- Email: [email protected]
Unexpected detection of DNA by nucleic acid sequence-based amplification technique
Molecular and Cellular Probes 18 (2004) 251–253 www.elsevier.com/locate/ymcpr
Unexpected detection of DNA by nucleic acid sequence-based amplification technique David Rodrı´guez-La`zaroa, Joy Lloydb, John Ikonomopoulosc, Maria Plaa,*, Nigel Cookb a
Institute of Food and Agricultural Technology (INTEA), University of Girona, Campus Montilivi s/n, E-17071 Girona, Spain b Central Science Laboratory (CSL), Sand Hutton, York YO41 1LZ, UK c Department of Anatomy-Physiology, School of Animal Science, Agricultural University of Athens, Athens, Greece Received 20 November 2003; accepted for publication 14 January 2004
Abstract Nucleic acid sequence-based amplification (NASBA) is a technique that has been previously shown to selectively mediate the detection of RNA in microbial cells. In a series of tests, nucleic acids were extracted from Salmonella enterica serotype Typhimurium and Mycobacterium avium subsp. paratuberculosis, and subjected to four enzymatic treatments prior to NASBA. These enzymatic treatments were DNase, RNase, S1 nuclease, and RNase/S1 nuclease. The results obtained were different for the two bacteria. With S. enterica serotype Typhimurium, RNase and RNase/S1 nuclease abolished the NASBA signal, as expected. But with M. avium subsp. paratuberculosis RNase, S1 nuclease, and RNase/S1 nuclease had no effect on the NASBA signal, whereas DNase treatment abolished it. This indicates that in the latter bacterium, NASBA can detect DNA, and demonstrates the necessity of verifying the nucleic acid origin of a NASBA signal if detection of RNA is objective. q 2004 Elsevier Ltd. All rights reserved. Keywords: Nucleic acid sequence-based amplification; DNA; RNA
Nucleic acid sequence-based amplification (NASBA) is specifically designed to detect RNA. At 41 8C, the standard incubation temperature of NASBA, genomic DNA should remain double-stranded and not become a substrate for amplification. This should eliminate the necessity for DNase treatment, which is required when using RT-PCR to try to ensure that the signal comes only from amplification of RNA [1]. In fact, NASBA has been shown [2] to selectively amplify mRNA sequences when genomic DNA is present. We show here that this approach must be very carefully evaluated, by presenting a comparison of NASBA results from two target microorganisms, Salmonella enterica serotype Typhimurium (SET) and Mycobacterium avium subsp. paratuberculosis (MAP). Simpkins et al. [2] used mRNA from the SET heat-shock gene dnaK as target for NASBA. In the current study, a NASBA was designed to target the transcript of an analogous gene, hsp70 [3] in MAP. In accordance with NASBA principles [4], the forward, reverse and capture probe sequences were, * Corresponding author. Tel.: þ 34-972-419852; fax: þ 34-972-418399. E-mail address: [email protected] (M. Pla). 0890-8508/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.mcp.2004.01.001
respectively, 50 -gatgcaaggtcgcatatgagggaatcgcttgtctaccaga-30 , 50 -aattctaatacgactcactatagggagaagggagcttctccatcgccgacttga-30 , and 50 -biotin-tcccgaggagacgctgtc-30 . Nucleic acids were obtained from MAP strain ATCC 19698 and SET strain FMG2 using NucliSensw Basic Kit (bioMe´rieux bv, The Netherlands). Nucleic acid concentration and quality was determined using spectrophotometry. MAP and SET extracts containing 100 ng/ml nucleic acids (OD260/OD280 ¼ 2.00) were produced. NASBA reactions were performed as described in Ref. [2]. Each reaction contained a maximum of 10 ng nucleic acids. Before addition of the enzymatic mix, reactions were incubated at 65 8C for 5 min, and then cooled to 41 8C to allow primer annealing. The amplification reaction was carried out for 90 min at 41 8C, and the amplicons detected using a NucliSensw ECL Reader (bioMe´rieux bv, The Netherlands). Positive amplification signals were obtained for each bacterium. To confirm that the origin of the NASBA signals was indeed mRNA, prior to amplification, 3.5 mg MAP and SET nucleic acid extracts were subjected to four different enzymatic treatments (RNase, Dnase, S1 nuclease, Rnase þ S1 nuclease) according to Ausubel et al. [5].
D. Rodrı´guez-La`zaro et al. / Molecular and Cellular Probes 18 (2004) 251–253
252
Table 1 Nucleic acid amplification following different enzymatic treatments Bacterium
MAP
SET
Detection assay
NASBA PCR RT-PCR NASBA PCR RT-PCR
Enzymatic treatments Untreated
RNase
DNase
S1 nuclease
RNase þ S1 nuclease
þ þ þ þ þ þ
þ þ þ 2 þ þ
2 2 2 þ 2 þ
þ þ þ 2 þ þ
þ þ þ 2 þ þ
All tests were performed in triplicate. þ: positive amplification signal; 2: no signal.
After enzymatic treatment, purification of nucleic acids was performed according to Sambrook and Russell [6]. RNase treatment was performed with ribonuclease A (Sigma-Aldrich Co., Missouri, USA), treated to inactivate any contaminating DNase [6]. DNase and S1 nuclease treatments were performed using the RQ1 RNase-free DNase kit (Promega, Madison, USA) and the S1 Nuclease Kit (Promega, Madison, USA), respectively. Unexpectedly, treatment of the MAP nucleic acid extracts with RNase had no effect on the NASBA signal, whereas DNase treatment abolished it (Table 1). With SET nucleic acids, as expected, the converse was observed, i.e. RNase treatment abolished the NASBA signal whereas DNase treatment had no effect (Table 1). These results suggested that whereas SET RNA was amplified by NASBA, with MAP, NASBA was amplifying DNA. To confirm that the enzymatic treatments had been effective, the treated extracts were assayed by PCR, using the same primers. Reactions contained 1 £ GeneAmpw PCR buffer (Applied Biosystems, Branchburg, Germany), 200 mM each dNTP, 1 mM each primer, and 1 U of AmpliTaq Goldw DNA polymerase (Applied Biosystems, Branchburg, Germany). Reactions were performed as follows: 10 min at 95 8C, 45 cycles of 1 min at 94 8C, 30 s in 55 8C, 1 min at 72 8C and 5 min 72 8C. Products were analysed by 2% agarose gel electrophoresis and ethidium bromide staining. As expected, in each case, only DNase treatment abolished the PCR signal (Table 1). As a further test, reverse transcription (RT)-PCR was performed, using the Access RTPCR system (Promega, Madison, USA). Recreations were performed as follows: 48 8C for 45 min, 2 min at 94 8C, 45 cycles of 1 min at 94 8C, 30 s at 55 8C and 1 min at 68 8C, and 5 min at 68 8C. A signal was obtained from both MAP and SET RNasetreated nucleic acid extracts: this would have been due to the presence of genomic DNA. In contrast, when the DNasetreated nucleic acid extracts were analysed, a signal was obtained from SET samples, but not from MAP. This could indicate that MAP hsp70 transcripts were not abundantly present (perhaps attributable to the very slow growth rate of MAP) or that the gene had not been transcribed immediately prior to extraction. It might be argued that MAP mRNA was not recovered by the extraction procedure used; however,
Schuitema et al. [7] reported recovery of M. tuberculosis mRNA by the same procedure. The recovery of SET mRNA shows that the extraction procedure and the post-enzymatic treatment clean-up procedure were efficiently performed. NASBA amplification of DNA has been previously observed, with some human and viral targets [8], but the nature of the signal was not confirmed, i.e. was single or double-stranded DNA the origin? Deiman et al. [8] speculated that DNA amplification by NASBA was due to the target DNA being in excess in the reaction. However, in the study presented here, identical quantities of SET and MAP DNA were used, but the former did not elicit a signal at any time. Besides, Simpkins et al. [2] demonstrated, using SET nucleic acids, that NASBA selectively amplifies RNA in the presence of a DNA background. The amplification of DNA by NASBA has been demonstrated using primers directed against easily accessible DNA regions such as plasmid DNA, low-meltpoint sequences or single-stranded regions [8,9]. However, the target MAP sequence has a G –C ratio greater that 65%, and therefore, it was highly unlikely to have been denatured at the relatively low temperatures used in the NASBA reaction. Furthermore, the results were reproducible even when the initial 65 8C step was omitted from the NASBA reactions (data not shown). It was not single-stranded DNA regions which were being amplified, as by S1 nuclease treatment did not abolish the NASBA signal. Thus, we conclude that the origin of the NASBA signal in MAP was double-stranded DNA. This study shows, for the first time, NASBA amplification of bacterial genomic DNA. It is a salutary example to workers who are contemplating the use of NASBA for selective detection of RNA. After the unexpected but reproducible results of this study, the enzymatic tests should be applied in each case to conclusively demonstrate that the NASBA signal has indeed been generated only by RNA and not DNA.
Acknowledgements This work was supported by EU project ‘SACROHN’ N. QLK2-CT-2000-00928, and a grant from the National
D. Rodrı´guez-La`zaro et al. / Molecular and Cellular Probes 18 (2004) 251–253
Association for Crohn’s and Colitis. David Rodrı´guezLa´zaro was supported by a FEMS Research Fellowship. We thank John Hays, Milan Bartos and Ivo Pavlik for critical reading of the manuscript.
References [1] Klein PG, Kuneja VJ. Sensitive detection of viable Listeria monocytogenes by reverse trascription-PCR. Appl Environ Microbiol 1997;63:4441–8. [2] Simpkins SA, Chan AB, Hays J, Po¨pping B, Cook N. An RNA transcription-based amplification technique (NASBA) for the detection of viable Salmonella enterica. Lett Appl Microbiol 2000;30:75–9. [3] Koets AP, Rutten VP, de Boer M, Bakker D, Valentin-Weigand P, van Eden W. Differential changes in heat shock protein-, lipoarabinomannan-, and purified protein derivative-specific immunoglobulin G1 and G2 isotype responses during bovine Mycobacterium avium subsp. paratuberculosis infection. Infect Immun 2001;69:1492–8.
253
[4] van Gemen B, van Beuningen R, Nabble A, van Strijp D, Jurriaans S, Lens P, Kievits T. A one-tube quantitative HIV-1 RNA NASBA nucleic acid amplification assay using electrochemiluminescent (ECL) labelled probes. J Virol Meth 1994;49:157–67. [5] Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K, editors. Enzymatic manipulation of DNA and RNA. Current protocol in molecular biology, vol. 1. New York: Wiley; 1990. [6] Sambrook J, Russell D. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbour, NY: Cold Spring Harbour Laboratory; 2000. [7] Schuitema A, van Leewen J, van Aarle P, Sillekens P, Klaster P. Detection of Mycobacterium tuberculosis rRNA and mRNA using the Nuclisensw Basic Kit. J Microbiol Meth 1999;38:256. [8] Deiman B, van Aarle P, Sillekens P. Characteristics and applications of nucleic acid sequence-based amplification (NASBA). Mol Biotechnol 2002;20:163 –79. [9] Voisset C, Mandrand B, Paranhos-Baccala G. RNA amplification technique, NASBA, also amplifies homologous plasmid DNA in nondenaturing conditions. Biotechniques 2000;29:236–40.
Unexpected detection of DNA by nucleic acid sequence-based amplification technique David Rodrı´guez-La`zaroa, Joy Lloydb, John Ikonomopoulosc, Maria Plaa,*, Nigel Cookb a
Institute of Food and Agricultural Technology (INTEA), University of Girona, Campus Montilivi s/n, E-17071 Girona, Spain b Central Science Laboratory (CSL), Sand Hutton, York YO41 1LZ, UK c Department of Anatomy-Physiology, School of Animal Science, Agricultural University of Athens, Athens, Greece Received 20 November 2003; accepted for publication 14 January 2004
Abstract Nucleic acid sequence-based amplification (NASBA) is a technique that has been previously shown to selectively mediate the detection of RNA in microbial cells. In a series of tests, nucleic acids were extracted from Salmonella enterica serotype Typhimurium and Mycobacterium avium subsp. paratuberculosis, and subjected to four enzymatic treatments prior to NASBA. These enzymatic treatments were DNase, RNase, S1 nuclease, and RNase/S1 nuclease. The results obtained were different for the two bacteria. With S. enterica serotype Typhimurium, RNase and RNase/S1 nuclease abolished the NASBA signal, as expected. But with M. avium subsp. paratuberculosis RNase, S1 nuclease, and RNase/S1 nuclease had no effect on the NASBA signal, whereas DNase treatment abolished it. This indicates that in the latter bacterium, NASBA can detect DNA, and demonstrates the necessity of verifying the nucleic acid origin of a NASBA signal if detection of RNA is objective. q 2004 Elsevier Ltd. All rights reserved. Keywords: Nucleic acid sequence-based amplification; DNA; RNA
Nucleic acid sequence-based amplification (NASBA) is specifically designed to detect RNA. At 41 8C, the standard incubation temperature of NASBA, genomic DNA should remain double-stranded and not become a substrate for amplification. This should eliminate the necessity for DNase treatment, which is required when using RT-PCR to try to ensure that the signal comes only from amplification of RNA [1]. In fact, NASBA has been shown [2] to selectively amplify mRNA sequences when genomic DNA is present. We show here that this approach must be very carefully evaluated, by presenting a comparison of NASBA results from two target microorganisms, Salmonella enterica serotype Typhimurium (SET) and Mycobacterium avium subsp. paratuberculosis (MAP). Simpkins et al. [2] used mRNA from the SET heat-shock gene dnaK as target for NASBA. In the current study, a NASBA was designed to target the transcript of an analogous gene, hsp70 [3] in MAP. In accordance with NASBA principles [4], the forward, reverse and capture probe sequences were, * Corresponding author. Tel.: þ 34-972-419852; fax: þ 34-972-418399. E-mail address: [email protected] (M. Pla). 0890-8508/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.mcp.2004.01.001
respectively, 50 -gatgcaaggtcgcatatgagggaatcgcttgtctaccaga-30 , 50 -aattctaatacgactcactatagggagaagggagcttctccatcgccgacttga-30 , and 50 -biotin-tcccgaggagacgctgtc-30 . Nucleic acids were obtained from MAP strain ATCC 19698 and SET strain FMG2 using NucliSensw Basic Kit (bioMe´rieux bv, The Netherlands). Nucleic acid concentration and quality was determined using spectrophotometry. MAP and SET extracts containing 100 ng/ml nucleic acids (OD260/OD280 ¼ 2.00) were produced. NASBA reactions were performed as described in Ref. [2]. Each reaction contained a maximum of 10 ng nucleic acids. Before addition of the enzymatic mix, reactions were incubated at 65 8C for 5 min, and then cooled to 41 8C to allow primer annealing. The amplification reaction was carried out for 90 min at 41 8C, and the amplicons detected using a NucliSensw ECL Reader (bioMe´rieux bv, The Netherlands). Positive amplification signals were obtained for each bacterium. To confirm that the origin of the NASBA signals was indeed mRNA, prior to amplification, 3.5 mg MAP and SET nucleic acid extracts were subjected to four different enzymatic treatments (RNase, Dnase, S1 nuclease, Rnase þ S1 nuclease) according to Ausubel et al. [5].
D. Rodrı´guez-La`zaro et al. / Molecular and Cellular Probes 18 (2004) 251–253
252
Table 1 Nucleic acid amplification following different enzymatic treatments Bacterium
MAP
SET
Detection assay
NASBA PCR RT-PCR NASBA PCR RT-PCR
Enzymatic treatments Untreated
RNase
DNase
S1 nuclease
RNase þ S1 nuclease
þ þ þ þ þ þ
þ þ þ 2 þ þ
2 2 2 þ 2 þ
þ þ þ 2 þ þ
þ þ þ 2 þ þ
All tests were performed in triplicate. þ: positive amplification signal; 2: no signal.
After enzymatic treatment, purification of nucleic acids was performed according to Sambrook and Russell [6]. RNase treatment was performed with ribonuclease A (Sigma-Aldrich Co., Missouri, USA), treated to inactivate any contaminating DNase [6]. DNase and S1 nuclease treatments were performed using the RQ1 RNase-free DNase kit (Promega, Madison, USA) and the S1 Nuclease Kit (Promega, Madison, USA), respectively. Unexpectedly, treatment of the MAP nucleic acid extracts with RNase had no effect on the NASBA signal, whereas DNase treatment abolished it (Table 1). With SET nucleic acids, as expected, the converse was observed, i.e. RNase treatment abolished the NASBA signal whereas DNase treatment had no effect (Table 1). These results suggested that whereas SET RNA was amplified by NASBA, with MAP, NASBA was amplifying DNA. To confirm that the enzymatic treatments had been effective, the treated extracts were assayed by PCR, using the same primers. Reactions contained 1 £ GeneAmpw PCR buffer (Applied Biosystems, Branchburg, Germany), 200 mM each dNTP, 1 mM each primer, and 1 U of AmpliTaq Goldw DNA polymerase (Applied Biosystems, Branchburg, Germany). Reactions were performed as follows: 10 min at 95 8C, 45 cycles of 1 min at 94 8C, 30 s in 55 8C, 1 min at 72 8C and 5 min 72 8C. Products were analysed by 2% agarose gel electrophoresis and ethidium bromide staining. As expected, in each case, only DNase treatment abolished the PCR signal (Table 1). As a further test, reverse transcription (RT)-PCR was performed, using the Access RTPCR system (Promega, Madison, USA). Recreations were performed as follows: 48 8C for 45 min, 2 min at 94 8C, 45 cycles of 1 min at 94 8C, 30 s at 55 8C and 1 min at 68 8C, and 5 min at 68 8C. A signal was obtained from both MAP and SET RNasetreated nucleic acid extracts: this would have been due to the presence of genomic DNA. In contrast, when the DNasetreated nucleic acid extracts were analysed, a signal was obtained from SET samples, but not from MAP. This could indicate that MAP hsp70 transcripts were not abundantly present (perhaps attributable to the very slow growth rate of MAP) or that the gene had not been transcribed immediately prior to extraction. It might be argued that MAP mRNA was not recovered by the extraction procedure used; however,
Schuitema et al. [7] reported recovery of M. tuberculosis mRNA by the same procedure. The recovery of SET mRNA shows that the extraction procedure and the post-enzymatic treatment clean-up procedure were efficiently performed. NASBA amplification of DNA has been previously observed, with some human and viral targets [8], but the nature of the signal was not confirmed, i.e. was single or double-stranded DNA the origin? Deiman et al. [8] speculated that DNA amplification by NASBA was due to the target DNA being in excess in the reaction. However, in the study presented here, identical quantities of SET and MAP DNA were used, but the former did not elicit a signal at any time. Besides, Simpkins et al. [2] demonstrated, using SET nucleic acids, that NASBA selectively amplifies RNA in the presence of a DNA background. The amplification of DNA by NASBA has been demonstrated using primers directed against easily accessible DNA regions such as plasmid DNA, low-meltpoint sequences or single-stranded regions [8,9]. However, the target MAP sequence has a G –C ratio greater that 65%, and therefore, it was highly unlikely to have been denatured at the relatively low temperatures used in the NASBA reaction. Furthermore, the results were reproducible even when the initial 65 8C step was omitted from the NASBA reactions (data not shown). It was not single-stranded DNA regions which were being amplified, as by S1 nuclease treatment did not abolish the NASBA signal. Thus, we conclude that the origin of the NASBA signal in MAP was double-stranded DNA. This study shows, for the first time, NASBA amplification of bacterial genomic DNA. It is a salutary example to workers who are contemplating the use of NASBA for selective detection of RNA. After the unexpected but reproducible results of this study, the enzymatic tests should be applied in each case to conclusively demonstrate that the NASBA signal has indeed been generated only by RNA and not DNA.
Acknowledgements This work was supported by EU project ‘SACROHN’ N. QLK2-CT-2000-00928, and a grant from the National
D. Rodrı´guez-La`zaro et al. / Molecular and Cellular Probes 18 (2004) 251–253
Association for Crohn’s and Colitis. David Rodrı´guezLa´zaro was supported by a FEMS Research Fellowship. We thank John Hays, Milan Bartos and Ivo Pavlik for critical reading of the manuscript.
References [1] Klein PG, Kuneja VJ. Sensitive detection of viable Listeria monocytogenes by reverse trascription-PCR. Appl Environ Microbiol 1997;63:4441–8. [2] Simpkins SA, Chan AB, Hays J, Po¨pping B, Cook N. An RNA transcription-based amplification technique (NASBA) for the detection of viable Salmonella enterica. Lett Appl Microbiol 2000;30:75–9. [3] Koets AP, Rutten VP, de Boer M, Bakker D, Valentin-Weigand P, van Eden W. Differential changes in heat shock protein-, lipoarabinomannan-, and purified protein derivative-specific immunoglobulin G1 and G2 isotype responses during bovine Mycobacterium avium subsp. paratuberculosis infection. Infect Immun 2001;69:1492–8.
253
[4] van Gemen B, van Beuningen R, Nabble A, van Strijp D, Jurriaans S, Lens P, Kievits T. A one-tube quantitative HIV-1 RNA NASBA nucleic acid amplification assay using electrochemiluminescent (ECL) labelled probes. J Virol Meth 1994;49:157–67. [5] Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K, editors. Enzymatic manipulation of DNA and RNA. Current protocol in molecular biology, vol. 1. New York: Wiley; 1990. [6] Sambrook J, Russell D. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbour, NY: Cold Spring Harbour Laboratory; 2000. [7] Schuitema A, van Leewen J, van Aarle P, Sillekens P, Klaster P. Detection of Mycobacterium tuberculosis rRNA and mRNA using the Nuclisensw Basic Kit. J Microbiol Meth 1999;38:256. [8] Deiman B, van Aarle P, Sillekens P. Characteristics and applications of nucleic acid sequence-based amplification (NASBA). Mol Biotechnol 2002;20:163 –79. [9] Voisset C, Mandrand B, Paranhos-Baccala G. RNA amplification technique, NASBA, also amplifies homologous plasmid DNA in nondenaturing conditions. Biotechniques 2000;29:236–40.