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Novel strategy combining SYBR Green I with carbon nanotubes for highly sensitive detection of Salmonella typhimurium DNA
Enzyme and Microbial Technology 54 (2014) 15–19
Contents lists available at ScienceDirect
Enzyme and Microbial Technology journal homepage: www.elsevier.com/locate/emt
Novel strategy combining SYBR Green I with carbon nanotubes for highly sensitive detection of Salmonella typhimurium DNA Pingdao Mao a,1 , Yi Ning a,1 , Wenkai Li a,b , Zhihui Peng a , Yongzhe Chen a , Le Deng a,b,∗ a The Co-construction Laboratory of Microbial Molecular Biology of Province and Ministry of Science and Technology, College of Life Science, Hunan Normal University, Changsha, Hunan, People’s Republic of China b College of Life Science, Central South University, Changsha, Hunan, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 24 July 2012 Received in revised form 22 September 2013 Accepted 23 September 2013 Keywords: Salmonella typhimurium SYBR Green I SWNTs Nucleic acid detection
a b s t r a c t A simple, selective, sensitive and label-free fluorescent method for detecting trpS-harboring Salmonella typhimurium was developed in this study. This assay used the non-covalent interaction of single-stranded DNA (ssDNA) probes with SWNTs, since SWNTs can quench fluorescence. Fluorescence recovery (78% with 1.8 nM target DNA) was detected in the presence of target DNA as ssDNA probes detached from SWNTs hybridized with target DNA, and the resulting double-stranded DNA (dsDNA) intercalated with SYBR Green I (SG) dyes. The increasing fluorescence intensity reached 4.54-fold. In contrast, mismatched oligonucleotides (1- or 3-nt difference to the target DNA) did not contribute to significant fluorescent recovery, which demonstrated the specificity of the assay. The increasing fluorescence intensity increased 3.15-fold when purified PCR products containing complementary sequences of trpS gene were detected. These results confirmed the ability to use this assay for detecting real samples. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Detection of the serotype of Salmonella often requires extensive resources for developing countries. Traditionally, diagnosis and monitoring depended on assays—such as the Widal test and other serological tests like DOT enzyme immunoassay, dip stick assays, semiquantitative tube agglutination test, and blood culture test—that required antibodies that are specific to Salmonella enterica subs. [1,2]. These classical methods require high-quality O-grouping and H-typing antisera, and special reagents have low specificity and are time-consuming. An alternative is PCR-based systems, which improve sensitivity and specificity by targeting the Vi-antigen-encoding, the flagellin, and the pathogenicity island 2 genes [3–7]. Although these genes present differently in Salmonella serotypes, they share certain similar nucleotides [8]. The differences, in some conditions, may lead to miscellaneous PCR amplification products, which results in falsepositive results. However, this problem can be solved by isolating
Abbreviations: ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; SG, SYBR Green I; EB, ethidium bromide; SWNTs, single wall carbon nanotubes; nIR, near-infrared. ∗ Corresponding author at: The Co-construction Laboratory of Microbial Molecular Biology of Province and Ministry of Science and Technology, College of Life Science, Hunan Normal University, Changsha, Hunan 410081, People’s Republic of China. Tel.: +86 0731 88872927; fax: +86 0731 88883310. E-mail addresses: [email protected], [email protected] (L. Deng). 1 These authors contributed equally to this work. 0141-0229/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.enzmictec.2013.09.011
regions unique to S. typhimurium. For this study, a minor region of the S. enterica core locus trpS, which encodes tryptophanyl-tRNA synthetase, has been chosen by comparing the genomic analysis of the loci of core bacterial genetic determinants [7]. Nucleic acid detection has been important for a variety of analytic and diagnostic applications that used fluorescent dyes. Early attempts focused on the design of a single-stranded DNA (ssDNA) probe with a dye–quencher pair [9–12]. However, the design and synthesis of these probes have limited their application to common bio-sensors. The use of DNA-intercalating dyes is an alternative strategy. The monomeric cyanine dye, SYBR Green I (SG), has been applied to the measurement of nucleic acids since its introduction in the early 1990s [13]. Unlike ethidium bromide (EB), which is toxic and mutagenic, SG exhibits high affinity for double-stranded DNA (dsDNA). Its fluorescence enhanced significantly when bound to dsDNA at least 11-fold higher than binding to ssDNA [14]. Furthermore, the fluorescence signal was not greatly affected by assay conditions (pH, volume, and time) [15]. These benefits make it suitable for measuring low concentrations of DNA sample or DNA amplification products, even in the presence of other contaminants (RNA, ssDNA, nucleotides). Inorganic nanomaterials that possess special structural characteristics, such as nanocrystals, nanotubes, and nanowires, have shown great capacity for detecting molecules, including nucleic acids and proteins [16–18]. ssDNA has recently been demonstrated to interact non-covalently with single wall carbon nanotubes (SWNTs) by means of aromatic interactions between the nucleotide bases and the SWNT sidewalls resulting in a stable suspension
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P. Mao et al. / Enzyme and Microbial Technology 54 (2014) 15–19
Fig. 1. (A) Schematic representation of DNA fluorescent assay based on SWNTs. (I) The binding of probe DNA with SWNTs results in quenching of the dye fluorescence. (II) Target DNA binding with the probe DNA disturbs the absorption between dye and SWNTs, resulting in the restoration of the dye fluorescence. (B) Structures of SG, SWNTs, probe DNA, and target DNA.
[19,20]. SWNTs are also resistant to photo-bleaching [21] and fluorescence at near-infrared (nIR) wavelengths [22,23]. Furthermore, nanotubes have been successfully implanted in cells, allowing for potential in vivo applications [24]. Many nano-based bacterial detection systems have utilized the conductive/amplification properties of the nanotubes [25–28] and further research into PCR amplification without the use of PCR continues in an effort to eliminate the need for highly trained operators [29]. In this study, a novel assay was designed to detect trpSharboring S. typhimurium using fluorescence quenching and recovering (Fig. 1). Significant sensitivity and selectivity could be observed through the interactions between SGs, SWNTs, and DNA. SG fluorescence signal alone was weak but could be detected (485 nm absorption, 520 nm emission) once ssDNA was added into the solution since SGs bound to ssDNAs. With the addition of SWNTs, ssDNAs extracted from the SG–ssDNA complex and absorbed to the SWNTs, and thereby the fluorescence was quenched indirectly. When target DNA was then added, dsDNAs would quickly form and rebind with SGs, which resulted in a recovery of fluorescence that was much higher than that of SG–ssDNA. It was also demonstrated that the asymmetric PCR products containing complementary sequences of trpS gene derived from bacteria can be easily detected by this system. This strategic assay can provide a rapid and highly sensitive and specific method for detecting trpS-containing S. typhimurium and has great significance in pathogen detection, medical research, and clinical diagnosis and treatment. 2. Materials and methods 2.1. Materials SG was purchased from Fanbo Biochemical Company (Beijing, China). Carboxylmodified SWNTs were purchased from Chengdu Organic Chemicals Company (Chengdu, China). The S. typhimurium strain originated from Xiangya Hospital of Central South University (Changsha, China). The species-specific trps gene of S. typhimurium was obtained through Gene Bank (NC-003197). The ssDNA probe was prepared following a previously described method [7]. Bacteria strains were cultivated in a 100 ml flask with 50 ml of Luria Bertani (LB) medium (beef extract 1%,
yeast extract 0.5%, NaCl 1%, pH 7.4) at 37 ◦ C for 24 h, with shaking at 100 rpm. Purified DNA stocks of bacteria strains used in this study were prepared by the TIANamp Bacteria DNA Kit from Tiangen Biotech Company (Beijing, China). The purified PCR products DNA stocks were prepared by the Gel Extraction Kit from Dongsheng Biotech Company (Guangzhou, China). The anhydrous N,N-dimethylformamide (DMF), oligonucleotides (Table 1), and primers (Table 1) used in these experiments were synthesized by Sangon Biotechnology Company (Shanghai, China). Other reagents were purchased from Peng Cheng Company (Changsha, China). TE buffer (10 mM Tris (hydroxymethyl)-aminomethane (Tris), 1 mM ethylenediaminetetraacetic acid (EDTA), pH 7.5) was prepared with ultrapure Mill-Q water (electric resistivity 18 M cm−1 ). All the water used in this experiment was purified with the Millipore-Q system (Millipore Inc., America). 2.2. Instruments Fluorescence spectra were measured using a LS55 luminescence spectrometer (PerkinElmer [ADS16], UK) at 28 ◦ C. Fluorescence signals were recorded in the range 200–800 nm when solutions were excited at 485 nm. Excitation and emission spectrometer slits were set at 10.0 nm. The scan rate was 1500 nm s−1 . Purification was achieved using a centrifuge (Hettich Universal 32R [ADS17], UK). A UV spectrophotometer (Tianmei Techcomp, Shanghai, China) was used for absorbance detection of DNA at 260 nm or 280 nm. PCR was performed with an Authorized Thermal Cycler from Eppendorf. 2.3. SWNTs purification SWNTs were first sonicated for 8 h in an ice bath before being filtered through a 220 nm Millipore membrane with the aid of a pump. They were then thoroughly
Table 1 Oligonucleotide sequences of probe,a target, mismatch DNAs,b and PCR primers.c Oligonucleotide
Sequence (5 –3 )
Probe (S. typhimurium) Target One base mismatch (S. paratyphi A) Three bases mismatch (S. typhi) Forward primer Reverse primer
CGTGATCGGCTTGCTGG CCAGCAAGCCGATCACG CCAGCAGGCCGATCACG CCAACAGGCCGATTACG GCTGGAGCCGACAAAGA CCGATTTCGGATCTTCC
a Note that nucleotide sequence selected for probe is the 615–631st nucleotide sequence of trps gene. b The underlined bases indicate the mismatch base. c Primers used in the asymmetric PCR.
P. Mao et al. / Enzyme and Microbial Technology 54 (2014) 15–19
Fig. 2. The influences of different concentrations of SWNTs on the detection efficiency. (1) SG (300 nM) + probe (1.8 nM); (2) SG (300 nM) + probe (1.8 nM) + SWNTs (10 g/ml); (3) SG (300 nM) + probe (1.8 nM) + SWNTs (20 g/ml); (4) SG (300 nM) + probe (1.8 nM) + SWNTs (40 g/ml); (5) SG (300 nM) + probe (1.8 nM) + SWNTs (50 g/ml); (control) SG (300 nM). F is SG fluorescence intensity in different conditions. F0 and F1 are SG fluorescence intensities in control with only SG and in SG–probe solution without SWNTs, respectively. Error bars SD (n = 3).
washed with ultrapure water to obtain a neutral state and dried overnight in vacuum at 60 ◦ C. 2.4. Detection of trpS gene, mismatched DNA, and asymmetric PCR products of bacteria The purified SWNTs were sonicated in DMF for 5 h to form a homogeneous black solution and then stored for use. The SG working solution was obtained by diluting the stock solution to a concentration of 300 nM with 10 mM TE buffer (10 mM Tris (hydroxymethyl)-aminomethane (Tris), 1 mM ethylenediaminetetraacetic acid (EDTA), pH 7.5). For the trpS gene assay, appropriate concentrations of probe stock (1 mM), target stock (1 mM), and SWNTs solutions (100 g/ml) were mixed into the working solution and incubated for 20 min at 37 ◦ C before the fluorescence intensity was measured. Fluorescence intensity was recorded at 520 nm with an excitation wavelength of 485 nm. Two mismatched DNAs were tested as controls using the same condition. The complete bacterial DNA was obtained using TIANamp Bacteria DNA Kit. Asymmetric PCR products containing complementary sequences of trpS gene were tested, and the primers used in the experiments were shown in Table 1. The concentrations of mismatched DNA (1.5 nM) and PCR products (1.5 nM) were the same as that of the target DNA (1.5 nM).
3. Results and discussion 3.1. Fluorescence quenching by SWNTs Different SWNT concentrations and their ability to quench the fluorescence of 300 nM SG with 1.8 nM ssDNA were investigated. Fig. 2 shows the fluorescence emission intensities of the different SWNT concentrations with the SG–ssDNA complex in 10 mM TE buffer when excited at the maximal absorption (485 nm) and emission wavelengths (520 nm) of SG. Fluorescence intensities decreased proportionally to increasing SWNTs. More than 61.5% of fluorescence was quenched by 20 g/ml SWNTs in the solution (Fig. 2). Complete quenching required larger amounts of SWNTs, but fluorescence was then difficult to recover once the target was added. This indicated that SWNTs could efficiently adsorb to the probe, and thereby, indirectly quench the fluorescence. 3.2. Fluorescence recovery by target sequence Fig. 3 shows the fluorescence emission spectra of SG in different conditions. The fluorescence of 300 nM SG alone was very weak, nearly equal to the buffer. When 1.8 nM ssDNA probe was added, the fluorescence increased because of the high affinity between SGs and ssDNAs. Further increases in fluorescence were seen when 1.8 nM target DNA was added and SG–dsDNA complexes formed (Fig. 3A). When 20 g/ml SWNTs was added to SGs alone, fluorescence was nearly equal to the buffer. The fluorescence remained unchanged when 1.5 nM ssDNA probe was added as SWNTs adsorbed the ssDNA. When 1.5 nM of target DNA was added,
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however, the fluorescence emission increased as the ssDNA desorbed from the SWNTs, annealed to the target DNA to form dsDNA, and subsequently bound to the SG (Fig. 3B). Fluorescence recovery in the presence of SWNTs reached 78% of the system compared with no SWNTs. The increasing fluorescence intensity (F2 − F0 )/(F1 − F0 ) of the SG system was found to be smaller than that of the SG–SWNTs system, where F0 , F1 , and F2 were the SG intensities at 520 nm in the SG, ssDNA–SG, and dsDNA–SG systems, respectively. The (F2 − F0 )/(F1 − F0 ) of the SG system was 2.51-fold and the SG–SWNTs system 4.54-fold with the same concentration of dsDNAs. These results clearly demonstrated that SG–SWNTs could be used as a sensitive approach for DNA fluorescent detection. 3.3. Studies on mechanism To determine whether SG bound with the ssDNA probe in solution or on the surface of SWNTs, two tubes (tube 1 and tube 2) containing 1.8 nM ssDNA probe, 300 nM SG, and 20 g/ml SWNTs were prepared and centrifuged. The supernatant of tube 1 was transferred to a clean tube 3. TE buffer was added to the precipitate in tube 1. The fluorescence intensities of both solutions in tube 1 and tube 3 were measured with and without the addition of the target DNAs (1.8 nM). SG alone in buffer was used as the control. As shown in Fig. 4, the fluorescence intensities of the supernatant (tube 3, b) and precipitate (tube 1, c) without target DNA, and the precipitate with target DNA (tube 1, e) was about 50 a.u., which was similar to the blank containing only SG (a). The intensity of the supernatant with target DNA (tube 3, d) was 97.70 a.u., which was about twice the control, and tube 2 containing the supernatant (f) reached 141.70 (a.u.) after addition of target DNA. These results were similar to Fig. 3A and B. After centrifugation, the SWNTs and ssDNA probe were in the precipitate, while the SG was in the supernatant. The fluorescence of both the supernatant (b) and precipitate (c) without target DNA would be similar to the blank containing only SG (a). The precipitate with target DNA (e) would also mirror the blank (a) as SG remained in the supernatant. With the ssDNA probe in the precipitate, the fluorescence of the supernatant with target DNA (d) and SG would be lower than that of the solution contained dsDNA probe–target and SG (f). These results revealed that SG could bind with ssDNA probe in solution but not on the surface of SWNTs. SWNTs could selectively bind to the ssDNA probe and indirectly quenched the SG. 3.4. Specificity of mismatch DNA detection The main disadvantage of using the intercalating dye SG for detecting DNA is its lack of specificity. Binding SWNTs with the ssDNA probe can improve the selectivity of DNA hybridization. As Salmonella serotypes often possess closely related sequences with high homology (1- or 3-nt difference), the serotypes are useful for evaluating the sequence specificity of this approach. Complementary and non-complementary DNA with one and three mismatched bases were used to assess the ability of this system to detect target DNA (Fig. 5). Solutions containing SG and ssDNA probe–SWNTs complexes were prepared, and 1.8 nM target DNA and 1.8 nM mismatched DNA were added to separate solutions. SG alone in buffer was used as the control. All solutions were excited at the maximal absorption wavelength (485 nm), and the emission wavelength (520 nm) was recorded. The increasing fluorescence intensities (F2 − F0 )/(F1 − F0 ) of target DNA sequences were much higher than that of mismatched sequences, where F0 , F1 , and F2 were the SG intensities at 520 nm of the SG–SWNTs, probe–SG–SWNTs, and hybridization solutions, respectively. The (F2 − F0 )/(F1 − F0 ) of target–probe hybridization was 4.54-fold, while one and three mismatched sequence hybridizations were
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P. Mao et al. / Enzyme and Microbial Technology 54 (2014) 15–19
Fig. 3. Fluorescence emission spectra of SG in the Tris–EDTA buffer solution in the absence (A) and the presence (B) of SWNTs. All spectra were obtained under different conditions. From bottom to top: buffer, SG (300 nM), SG (300 nM) + probe (1.8 nM), and SG (300 nM) + probe (1.8 nM) + target (1.8 nM), respectively. ex = 485 nm.
Fig. 4. Studies on the mechanism of this system. (a) Buffer, (b) the supernate after centrifugation, (c) the precipitate after centrifugation, (d) target + the supernate, (e) target + the precipitate, (f) target + reaction solution without centrifugation. Error bars SD (n = 3).
significantly lower at 2.08-fold and 1.46-fold, respectively. These results clearly demonstrated that this approach could be used as a sensitive detection method for DNA.
3.5. Sensitive detection of the trpS-containing S. typhimurium
Fig. 5. The different recovery values of PL intensity of SG with the addition of target DNA and mismatch DNAs in the presence of SWNTs. From bottom to top: SG (300 nM), SG (300 nM) + probe (1.8 nM) + SWNTs (20 g/ml), SG (300 nM) + probe (1.8 nM) + SWNTs (20 g/ml) + three bases mismatch (1.8 nM), SG (300 nM) + probe (1.8 nM) + SWNTs (20 g/ml) + one base mismatch (1.8 nM), SG (300 nM) + probe (1.8 nM) + SWNTs (20 g/ml) + target (1.8 nM), respectively. ex = 485 nm.
Bacteria carrying the trpS gene were used to assess the sensitivity of the system. The total bacterial DNA was extracted using TIANamp Bacteria DNA Kit and the complementary sequence of the trpS gene was obtained using asymmetric PCR and carefully chosen primers. The PCR products were purified by Gel Extraction Kit. UV absorbance of the purified PCR products at 260/280 nm was measured to determine their concentration and purity. A solution containing SG and probe–SWNTs complex was obtained, and 1.8 nM of the purified PCR products of the extracted bacterial DNAs were added. SG alone in buffer was used as the control. All solutions were excited at the maximal absorption wavelength (485 nm), and the emission wavelength (520 nm) was recorded. The increasing fluorescence intensity (F2 − F0 )/(F1 − F0 ) was 3.15-fold (Fig. 6), which was still 1.07-fold and 1.69-fold higher than those of the mismatched oligonucleotides (Fig. 5). Because the PCR sequences containing the target gene were longer than the target gene itself, 17 dsDNA base pairs were left free with SG, and the remaining 63 bases to SWNTS. This resulted in an increasing fluorescence intensity that was lower than that of the target–probe
P. Mao et al. / Enzyme and Microbial Technology 54 (2014) 15–19
Fig. 6. Fluorescence emission spectra of SG in the detection of the PCR products of S. typhimurium in the presence of SWNTs. From bottom to top: SG (300 nM), SG (300 nM) + probe (1.8 nM) + SWNTs (20 g/ml), SG (300 nM) + probe (1.8 nM) + SWNTs (20 g/ml) + PCR products (1.8 nM), respectively. ex = 485 nm.
DNA complexes (Fig. 5). These results confirmed that this approach had promise in cellular assays because it could sensitively and specifically detect trpS-harboring S. typhimurium. 4. Conclusions We successfully developed a simple, selective, sensitive and label-free fluorescent method for detecting trpS-harboring S. typhimurium. This approach was based on the non-covalent stacking interaction of ssDNA probe with SWNTs. SG was used to avoid labeling the probe DNA or the target DNA, and it remained superior to DNA probes with dye–quencher pairs. With our approach, the fluorescence recovery of the trpS gene with 1.8 nM target DNA was 78%. The increasing fluorescence intensity reached 4.54-fold. Sequences with high homology (1- or 3-nt difference) could also be distinguished. Prominent fluorescence signals and a 3.15-fold increase in fluorescence intensity were obtained for trpS-containing S. typhimurium. These results confirmed that this system could simply, quickly, selectively, and sensitively detect trpS genes and trpS-containing S. typhimurium. Because these bacteria are becoming a global problem, this method can be used to identify and detect trps-harboring S. typhimurium. It can also be extended to other DNA, virus or protein sensing applications. Its simplicity, sensitivity and specificity will make it a promising candidate for applications in biomolecular interaction studies and their relative detections. Acknowledgements We would like to thank the National Natural Science Foundation (81271660), Ministry of Education for the Doctoral Program of Higher Education (20114306110006), and A Project Supported by Scientific Research Fund of Hunan Provincial Education Department (12K032) for financial support. References [1] Olsen SJ, Pruckler J, Bibb W, Nguyen TM, Tran MT, Nguyen TM, et al. Evaluation of rapid diagnostic tests for typhoid fever. J Clin Microbiol 2004;42:1885–9.
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Contents lists available at ScienceDirect
Enzyme and Microbial Technology journal homepage: www.elsevier.com/locate/emt
Novel strategy combining SYBR Green I with carbon nanotubes for highly sensitive detection of Salmonella typhimurium DNA Pingdao Mao a,1 , Yi Ning a,1 , Wenkai Li a,b , Zhihui Peng a , Yongzhe Chen a , Le Deng a,b,∗ a The Co-construction Laboratory of Microbial Molecular Biology of Province and Ministry of Science and Technology, College of Life Science, Hunan Normal University, Changsha, Hunan, People’s Republic of China b College of Life Science, Central South University, Changsha, Hunan, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 24 July 2012 Received in revised form 22 September 2013 Accepted 23 September 2013 Keywords: Salmonella typhimurium SYBR Green I SWNTs Nucleic acid detection
a b s t r a c t A simple, selective, sensitive and label-free fluorescent method for detecting trpS-harboring Salmonella typhimurium was developed in this study. This assay used the non-covalent interaction of single-stranded DNA (ssDNA) probes with SWNTs, since SWNTs can quench fluorescence. Fluorescence recovery (78% with 1.8 nM target DNA) was detected in the presence of target DNA as ssDNA probes detached from SWNTs hybridized with target DNA, and the resulting double-stranded DNA (dsDNA) intercalated with SYBR Green I (SG) dyes. The increasing fluorescence intensity reached 4.54-fold. In contrast, mismatched oligonucleotides (1- or 3-nt difference to the target DNA) did not contribute to significant fluorescent recovery, which demonstrated the specificity of the assay. The increasing fluorescence intensity increased 3.15-fold when purified PCR products containing complementary sequences of trpS gene were detected. These results confirmed the ability to use this assay for detecting real samples. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Detection of the serotype of Salmonella often requires extensive resources for developing countries. Traditionally, diagnosis and monitoring depended on assays—such as the Widal test and other serological tests like DOT enzyme immunoassay, dip stick assays, semiquantitative tube agglutination test, and blood culture test—that required antibodies that are specific to Salmonella enterica subs. [1,2]. These classical methods require high-quality O-grouping and H-typing antisera, and special reagents have low specificity and are time-consuming. An alternative is PCR-based systems, which improve sensitivity and specificity by targeting the Vi-antigen-encoding, the flagellin, and the pathogenicity island 2 genes [3–7]. Although these genes present differently in Salmonella serotypes, they share certain similar nucleotides [8]. The differences, in some conditions, may lead to miscellaneous PCR amplification products, which results in falsepositive results. However, this problem can be solved by isolating
Abbreviations: ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; SG, SYBR Green I; EB, ethidium bromide; SWNTs, single wall carbon nanotubes; nIR, near-infrared. ∗ Corresponding author at: The Co-construction Laboratory of Microbial Molecular Biology of Province and Ministry of Science and Technology, College of Life Science, Hunan Normal University, Changsha, Hunan 410081, People’s Republic of China. Tel.: +86 0731 88872927; fax: +86 0731 88883310. E-mail addresses: [email protected], [email protected] (L. Deng). 1 These authors contributed equally to this work. 0141-0229/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.enzmictec.2013.09.011
regions unique to S. typhimurium. For this study, a minor region of the S. enterica core locus trpS, which encodes tryptophanyl-tRNA synthetase, has been chosen by comparing the genomic analysis of the loci of core bacterial genetic determinants [7]. Nucleic acid detection has been important for a variety of analytic and diagnostic applications that used fluorescent dyes. Early attempts focused on the design of a single-stranded DNA (ssDNA) probe with a dye–quencher pair [9–12]. However, the design and synthesis of these probes have limited their application to common bio-sensors. The use of DNA-intercalating dyes is an alternative strategy. The monomeric cyanine dye, SYBR Green I (SG), has been applied to the measurement of nucleic acids since its introduction in the early 1990s [13]. Unlike ethidium bromide (EB), which is toxic and mutagenic, SG exhibits high affinity for double-stranded DNA (dsDNA). Its fluorescence enhanced significantly when bound to dsDNA at least 11-fold higher than binding to ssDNA [14]. Furthermore, the fluorescence signal was not greatly affected by assay conditions (pH, volume, and time) [15]. These benefits make it suitable for measuring low concentrations of DNA sample or DNA amplification products, even in the presence of other contaminants (RNA, ssDNA, nucleotides). Inorganic nanomaterials that possess special structural characteristics, such as nanocrystals, nanotubes, and nanowires, have shown great capacity for detecting molecules, including nucleic acids and proteins [16–18]. ssDNA has recently been demonstrated to interact non-covalently with single wall carbon nanotubes (SWNTs) by means of aromatic interactions between the nucleotide bases and the SWNT sidewalls resulting in a stable suspension
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Fig. 1. (A) Schematic representation of DNA fluorescent assay based on SWNTs. (I) The binding of probe DNA with SWNTs results in quenching of the dye fluorescence. (II) Target DNA binding with the probe DNA disturbs the absorption between dye and SWNTs, resulting in the restoration of the dye fluorescence. (B) Structures of SG, SWNTs, probe DNA, and target DNA.
[19,20]. SWNTs are also resistant to photo-bleaching [21] and fluorescence at near-infrared (nIR) wavelengths [22,23]. Furthermore, nanotubes have been successfully implanted in cells, allowing for potential in vivo applications [24]. Many nano-based bacterial detection systems have utilized the conductive/amplification properties of the nanotubes [25–28] and further research into PCR amplification without the use of PCR continues in an effort to eliminate the need for highly trained operators [29]. In this study, a novel assay was designed to detect trpSharboring S. typhimurium using fluorescence quenching and recovering (Fig. 1). Significant sensitivity and selectivity could be observed through the interactions between SGs, SWNTs, and DNA. SG fluorescence signal alone was weak but could be detected (485 nm absorption, 520 nm emission) once ssDNA was added into the solution since SGs bound to ssDNAs. With the addition of SWNTs, ssDNAs extracted from the SG–ssDNA complex and absorbed to the SWNTs, and thereby the fluorescence was quenched indirectly. When target DNA was then added, dsDNAs would quickly form and rebind with SGs, which resulted in a recovery of fluorescence that was much higher than that of SG–ssDNA. It was also demonstrated that the asymmetric PCR products containing complementary sequences of trpS gene derived from bacteria can be easily detected by this system. This strategic assay can provide a rapid and highly sensitive and specific method for detecting trpS-containing S. typhimurium and has great significance in pathogen detection, medical research, and clinical diagnosis and treatment. 2. Materials and methods 2.1. Materials SG was purchased from Fanbo Biochemical Company (Beijing, China). Carboxylmodified SWNTs were purchased from Chengdu Organic Chemicals Company (Chengdu, China). The S. typhimurium strain originated from Xiangya Hospital of Central South University (Changsha, China). The species-specific trps gene of S. typhimurium was obtained through Gene Bank (NC-003197). The ssDNA probe was prepared following a previously described method [7]. Bacteria strains were cultivated in a 100 ml flask with 50 ml of Luria Bertani (LB) medium (beef extract 1%,
yeast extract 0.5%, NaCl 1%, pH 7.4) at 37 ◦ C for 24 h, with shaking at 100 rpm. Purified DNA stocks of bacteria strains used in this study were prepared by the TIANamp Bacteria DNA Kit from Tiangen Biotech Company (Beijing, China). The purified PCR products DNA stocks were prepared by the Gel Extraction Kit from Dongsheng Biotech Company (Guangzhou, China). The anhydrous N,N-dimethylformamide (DMF), oligonucleotides (Table 1), and primers (Table 1) used in these experiments were synthesized by Sangon Biotechnology Company (Shanghai, China). Other reagents were purchased from Peng Cheng Company (Changsha, China). TE buffer (10 mM Tris (hydroxymethyl)-aminomethane (Tris), 1 mM ethylenediaminetetraacetic acid (EDTA), pH 7.5) was prepared with ultrapure Mill-Q water (electric resistivity 18 M cm−1 ). All the water used in this experiment was purified with the Millipore-Q system (Millipore Inc., America). 2.2. Instruments Fluorescence spectra were measured using a LS55 luminescence spectrometer (PerkinElmer [ADS16], UK) at 28 ◦ C. Fluorescence signals were recorded in the range 200–800 nm when solutions were excited at 485 nm. Excitation and emission spectrometer slits were set at 10.0 nm. The scan rate was 1500 nm s−1 . Purification was achieved using a centrifuge (Hettich Universal 32R [ADS17], UK). A UV spectrophotometer (Tianmei Techcomp, Shanghai, China) was used for absorbance detection of DNA at 260 nm or 280 nm. PCR was performed with an Authorized Thermal Cycler from Eppendorf. 2.3. SWNTs purification SWNTs were first sonicated for 8 h in an ice bath before being filtered through a 220 nm Millipore membrane with the aid of a pump. They were then thoroughly
Table 1 Oligonucleotide sequences of probe,a target, mismatch DNAs,b and PCR primers.c Oligonucleotide
Sequence (5 –3 )
Probe (S. typhimurium) Target One base mismatch (S. paratyphi A) Three bases mismatch (S. typhi) Forward primer Reverse primer
CGTGATCGGCTTGCTGG CCAGCAAGCCGATCACG CCAGCAGGCCGATCACG CCAACAGGCCGATTACG GCTGGAGCCGACAAAGA CCGATTTCGGATCTTCC
a Note that nucleotide sequence selected for probe is the 615–631st nucleotide sequence of trps gene. b The underlined bases indicate the mismatch base. c Primers used in the asymmetric PCR.
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Fig. 2. The influences of different concentrations of SWNTs on the detection efficiency. (1) SG (300 nM) + probe (1.8 nM); (2) SG (300 nM) + probe (1.8 nM) + SWNTs (10 g/ml); (3) SG (300 nM) + probe (1.8 nM) + SWNTs (20 g/ml); (4) SG (300 nM) + probe (1.8 nM) + SWNTs (40 g/ml); (5) SG (300 nM) + probe (1.8 nM) + SWNTs (50 g/ml); (control) SG (300 nM). F is SG fluorescence intensity in different conditions. F0 and F1 are SG fluorescence intensities in control with only SG and in SG–probe solution without SWNTs, respectively. Error bars SD (n = 3).
washed with ultrapure water to obtain a neutral state and dried overnight in vacuum at 60 ◦ C. 2.4. Detection of trpS gene, mismatched DNA, and asymmetric PCR products of bacteria The purified SWNTs were sonicated in DMF for 5 h to form a homogeneous black solution and then stored for use. The SG working solution was obtained by diluting the stock solution to a concentration of 300 nM with 10 mM TE buffer (10 mM Tris (hydroxymethyl)-aminomethane (Tris), 1 mM ethylenediaminetetraacetic acid (EDTA), pH 7.5). For the trpS gene assay, appropriate concentrations of probe stock (1 mM), target stock (1 mM), and SWNTs solutions (100 g/ml) were mixed into the working solution and incubated for 20 min at 37 ◦ C before the fluorescence intensity was measured. Fluorescence intensity was recorded at 520 nm with an excitation wavelength of 485 nm. Two mismatched DNAs were tested as controls using the same condition. The complete bacterial DNA was obtained using TIANamp Bacteria DNA Kit. Asymmetric PCR products containing complementary sequences of trpS gene were tested, and the primers used in the experiments were shown in Table 1. The concentrations of mismatched DNA (1.5 nM) and PCR products (1.5 nM) were the same as that of the target DNA (1.5 nM).
3. Results and discussion 3.1. Fluorescence quenching by SWNTs Different SWNT concentrations and their ability to quench the fluorescence of 300 nM SG with 1.8 nM ssDNA were investigated. Fig. 2 shows the fluorescence emission intensities of the different SWNT concentrations with the SG–ssDNA complex in 10 mM TE buffer when excited at the maximal absorption (485 nm) and emission wavelengths (520 nm) of SG. Fluorescence intensities decreased proportionally to increasing SWNTs. More than 61.5% of fluorescence was quenched by 20 g/ml SWNTs in the solution (Fig. 2). Complete quenching required larger amounts of SWNTs, but fluorescence was then difficult to recover once the target was added. This indicated that SWNTs could efficiently adsorb to the probe, and thereby, indirectly quench the fluorescence. 3.2. Fluorescence recovery by target sequence Fig. 3 shows the fluorescence emission spectra of SG in different conditions. The fluorescence of 300 nM SG alone was very weak, nearly equal to the buffer. When 1.8 nM ssDNA probe was added, the fluorescence increased because of the high affinity between SGs and ssDNAs. Further increases in fluorescence were seen when 1.8 nM target DNA was added and SG–dsDNA complexes formed (Fig. 3A). When 20 g/ml SWNTs was added to SGs alone, fluorescence was nearly equal to the buffer. The fluorescence remained unchanged when 1.5 nM ssDNA probe was added as SWNTs adsorbed the ssDNA. When 1.5 nM of target DNA was added,
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however, the fluorescence emission increased as the ssDNA desorbed from the SWNTs, annealed to the target DNA to form dsDNA, and subsequently bound to the SG (Fig. 3B). Fluorescence recovery in the presence of SWNTs reached 78% of the system compared with no SWNTs. The increasing fluorescence intensity (F2 − F0 )/(F1 − F0 ) of the SG system was found to be smaller than that of the SG–SWNTs system, where F0 , F1 , and F2 were the SG intensities at 520 nm in the SG, ssDNA–SG, and dsDNA–SG systems, respectively. The (F2 − F0 )/(F1 − F0 ) of the SG system was 2.51-fold and the SG–SWNTs system 4.54-fold with the same concentration of dsDNAs. These results clearly demonstrated that SG–SWNTs could be used as a sensitive approach for DNA fluorescent detection. 3.3. Studies on mechanism To determine whether SG bound with the ssDNA probe in solution or on the surface of SWNTs, two tubes (tube 1 and tube 2) containing 1.8 nM ssDNA probe, 300 nM SG, and 20 g/ml SWNTs were prepared and centrifuged. The supernatant of tube 1 was transferred to a clean tube 3. TE buffer was added to the precipitate in tube 1. The fluorescence intensities of both solutions in tube 1 and tube 3 were measured with and without the addition of the target DNAs (1.8 nM). SG alone in buffer was used as the control. As shown in Fig. 4, the fluorescence intensities of the supernatant (tube 3, b) and precipitate (tube 1, c) without target DNA, and the precipitate with target DNA (tube 1, e) was about 50 a.u., which was similar to the blank containing only SG (a). The intensity of the supernatant with target DNA (tube 3, d) was 97.70 a.u., which was about twice the control, and tube 2 containing the supernatant (f) reached 141.70 (a.u.) after addition of target DNA. These results were similar to Fig. 3A and B. After centrifugation, the SWNTs and ssDNA probe were in the precipitate, while the SG was in the supernatant. The fluorescence of both the supernatant (b) and precipitate (c) without target DNA would be similar to the blank containing only SG (a). The precipitate with target DNA (e) would also mirror the blank (a) as SG remained in the supernatant. With the ssDNA probe in the precipitate, the fluorescence of the supernatant with target DNA (d) and SG would be lower than that of the solution contained dsDNA probe–target and SG (f). These results revealed that SG could bind with ssDNA probe in solution but not on the surface of SWNTs. SWNTs could selectively bind to the ssDNA probe and indirectly quenched the SG. 3.4. Specificity of mismatch DNA detection The main disadvantage of using the intercalating dye SG for detecting DNA is its lack of specificity. Binding SWNTs with the ssDNA probe can improve the selectivity of DNA hybridization. As Salmonella serotypes often possess closely related sequences with high homology (1- or 3-nt difference), the serotypes are useful for evaluating the sequence specificity of this approach. Complementary and non-complementary DNA with one and three mismatched bases were used to assess the ability of this system to detect target DNA (Fig. 5). Solutions containing SG and ssDNA probe–SWNTs complexes were prepared, and 1.8 nM target DNA and 1.8 nM mismatched DNA were added to separate solutions. SG alone in buffer was used as the control. All solutions were excited at the maximal absorption wavelength (485 nm), and the emission wavelength (520 nm) was recorded. The increasing fluorescence intensities (F2 − F0 )/(F1 − F0 ) of target DNA sequences were much higher than that of mismatched sequences, where F0 , F1 , and F2 were the SG intensities at 520 nm of the SG–SWNTs, probe–SG–SWNTs, and hybridization solutions, respectively. The (F2 − F0 )/(F1 − F0 ) of target–probe hybridization was 4.54-fold, while one and three mismatched sequence hybridizations were
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Fig. 3. Fluorescence emission spectra of SG in the Tris–EDTA buffer solution in the absence (A) and the presence (B) of SWNTs. All spectra were obtained under different conditions. From bottom to top: buffer, SG (300 nM), SG (300 nM) + probe (1.8 nM), and SG (300 nM) + probe (1.8 nM) + target (1.8 nM), respectively. ex = 485 nm.
Fig. 4. Studies on the mechanism of this system. (a) Buffer, (b) the supernate after centrifugation, (c) the precipitate after centrifugation, (d) target + the supernate, (e) target + the precipitate, (f) target + reaction solution without centrifugation. Error bars SD (n = 3).
significantly lower at 2.08-fold and 1.46-fold, respectively. These results clearly demonstrated that this approach could be used as a sensitive detection method for DNA.
3.5. Sensitive detection of the trpS-containing S. typhimurium
Fig. 5. The different recovery values of PL intensity of SG with the addition of target DNA and mismatch DNAs in the presence of SWNTs. From bottom to top: SG (300 nM), SG (300 nM) + probe (1.8 nM) + SWNTs (20 g/ml), SG (300 nM) + probe (1.8 nM) + SWNTs (20 g/ml) + three bases mismatch (1.8 nM), SG (300 nM) + probe (1.8 nM) + SWNTs (20 g/ml) + one base mismatch (1.8 nM), SG (300 nM) + probe (1.8 nM) + SWNTs (20 g/ml) + target (1.8 nM), respectively. ex = 485 nm.
Bacteria carrying the trpS gene were used to assess the sensitivity of the system. The total bacterial DNA was extracted using TIANamp Bacteria DNA Kit and the complementary sequence of the trpS gene was obtained using asymmetric PCR and carefully chosen primers. The PCR products were purified by Gel Extraction Kit. UV absorbance of the purified PCR products at 260/280 nm was measured to determine their concentration and purity. A solution containing SG and probe–SWNTs complex was obtained, and 1.8 nM of the purified PCR products of the extracted bacterial DNAs were added. SG alone in buffer was used as the control. All solutions were excited at the maximal absorption wavelength (485 nm), and the emission wavelength (520 nm) was recorded. The increasing fluorescence intensity (F2 − F0 )/(F1 − F0 ) was 3.15-fold (Fig. 6), which was still 1.07-fold and 1.69-fold higher than those of the mismatched oligonucleotides (Fig. 5). Because the PCR sequences containing the target gene were longer than the target gene itself, 17 dsDNA base pairs were left free with SG, and the remaining 63 bases to SWNTS. This resulted in an increasing fluorescence intensity that was lower than that of the target–probe
P. Mao et al. / Enzyme and Microbial Technology 54 (2014) 15–19
Fig. 6. Fluorescence emission spectra of SG in the detection of the PCR products of S. typhimurium in the presence of SWNTs. From bottom to top: SG (300 nM), SG (300 nM) + probe (1.8 nM) + SWNTs (20 g/ml), SG (300 nM) + probe (1.8 nM) + SWNTs (20 g/ml) + PCR products (1.8 nM), respectively. ex = 485 nm.
DNA complexes (Fig. 5). These results confirmed that this approach had promise in cellular assays because it could sensitively and specifically detect trpS-harboring S. typhimurium. 4. Conclusions We successfully developed a simple, selective, sensitive and label-free fluorescent method for detecting trpS-harboring S. typhimurium. This approach was based on the non-covalent stacking interaction of ssDNA probe with SWNTs. SG was used to avoid labeling the probe DNA or the target DNA, and it remained superior to DNA probes with dye–quencher pairs. With our approach, the fluorescence recovery of the trpS gene with 1.8 nM target DNA was 78%. The increasing fluorescence intensity reached 4.54-fold. Sequences with high homology (1- or 3-nt difference) could also be distinguished. Prominent fluorescence signals and a 3.15-fold increase in fluorescence intensity were obtained for trpS-containing S. typhimurium. These results confirmed that this system could simply, quickly, selectively, and sensitively detect trpS genes and trpS-containing S. typhimurium. Because these bacteria are becoming a global problem, this method can be used to identify and detect trps-harboring S. typhimurium. It can also be extended to other DNA, virus or protein sensing applications. Its simplicity, sensitivity and specificity will make it a promising candidate for applications in biomolecular interaction studies and their relative detections. Acknowledgements We would like to thank the National Natural Science Foundation (81271660), Ministry of Education for the Doctoral Program of Higher Education (20114306110006), and A Project Supported by Scientific Research Fund of Hunan Provincial Education Department (12K032) for financial support. References [1] Olsen SJ, Pruckler J, Bibb W, Nguyen TM, Tran MT, Nguyen TM, et al. Evaluation of rapid diagnostic tests for typhoid fever. J Clin Microbiol 2004;42:1885–9.
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