromosome atlas is adapted from Genome Atlas Database at www.cbs.dtu.dk.
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Fig. 2. Multiple displacement amplification of genomic DNA from C. burnetii using REPLI-g Mini Kit. Genomic DNA template from C. burnetii RSA 493 was used at different concentrations (2.5 μl per reaction) for MDA amplification. Lane 1: Un-amplified genomic DNA 2.5 μl (90 ng ml−1); lanes 2–4: REPLI-g amplification using 2.5 μl amplified genomic DNA at dilutions 1:100; 1:200; and 1:400.
References Amemiya, C.T., Zhong, T.P., Silverman, G.A., Fishman, M.C., 1999. The zebrafish, genomics and genetics. In: Detrich, W., Westerfield, M., Zon, L. (Eds.), Methods in Cell Biology. Academic Press, New York, pp. 235–258. Angelakis, E., Raoult, D., 2010. Q fever. Vet. Microbiol. 140, 297–309. Blanco, L., Bernard, A., Lazaro, J.M., Martin, G., Garmendia, C., Salas, M., 1989. Highly efficient DNA synthesis by the phage phi29 DNA polymerase. Symmetrical mode of DNA replication. J. Biol. Chem. 264, 8935–8940. Dean, F.B., Nelson, J.R., Giesler, T.L., Lasken, R.S., 2001. Rapid amplification of plasmid and phage DNA using phi29 DNA polymerase and multiply-primed rolling circle amplification. Genome Res. 11, 1095–1099. Dean, F.B., Hosono, S., Fang, L., Wu, Z., Faruqi, A.F., Bray-Ward, P., Sun, Z., Zong, Q., Du, Y., Du, J., Driscoll, M., Song, W., Kingsmore, S.F., Egholm, M., Lasken, R.S., 2002. Comprehensive human genome amplification using multiple displacement amplification. Proc. Natl. Acad. Sci. U. S. A. 99, 5261–5266. Heinzen, R.A., Scidmore, M.A., Rockey, D.D., Hackstadt, T., 1996. Differential interaction with endocytic and exocytic pathways distinguish parasitophorous vacuoles of Coxiella burnetii and Chlamydia trachomatis. Infect. Immun. 64, 796–809. Hellani, A., Coskun, S., Benkhalifa, M., Tbakhi, A., Sakati, N., Al-Odaib, A., Ozand, P., 2004. Multiple displacement amplification on a single cell and possible PGD applications. Mol. Hum. Reprod. 10, 847–852. Hosono, S., Faruqi, A.F., Dean, F.B., Du, Y., Sun, Z., Wu, X., Du, J., Kingsmore, S.F., Egholm, M., Larken, R.S., 2003. Unbiased whole-genome amplification directly from clinical samples. Genome Res. 13, 954–964. Jeyaprakash, A., Hoy, M.A., 2004. Multiple displacement amplification in combination with high-fidelity PCR improves detection of bacteria from single females or eggs of Metaseiulus occidentalis (Nesbitt) (Acari: Phytoseiidae). J. Invertebr. Pathol. 86, 111–116. Kwon, Y.M., Cox, M.M., 2004. Improved efficacy of whole genome amplification from bacterial cells. Biotechniques 37, 1–3. Lipski, A., Friedrich, U., Altendorf, K., 2001. Application of rRNA targeted oligonucleotide probes in biotechnology. Appl. Microbiol. Biotechnol. 56, 40–57. Maurin, M., Raoult, D., 1999. Q fever. Clin. Microbiol. Rev. 12, 518–553.
Fig. 3. Comparison of PCR amplification of 20 target genes using specific primers and unamplified (left) and MDA amplified template genomic DNA from C. burnetii. A representative image of PCR product for one dilution (from a range of two-fold serially diluted templates) of un-amplified and MDA-amplified template has been shown for comparison. Dilutions of the two templates were made to determine the limiting dilution for PCR amplification of target genes as reflected in Table 2. Osoegawa, K., Mammoser, A.G., Wu, C., Frengen, E., Zeng, C., Catanese, J.J., de Jong, P.J., 2001. A bacterial artificial chromosome library for sequencing the complete human genome. Genome Res. 11, 483–496. Paez, J.G., Lin, M., Beroukhim, R., Lee, J.C., Zhao, X., Richter, D.J., Gabriel, S., Herman, P., Sasaki, H., Altshuler, D., Li, C., Meyerson, M., Sellers, W.R., 2004. Genome coverage and sequence fidelity of phi29 polymerase based multiple strand displacement whole genome amplification. Nucleic Acids Res. 32, 71. Rodolakis, A., 2006. Q fever, state of art: epidemiology, diagnosis and prophylaxis. Small Rumin. Res. 62, 121–124. Willems, H., Thiele, D., Frohlich-Ritter, R., Krauss, H., 1994. Detection of Coxiella burnetii in cow’s milk using the polymerase chain reaction (PCR). J. Vet. Med. Ser. B 41, 580–587.
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Table 2 Limiting dilution for amplification of target genes using un-amplified and amplified genome of C. burnetii. ORF no.
CBU_0307 CBU_0091 CBU_0952 CBU_0090 CBU_2079 CBU_0891 CBU_1853 CBU_0754 CBU_1869 CBU_1967 CBU_1184 CBU_0718 CBU_0781 CBU_0898 CBU_1865 CBU_0658 CBU_1754 CBU_0532 CBU_1626 CBU_0006 a b
Dilution of un-amplified genomic template (×10−3)a
Dilution of MDA-amplified genomic template (×10−5)
Fold amplification
2
8
32
128
512
2048
8192
1.5
6
24
96
384
1536
6144
+ −b + + + + + + + −b + −b + + + −b + + + +
− − − − − − − − − − − − + − + − + + − −
− − − − − − − − − − − − − − − − − + − −
− − − − − − − − − − − − − − − − − + − −
− − − − − − − − − − − − − − − − − − − −
− − − − − − − − − − − − − − − − − − − −
− − − − − − − − − − − − − − − − − − − −
+ + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + +
+ − + − + + + + − − + + + + + + + + + +
− − − + − + + − − + + + + + − + + + +
− − − + − − − − − − − + + + − + + + −
− − − − − − − − − − − − − + − + − − −
− − − − − − − − − − − − − − − − − − −
The starting dilution 2 × 10−3–9 pg/μl of template DNA. Amplification was observed only at a lower dilution using un-amplified genomic DNA as template.
12 × 102 b3 × 102 12 × 102 3 × 102 192 × 102 12 × 102 48 × 102 48 × 102 3 × 102 b3 × 102 48 × 102 b48 × 102 48 × 102 192 × 102 192 × 102 b12 × 102 192 × 102 3 × 102 192 × 102 48 × 102
