Luciferase-Zinc-Finger System for the Rapid Detection of Pathogenic

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Luciferase-Zinc-Finger System for the Rapid Detection of Pathogenic Bacteria Chu Shi,†,∥ Qing Xu,‡,∥ Yue Ge,† Ling Jiang,*,§ and He Huang*,‡ †

College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 30, Puzhu Road, Nanjing 211800, China State Key Laboratory of Material-Oriented Chemical Engineering, School of Pharmaceutical Sciences, Nanjing Tech University, No. 30 Puzhu Road, Nanjing 211800, China § College of Food Science and Light Industry, Nanjing Tech University, No. 30, Puzhu Road, Nanjing 211800, China ‡

ABSTRACT: Rapid and reliable detection of pathogenic bacteria is crucial for food safety control. Here, we present a novel luciferase-zinc finger system for the detection of pathogens that offers rapid and specific profiling. The system, which uses a zincfinger protein domain to probe zinc finger recognition sites, was designed to bind the amplified conserved regions of 16S rDNA, and the obtained products were detected using a modified luciferase. The luciferase-zinc finger system not only maintained luciferase activity but also allowed the specific detection of different bacterial species, with a sensitivity as low as 10 copies and a linear range from 10 to 104 copies per microliter of the specific PCR product. Moreover, the system is robust and rapid, enabling the simultaneous detection of 6 species of bacteria in artificially contaminated samples with excellent accuracy. Thus, we envision that our luciferase-zinc finger system will have far-reaching applications. KEYWORDS: luciferase, zinc-finger protein, pathogenic bacteria, detection

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INTRODUCTION The detection of pathogenic bacteria is crucial for disease prevention and identification of the sources of infection in the food and the environment.1,2 Until now, many molecular identification methods have been developed, and technologies based on polymerase chain reaction (PCR) are considered to be the most promising.3−7 Generally, the amplified PCR products can be confirmed by gel electrophoresis. However, gel electrophoresis is not sensitive and specific enough for some applications. To reduce the impact of these limitations, DNA probe hybridization is generally performed.8 However, DNA probe hybridization is based on single-stranded DNA (ssDNA), and the process requires the generation of single-stranded material from the original double-stranded DNA (dsDNA) to hybridize the DNA probe with the target sequence, which is complicated and inefficient.9 Thus, the direct and specific detection of dsDNA might be a more promising approach for the specific detection of pathogens. Zinc-finger proteins (ZF) are the most common DNA-binding proteins in mammals and as such can recognize and bind to specific dsDNA sequences.10−12 With recent advances in dsDNA detection, zinc-finger proteins, which represent one of the most compact DNA-recognition motifs in nature, are being developed as an alternative to the transcription activator-like effector nucleases (TALEN) since TALEN’s large molecular weight may limit their application scope.13 Thus, using a zinc-finger protein as a recognition element, a rapid, simple, and accurate detection system for PCR products can be constructed. For example, by fusing a zinc-finger protein to a biosensor, Osawa et al. developed a simple system to detect specific double-stranded PCR products from pathogenic microorganisms.14−16 However, these systems rely on ELISA, which means that the detection procedure is complicated and time-consuming. © 2017 American Chemical Society

Firefly luciferase, which can emit high-intensity light signals in the presence of D-luciferin and ATP with high sensitivity and low amount of side-signals,17 is an attractive candidate biosensor for the sensitive detection of biomolecules.18,19 However, in previous studies, the activity of luciferase was significantly decreased when it was fused to other proteins,20,21 which hampers its application in the detection of pathogenic bacteria. In this work, we aimed to construct a highly sensitive sensor. We first modified firefly luciferase and fused it with a zinc-finger protein to implement the detection principle as illustrated in Figure 1. This detection procedure consists of two steps: PCR

Figure 1. Detection of bacterial 16S rDNA using the luciferase-ZF system. Schematic diagram of the experimental process. Bacterial genomic DNA is extracted from the sample, and 16S rDNA is specifically amplified by PCR. Biotinylated dsDNA of the amplified PCR product is then captured by MPG streptavidin. Samples are subsequently analyzed using a fluorescence photometer. Received: Revised: Accepted: Published: 6674

May 10, 2017 July 11, 2017 July 13, 2017 July 13, 2017 DOI: 10.1021/acs.jafc.7b02204 J. Agric. Food Chem. 2017, 65, 6674−6681

Article

Journal of Agricultural and Food Chemistry

Design of the Primer Set for the Specific Detection of Bacteria. We used the 16S rDNA gene sequences of different bacterial genera (obtained from the NCBI database) to align and select the target region for amplification and hybridization. All sequences were aligned using Vector NTI software. A high-specificity region was selected for the differential target sequence. For the 16S rDNA sequences of the 6 different genera Staphylococcus, Enterobacter, Proteus, Escherichia, Pseudomonas, and Klebsiella, the zif268 protein recognition site 5′-GCGTGGGCG-3′ was added to the 5′ end of the antisense primer of the sequences amplified from each specific region. The primers were designed so that all amplicons (65−75 nucleotides in length) could be used to amplify the corresponding target regions. We also checked every amplicon to ensure the species specificity of each sequence using NCBI Nucleotide BLAST. All oligonucleotides used as primers were customized and supplied by GENEWIZ. Detection of PCR Products Using the Luciferase-ZF Assay. All bacterial strains were grown in 250 mL shake flasks comprising 50 mL of liquid LB medium at 37 °C and 200 rpm, until reaching an OD600 of 1.0. The TaKaRa MiniBEST Bacterial Genomic DNA Extraction Kit version 3.0 was used to obtain genomic DNA, after which the target region was amplified via PCR in 25 μL reactions. PCR was carried out with 1.0 × 107 copies of genomic DNA as template, and the primers used for amplification contained biotin on the forward primer at the 5′ end and zinc finger recognition sites on the reverse primer at the 5′ end under the following conditions: 95 °C for 5 min, followed by 35 cycles comprising 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 10 s, and a final extension step at 72 °C for 10 min. The final DNA concentration was measured using a Nanodrop 1000 instrument (Thermo Scientific, USA), and the PCR products were validated using agarose gel electrophoresis (Figure 4). In order to bind DNA to magnetic beads, 100 μL (1 mg) of MPG streptavidin was added to a 1.5 mL tube and placed on the magnetic stand for 1 min, after which the supernatant was removed. Subsequently, an aliquot comprising 100 μL of a mixed biotin-labeled DNA solution and the same volume of 2× binding buffer (20 mM Tris-HCl (pH 8.0), 2 mM EDTA, 2 M NaCl, and 0.2% Triton X-100) was added into the tube containing the particles. After mixing, the tube was left at room temperature for 10 min, after which it was placed on the magnetic stand for 1 min and the supernatant removed, followed by the addition of 200 μL of 1× binding buffer and mixing. Finally, the tube was placed on the magnetic stand for 1 min, and the supernatant was removed, at which point the DNA was firmly bound to the magnetic beads. Next, an aliquot comprising 200 μL of zinc finger binding buffer (PBS, pH 7.5, 90 μM ZnCl2) was added into the mixture and the solution removed, after which 100 μL of purified luciferase-ZF (0.1 g/L) in 50 mM PBS pH 7.5 was added to the beads, and the resulting suspension incubated for 10 min at room temperature. After incubation for 10 min, MPG streptavidin was washed 3 times using zinc-finger binding buffer. Finally, the supernatant was discarded. Luciferase activity was measured using a GloMaxTM 20/20 luminometer (Promega, USA) at room temperature. The assay was initiated by injecting 100 μL of complex solution (0.47 mM luciferin, 1.0 mM ATP, 10 mM MgSO4, 10 mM DL-dithiothreitol (DTT), and 25 mM tricine, pH 7.5) into the MPG streptavidin sample. The relative light units (RLUs) of the luminometer over 10 s were used to express the enzymatic activity.23 Real-Time PCR. The genomic DNA from each bacterial strain or DNA from the artificially contaminated samples was extracted using the TaKaRa MightyPrep reagent for DNA. For real-time PCR (quantitative real-time PCR, qPCR), DNA was amplified using TaKaRa E × Taq HS DNA polymerase and specific primers for each bacterial strain (Table 2). The following thermal cycling conditions were used: denaturation (95 °C, 30 s) and 40 cycles of PCR reaction (95 °C, 5 s; 60 °C, 30 s). Each sample was amplified in three parallel reactions. The final qPCR products were validated by TaKaRa PCR Thermal Cycler Dice Real Time System. Detection of Six Species of Bacteria in Artificially Contaminated Samples. Six species of bacteria (dilution factor 105) were introduced into 1 mL of sterilized water, whereby each sample contained a single species of bacterium, and an additional mixed sample contained all six species of bacteria (dilution factor 105). A sterilized sample without added bacteria was used as negative control. Subsequently, the

amplification of the specific region of 16S rDNA with synthetic oligonucleotides containing the recognition sequence of the zinc-finger protein, followed by capture and detection of the PCR products using the luciferase-zinc finger system. Using this system, we were able to detect various pathogenic bacteria with high sensitivity and specificity.

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MATERIALS AND METHODS

Materials. The zif268 gene sequence (Sequence ID: AB778505.1) was sourced from GenBank, and the gene was synthesized by GENEWIZ Corporation (Suzhou, China). The luciferase gene sequence (GenBank: KX650485) was from our own laboratory, as well as bacterial cultures of Staphylococcus aureus (CMCC(B)26003), Proteus mirabilis (CMCC(B)49005), Enterobacter aerogenes (ATCC13048), Escherichia coli (ATCC25922), Pseudomonas aeruginosa (CGMCC1.10452), and Klebsiella pneumoniae (ATCC43816) were purchased from microbiological culture collection. We used Escherichia coli strain BL21 (DE3) (Vazyme, China) to amplify recombinant plasmids and for luciferase-ZF protein expression. The plasmid pET22b (Novagen, USA) encoding a His6-tag sequence was used as a vector for gene expression. The magnetic stand for the adsorption of MPG streptavidin, the In-Fusion HD Cloning Kit, and affinity Ni-NTA spin column for His6-tagged proteins were from Clontech (USA). DNA purification and gel extraction kits were acquired from Oxygene (Hangzhou, China). 2× Phanta Max Master Mix for PCR-amplification was from Vazyme. Restriction enzymes and the MiniBEST Bacteria Genomic DNA Extraction Kit version 3.0 were obtained from Takara (Dalian, China). D-Luciferin potassium salt (LH2) was from Promega (Beijing, China). All other chemicals were obtained from Sigma-Aldrich (Shanghai, China). Plasmid Construction, Fusion Protein Expression, and Purification. The plasmid encoding a wild-type luciferase was sourced from our own laboratory. The luciferase gene was amplified from pET22b-luciferase, followed by cyclization with SpyTag and SpyCatcher. An Nde I recognition sequence was added to the forward primer 5′-AAGAAGGAGATATACATATGTCGTACTACCATCACCATCA-3′, and a GGSGGGSGGS linker was added on the reverse primer 5′-AGAACCACCAGAACCACCACCAGAACCACCTTTGGTGGGTTTATATGCAT-3′. Then, the other forward primer 5′-ATGCATATAAACCCACCAAAGGTGGTTCTGGTGGTGGTTCTGGTGGTTCTGAACGCCCGTACGCT-3′ and the reverse primer 5′-TGGTGGTGGTGGTGCTCGAGCTTGTCTTTTTGTCTTAAAT-3′, containing an Xho I cutting site, were used to clone the zif268 gene from PET22b-zif268. Finally, overlap extension PCR was used to generate the luciferase-ZF gene which was ligated into pET22b using the In-Fusion HD Cloning Kit. The inserted gene was verified by sequencing (GENEWIZ, Suzhou, China). The recombinant plasmid carrying the inserted gene was used to transform E. coli BL21 (DE3) for protein expression. The zif268 protein, luciferase, and luciferase-ZF producing strains were incubated in 50 mL of Luria−Bertani (LB) medium (10% sodium chloride, 10% tryptone, 0.5% yeast extract, and 100 μg/mL ampicillin) at 37 °C and 200 rpm until the OD600 reached 0.6−0.8, after which isopropyl-β-D-thiogalactoside was added to a final concentration of 0.5 mM to induce the expression of the fusion protein. Following expression at 30 °C and 200 rpm for 6 h,22 the bacteria were harvested by centrifugation at 5000g and 4 °C for 10 min and resuspended in 1 mL of 50 mM precooled phosphate-buffered saline (PBS, pH 7.5). The resuspended cells were sonicated and then centrifuged at 4 °C and 12,000g for 10 min to remove cell debris. The supernatant was mixed with Ni-NTA resin that was preequilibrated with 50 mM phosphate buffer (pH 7.5) containing 300 mM NaCl and 10 mM imidazole. Then, the Ni-NTA resin was washed with 50 mM phosphate buffer (pH 7.5) containing 300 mM NaCl and 20 mM imidazole in order to remove nonspecifically bound proteins. The target protein was collected by elution with 50 mM phosphate buffer (pH 7.5) containing 300 mM NaCl and 300 mM imidazole. The concentrations of all proteins were measured using the BIO-RAD Bradford protein analysis kit, according to the manufacturer’s instructions with BSA as protein standard. 6675

DOI: 10.1021/acs.jafc.7b02204 J. Agric. Food Chem. 2017, 65, 6674−6681

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Figure 2. SDS−PAGE of the purified zif268, luciferase, improved luciferase-ZF, and original luciferase-ZF. Lane M comprises the protein molecular standard weight marker. Lane zif268 comprises the zincfinger protein. Lanes 1, 2, 3, and 4 comprise luciferase-ZF, wild-type luciferase, original luciferase-ZF, and cyclized luciferase. The original luciferase-ZF comprises the zinc-finger protein directly fused to luciferase. Luciferase-ZF comprises the modified, cyclized luciferase. All proteins were purified using Ni-NTA resin.

Figure 4. Agarose gel (4%) electrophoresis of PCR amplicons. Lane L, molecular marker; 1, S. aureus; 2, E. aerogenes; 3, P. mirabilis; 4, E. coli; 5, P. aeruginosa; 6, K. pneumoniae. The specific 16S rDNA regions were amplified using the genomic DNA of the indicated bacteria as the template.

Figure 5. Comprehensive analysis of the binding capacity and specificity of the luciferase-ZF system to different PCR products. We used Staphylococcus aureus as the model organism, and the primer design is in Table 1. Figure 3. Thermostability of different luciferase forms. The relative activity of luciferase was determined after incubation at temperatures from 25 to 55 °C for 15 min. WT luciferase-ZF represents the zif268 protein fused with the wild-type linear luciferase, while luciferase-ZF represents the construct comprising the cyclized luciferase. The experiments were carried out in triplicate, and the error bars represent the standard deviations.

samples were transferred into 50 mL of sterilized LB medium and shaken for 10 min at 200 rpm, after which the samples were spread on LB agar plates, and the colonies enumerated after 12−24 h at 37 °C. The samples in the medium were incubated overnight at 37 °C. A 2 mL aliquot of the resulting bacterial culture liquid was taken from the sample medium and placed into a sterile 2 mL centrifugal tube for DNA extraction (described above) and subsequent detection.

Table 1. Sequences Used to Test the Specificity of Luciferase-ZFa

a

S. aureus was used as an example. bZinc-finger protein recognition sites are in red. 6676

DOI: 10.1021/acs.jafc.7b02204 J. Agric. Food Chem. 2017, 65, 6674−6681

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Journal of Agricultural and Food Chemistry Table 2. Sequences of Amplicons and Primers Used for Bacterial Detection

a

Zinc-finger protein recognition sites are in red.

Figure 6. Detection limit of PCR products using luciferase-ZF. (a−f) Different copy numbers of genomic DNA purified from each of the six species of bacteria (S. aureus, E. aerogenes, P. mirabilis, E. coli, P. aeruginosa, and K. pneumoniae) were added to the PCR solution, and the PCR products were analyzed. The lower detection limit for bacterial genomic 16S rDNA was evaluated using a series of different copy numbers of the genome.

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RESULTS AND DISCUSSION

original, nonfused construct. Since the signal intensity of the detection biosensor plays an important role in the accuracy of the results,18 this loss of activity was unacceptable. Therefore, to improve the stability of our system, we added SpyCatcher to the N terminus of luciferase and SpyTag to the C terminus, to generate a cyclized luciferase construct.24 We then fused the resulting cyclized luciferase with the zinc-finger protein.

Modification of the Biosensor-Luciferase. To develop an expression system for the luciferase-zinc finger fusion protein (luciferase-ZF), we first fused the wild-type firefly luciferase with the zinc-finger protein, but we found that the activity of the purified fusion luciferase decreased by half compared with the 6677

DOI: 10.1021/acs.jafc.7b02204 J. Agric. Food Chem. 2017, 65, 6674−6681

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Journal of Agricultural and Food Chemistry

Figure 7. Determination of the sensitivity of the luciferase-ZF system. (a) An example of bacterial detection using the luciferase-ZF system for Staphylococcus. The amplified product from Staphylococcus aureus 16S rDNA was distinguished from the negligible background signals from other species. (b) Relative activity of luciferase for differential detection of various species of bacteria, with high-specificity signals and lower background noise against other bacteria. All samples were tested in triplicate. (c) Determination of the sensitivity of the qPCR system.

The zif268 protein was about 18 kDa, and the luciferase was about 60 kDa, so that after fusion, the luciferase-ZF was expected to be over 78 kDa. SDS−PAGE was used to analyze the luciferase-ZF fusion proteins comprising the cyclized luciferase and wild-type luciferase. As shown in Figure 2, the wild-type luciferase-ZF fusion protein exhibited a band at about 90 kDa, while the cyclized luciferase-ZF fusion protein had an apparent size of about 118 kDa. Thus, the linear protein exhibited faster mobility than the circular form, which was consistent with the literature,24 demonstrating that the luciferase was successfully cyclized even when fused with zif268. When the activity of the fused and nonfused modified luciferase enzymes was compared (Figure 3), we found that after cyclization, the thermal stability of luciferase was improved, and no loss of activity was observed after fusion with the zinc-finger protein, which indicated that the cyclized luciferase would be a desirable biosensor for subsequent detection of specific DNA sequences. The expression of fusion proteins often leads to a reduction of the activity of the fused enzymes, which often limits the application of this technology in the field of multienzyme catalysis, heterologous pathway construction, and other fields.18

In this study, the stability of the fused enzyme was improved by cyclization, which might provide a new strategy to improve the enzyme’s characteristics after protein fusion. Binding Ability of the Luciferase-ZF System. The binding ability and specificity of the luciferase-ZF system toward different PCR products was comprehensively characterized. First, we designed a 29-bp synthetic oligonucleotide that contained a primer region for PCR amplification and a zif268recognition sequence. As negative controls, synthetic oligonucleotides with a mutation in the zif268-recognition sequence, 5′-GCGTGGTCG-3′, or a random DNA sequence instead of the zif268-recognition sequence, 5′-ACTGAGCCT-3′, were used (Table 1). The binding ability of luciferase-ZF was investigated using a bioluminescence assay. The results showed that luciferase-ZF could bind to both the target sequence (5′-GCGTGGGCG-3′) and the single-nucleotide mutated sequence (5′-GCGTGGTCG-3′) but that the affinity for the target sequence was much higher than that for the single nucleotide-mutated sequence (Figure 5). By contrast, little or no luminescent signal was observed with random DNA or samples without DNA, which indicated that the binding ability and 6678

DOI: 10.1021/acs.jafc.7b02204 J. Agric. Food Chem. 2017, 65, 6674−6681

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Journal of Agricultural and Food Chemistry

Figure 8. Detection of artificially contaminated sample with the luciferase-ZF system. Simulated contaminated water sample of six mixed species of pathogenic bacteria was used as the mixed sample. A sterilized sample without added bacteria was used as the negative control. Then, the samples were transferred into 50 mL of sterilized LB medium and incubated overnight at 37 °C, 200 rpm, and after that, extracted DNA and analyzed them with our luciferase-ZF system.

to target the conserved region of the 16S rDNA sequences from different bacterial species. An example of the detection of bacteria using the system is shown in Figure 7a. Using the designed probes, the luciferase-ZF system showed high selectivity with S. aureus and minimal nonspecific binding with the other bacterial species. Similarly, a strong luminescent signal was observed with specially designed probes for each type of bacteria, and a negligible background signal was observed with any of the nontarget species with the same probe. Moreover, the specificity of the luciferase-ZF system (Figure 7b) was compared to qPCR, and we found that the constructed system was superior to qPCR (Figure 7c). This result can be attributed to the highly conserved capture probes, high binding ability of the zinc-finger protein, simple analysis steps, and the stringent washing steps following each capture process, which allowed the removal of nonspecifically bound target and probes. The high specificity of the luciferase-ZF system thus enables the reliable and rapid detection of different bacterial species. While Zn-finger proteins have already been developed for the detection of obtained PCR products in earlier research, in these systems, the Zn-finger protein recognition site was amplified from the bacterial genome and detected directly.14−16,18,19,29 However, such systems have potential problems since their specificity remains to be improved due to the fact that different bacteria can have the same Zn-finger protein recognition site. Furthermore, these systems are complicated since bacteria can have different Zn-finger protein recognition sites so that different detection systems need to be constructed for different species. In our system, the conserved regions of 16S rDNA were used to differentiate between bacterial species, and the zinc-finger protein recognition site was added on the oligonucleotide, which means that only the oligonucleotide needs to be changed to detect different bacteria but not the detection system itself (luciferase-ZF), and simply changing a DNA sequence is much easier and more efficient.

specificity of the zinc-finger protein toward its target DNA remained intact after it was fused with luciferase, which means that the luciferase-ZF system can be used to detect target DNA from the genomes of pathogenic bacteria. Selection of Target Genes. With the development of nucleic acid sequencing technology, increasing numbers of 16S rDNA sequences of microorganisms have been determined and incorporated into the international gene database.25 The 16S rDNA of bacteria is significant because it can reflect the differences among different genera and species, and can be easily obtained by sequencing.26,27 Therefore, 16S rDNA can be used as a phylogenetic marker for bacterial community structure analysis. Hence, in this study, conserved and variable regions were both identified by aligning multiple 16S rDNA gene sequences from different bacterial species,28 and the conserved regions were selected as targets (Table 2). Two oligonucleotides that were complementary to the 5′ end and 3′ end of the target sequence were designed as probes, with a biotin label on the 5 ′end of one oligonucleotide and a recognition site for the zinc-finger protein on the other oligonucleotide. Validation of the Assay. We first investigated the sensitivity of the constructed luciferase-ZF system using 6 different genera Staphylococcus aureus, Proteus mirabilis, Enterobacter aerogenes, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae as the model organism. After amplification, different concentrations of the PCR product (0−104 copies/μL) were contacted with the luciferase-ZF system (Figure 6). To our delight, the luminescent signal was observed even at concentrations of the PCR product as low as 10 copies per microliter, and the signal increased with increasing DNA concentrations. However, at higher DNA concentrations errors were noted. These results indicate that the number of PCR cycles should be optimized to obtain a suitable dynamic concentration range for detection. In the next step, the specificity of the luciferase-ZF was comprehensively characterized. A series of probes were constructed 6679

DOI: 10.1021/acs.jafc.7b02204 J. Agric. Food Chem. 2017, 65, 6674−6681

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(6) Ottesen, E. A.; Hong, J. W.; Quake, S. R.; Leadbetter, J. R. Microfluidic digital PCR enables multigene analysis of individual environmental bacteria. Science 2006, 314, 1464−1467. (7) Rinttilä, T.; Kassinen, A.; Malinen, E.; Krogius, L.; Palva, A. Development of an extensive set of 16S rDNA-targeted primers for quantification of pathogenic and indigenous bacteria in faecal samples by real-time PCR. J. Appl. Microbiol. 2004, 97, 1166−1177. (8) Liong, M.; Hoang, A. N.; Chung, J.; Gural, N.; Ford, C. B.; Min, C.; Shah, R. R.; Ahmad, R.; Fernandez-Suarez, M.; Fortune, S. M.; Toner, M.; Lee, H.; Weissleder, R. Magnetic barcode assay for genetic detection of pathogens. Nat. Commun. 2013, 4, 1752. (9) Chhabra, P.; Vinjé, J. Molecular Detection Methods of Foodborne Viruses. Viruses in Foods; Springer International Publishing, 2016; pp 303−333. (10) Mandell, J. G.; Barbas, C. F. Zinc Finger Tools: custom DNAbinding domains for transcription factors and nucleases. Nucleic Acids Res. 2006, 34, W516−W523. (11) Schmitges, F. W.; Radovani, E.; Najafabadi, H. S.; Barazandeh, M.; Campitelli, L. F.; Yin, Y.; Jolma, A.; Zhong, G.; Guo, H.; Kanagalingam, T.; et al. Multiparameter functional diversity of human C2H2 zinc finger proteins. Genome Res. 2016, 26, 1742−1752. (12) Rohs, R.; Jin, X.; West, S. M.; Joshi, R.; Honig, B.; Mann, R. S. Origins of specificity in protein-DNA recognition. Annu. Rev. Biochem. 2010, 79, 233−269. (13) Abe, T. K. K.; Ikebukuro, W. Y. K. DNA Detection Technology Using Zinc Finger Protein. J. Microb. Biochem. Technol. 2015, 7, 278− 281. (14) Osawa, Y.; Ikebukuro, K.; Kumagai, T.; Motoki, H.; Matsuo, T.; Horiuchi, M.; Sode, K. Zn finger-based direct detection system for PCR products of Salmonella spp. and the Influenza A virus. Biotechnol. Lett. 2009, 31, 725−733. (15) Osawa, Y.; Ikebukuro, K.; Motoki, H.; Matsuo, T.; Horiuchi, M.; Sode, K. The simple and rapid detection of specific PCR products from bacterial genomes using Zn finger proteins. Nucleic Acids Res. 2008, 36, e68−10. (16) Osawa, Y.; Motoki, H.; Matsuo, T.; Horiuchi, M.; Sode, K.; Ikebukuro, K. Zinc finger protein-based detection system of PCR products for pathogen diagnosis. Nucleic Acids Symp. Ser. 2008, 52, 23− 24. (17) Deluca, M.; McElroy, W. [1] Purification and properties of firefly luciferase. Methods Enzymol. 1978, 57, 3−15. (18) Abe, K.; Kumagai, T.; Takahashi, C.; Kezuka, A.; Murakami, Y.; Osawa, Y.; Motoki, H.; Matsuo, T.; Horiuchi, M.; Sode, K.; Igimi, S.; Ikebukuro, K. Detection of pathogenic bacteria by using zinc finger protein fused with firefly luciferase. Anal. Chem. 2012, 84, 8028−8032. (19) Yoshida, W.; Kezuka, A.; Murakami, Y.; Lee, J.; Abe, K.; Motoki, H.; Matsuo, T.; Shimura, N.; Noda, M.; Igimi, S.; Ikebukuro, K. Automatic polymerase chain reaction product detection system for food safety monitoring using zinc finger protein fused to luciferase. Anal. Chim. Acta 2013, 801, 78−83. (20) George, R. A.; Heringa, J. An analysis of protein domain linkers: their classification and role in protein folding. Protein Eng., Des. Sel. 2002, 15, 871−879. (21) Chen, X.; Zaro, J. L.; Shen, W.-C. Fusion protein linkers: property, design and functionality. Adv. Drug Delivery Rev. 2013, 65, 1357−1369. (22) Studier, F. W. Protein production by auto-induction in highdensity shaking cultures. Protein Expression Purif. 2005, 41, 207−234. (23) Koksharov, M. I.; Ugarova, N. N. Thermostabilization of firefly luciferase by in vivo directed evolution. Protein Eng., Des. Sel. 2011, 24, 835−844. (24) Si, M.; Xu, Q.; Jiang, L.; Huang, H. SpyTag/SpyCatcher Cyclization Enhances the Thermostability of Firefly Luciferase. PLoS One 2016, 11, e0162318. (25) Yarza, P.; Yilmaz, P.; Pruesse, E.; Glöckner, F. O.; Ludwig, W.; Schleifer, K.-H.; Whitman, W. B.; Euzéby, J.; Amann, R.; Rosselló-Móra, R. Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences. Nat. Rev. Microbiol. 2014, 12, 635−645.

Detection of Bacteria in an Artificially Contaminated Sample. In order to demonstrate the practical application of our system, we simulated a contaminated water sample of mixed pathogenic bacteria and analyzed the sample using the luciferase-ZF system. Figure 8 shows the results obtained using our luciferase-ZF system. As can be seen in the figure, even though different bacteria were presented in the sample, no interference was observed between the species when using the luciferase-ZF system, demonstrating the system’s excellent accuracy. In conclusion, the luciferase-ZF system was successfully demonstrated as robust and rapid, offering high sensitivity and specificity. It therefore has the potential to be used for the rapid detection and identification of foodborne pathogenic bacteria. Moreover, our system showed considerable advantages over both conventional culture methods and qPCR-based detection methods, especially concerning the necessary assay time and overall cost. Furthermore, though various approaches for the rapid detection of bacteria have been developed,28,30 most of them required the generation of single-stranded DNA from asymmetric reverse transcription-PCR or the denaturation of dsDNA, which are complicated, while our luciferase-ZF system is much more simple. We therefore believe that our system will have far-reaching applications.

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AUTHOR INFORMATION

Corresponding Authors

*(L.J.) Tel/Fax: +86 25 58139942. E-mail: jiangling@njtech. edu.cn. *(H.H.) Tel/Fax: +86 25 58139942. E-mail: biotech@njtech. edu.cn. ORCID

He Huang: 0000-0003-2192-9620 Author Contributions ∥

C.S. and Q.X. contributed equally to this work.

Funding

This work was financially supported by the National Key Research and Development Plan of China (No. 2016YFC1201500) and subsidized by the Program for Innovative Research Team in University of Jiangsu Province. Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Nayak, M.; Kotian, A.; Marathe, S.; Chakravortty, D. Detection of microorganisms using biosensorsA smarter way towards detection techniques. Biosens. Bioelectron. 2009, 25, 661−667. (2) WHO Estimates of the Global Burden of Foodborne Diseases: Foodborne Disease Burden Epidemiology Reference Group 2007− 2015, http://www.who.int/foodsafety/publications/foodborne_disease/fergreport/en/ (accessed December 3, 2015). (3) McTaggart, L.; Richardson, S.; Witkowska, M.; Zhang, S. Phylogeny and identification of Nocardia species on the basis of multilocus sequence analysis. J. Clin. Microbiol. 2010, 48, 4525−4533. (4) Lazcka, O.; Del Campo, F. J.; Munoz, F. X. Pathogen detection: a perspective of traditional methods and biosensors. Biosens. Bioelectron. 2007, 22, 1205−1217. (5) Yan, H.; Shi, L.; Alam, M.; Li, L.; Yang, L.; Yamasaki, S. Usefulness of Sau-PCR for molecular epidemiology of nosocomial outbreaks due to Burkholderia cepacia which occurred in a local hospital in Guangzhou, China. Microbiol. Immunol. 2008, 52, 283−286. 6680

DOI: 10.1021/acs.jafc.7b02204 J. Agric. Food Chem. 2017, 65, 6674−6681

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Journal of Agricultural and Food Chemistry (26) Větrovský, T.; Baldrian, P. The variability of the 16S rRNA gene in bacterial genomes and its consequences for bacterial community analyses. PLoS One 2013, 8, e57923. (27) Sun, D. L.; Jiang, X.; Wu, Q. L.; Zhou, N. Y. Intragenomic heterogeneity of 16S rRNA genes causes overestimation of prokaryotic diversity. Appl. Environ. Microbiol. 2013, 79, 5962−5969. (28) Chung, H. J.; Castro, C. M.; Im, H.; Lee, H.; Weissleder, R. A magneto-DNA nanoparticle system for rapid detection and phenotyping of bacteria. Nat. Nanotechnol. 2013, 8, 369−375. (29) Lee, J.; Tatsumi, A.; Abe, K.; Yoshida, W.; Sode, K.; Ikebukuro, K. Electrochemical detection of pathogenic bacteria by using a glucose dehydrogenase fused zinc finger protein. Anal. Methods 2014, 6, 4991− 4994. (30) Abdalhai, M. H.; Maximiano Fernandes, A.; Bashari, M.; Ji, J.; He, Q.; Sun, X. Rapid and sensitive detection of foodborne pathogenic bacteria (staphylococcus aureus) using an electrochemical DNA genomic biosensor and its application in fresh beef. J. Agric. Food Chem. 2014, 62, 12659−12667.

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DOI: 10.1021/acs.jafc.7b02204 J. Agric. Food Chem. 2017, 65, 6674−6681