Suspension Bead Array of the Single-Stranded Multiplex Polymerase

Apr 29, 2013 - Rapid identification of single and multiple infectious agents is vital in clinical settings and during biothreat attack. This study ass...
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Suspension Bead Array of the Single-Stranded Multiplex Polymerase Chain Reaction Amplicons for Enhanced Identification and Quantification of Multiple Pathogens Hui-Ling Hsu,†,§ Hsin-Hsien Huang,§ Chung-Chih Liang,§ Hung-Chi Lin,§ Wen-Tssann Liu,§ Feng-Ping Lin,§ Jyh-Hwa Kau,*,§,∥ and Kuang-Hui Sun*,†,‡ †

Department of Biotechnology and Laboratory Science in Medicine, Infection and Immunity Center, National Yang-Ming University, Taipei, Taiwan, Republic of China ‡ Department of Education and Research, Taipei City Hospital, Taipei, Taiwan, Republic of China § Institute of Preventive Medicine, and ∥Department of Microbiology and Immunology, National Defense Medical Center, Taipei, Taiwan, Republic of China S Supporting Information *

ABSTRACT: Rapid identification of single and multiple infectious agents is vital in clinical settings and during biothreat attack. This study assesses the assay of single-stranded multiplex polymerase chain reaction (PCR) amplicons by suspension bead array (SSMP-SBA) for multiple pathogens identification in a single-tube reaction. A 15-plex assay for identification of 11 highly infectious pathogens was developed to evaluate the performance of SSMP-SBA. Pathogen-specific amplicons were obtained by sequential amplification of genomic DNAs using gene-specific primers tagged with artificial unique sequences and unique primers of which the reverse primer was modified by biotin and phosphorothioate. The SSMP products generated by T7 exonuclease-mediated DNA hydrolysis were hybridized to 15 sets of beads coupled with gene-specific and control oligonucleotide probes for pathogen identification and quantification by flow cytometry. This method was validated via assessment of 57 reference strains and one clinical bacterial isolate. All 11 pathogens can be detected by the 15-plex SSMP-SBA assay, and this design significantly enhanced the signal-to-noise ratio and improved the assay performance. This assay achieves similar sensitivity to our in-house real-time PCR system with the limit of detection equivalent to 5−100 genome copies and a linear dynamic range crossing three to five logs. In the validation assay, a 100% accuracy rate was achieved when the pathogens were among the target species. Notably, the species of pathogens were accurately identified from the samples with multiple infections. SSMP-SBA presents superior performance with multiplexing capability in a single-tube reaction and provides a new approach for detection and species identification of multiple pathogen infections.

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(PCR) is one of the most efficient and sensitive assays, but its multiplicity is limited to the number of fluorescence channels in instruments.2,3 The DNA-based microarray provides the highest multiplexing capability for microbiological detection. But the cost of the library establishment and microarray fabrication are always the major concerns.4,5 High-resolution melting (HRM) analysis of amplicons is also a rapid, sensitive, and specific method to identify multiple bacterial species and strains but is limited to one organism identification per assay.6 There are other multiplex assays that have been applied for detection of microbial pathogens or diagnosis of infectious diseases, such as PCR−electrospray ionization mass spectrometry (PCR−ESI/MS)7 and terminal-restriction fragment length

uring the past decade, there has been a rapid growth of diagnostic technology due to the occurrences of global epidemics and man-made biological disasters. The rapid and accurate diagnosis of pathogens is the most effective strategy for the appropriate treatment and prevention of the outbreak. The diagnosis and confirmation of pathogens can be achieved by conventional microbiological techniques that rely on cultivation and biochemical assay. However, it is time-consuming and difficult to differentiate with closely related pathogenic and nonpathogenic species. Immunoassay for direct detection of microorganisms is relatively rapid, but the sensitivity is limited.1 Nucleic-acid-based diagnostic assays are rapid, sensitive, highly specific, and can be applied not only identify microorganism species but to distinguish the pathogenicity associated with the presence of virulence factors. Multiplex detection of pathogens is a recent trend in molecular diagnosis. The real-time polymerase chain reaction © XXXX American Chemical Society

Received: March 15, 2013 Accepted: April 29, 2013

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polymorphism (T-RFLP).8 Nevertheless, to use HRM, PCR− ESI/MS, or T-RFLP, a database is required and the identification of multiple infections is limited due to the complexity of data interpretation. The Luminex suspension bead array (SBA) system is an open platform that offers an economical, flexible, and convenient technology for detecting multiple proteins or nucleic acids with many applications.9 It allows simultaneous detection up to 100−500 targets in one tube and is practical for microbiology applications based on cost, flexibility, and feasibility.5 SBA has been used for the detection of fungi, bacteria, and viruses with various strategies.10−14 These approaches are only applied in qualitative or semiquantitative analysis, and the analytical sensitivity is relatively decreased compared with real-time PCR. However, improving the analytical sensitivity and quantification of highly infectious pathogens is critical since most of them are highly pathogenic. In this study, we developed a novel assay of single-stranded multiplex PCR amplicons by suspension bead array (SSMPSBA) simultaneously targeting on 11 highly infectious agents, including Bacillus anthracis,15 Yersinia pestis,16 Francisella tularensis,17 Brucella species,18,19 Burkholderia mallei,20 Burkholderia pseudomallei,21 Coxiella burnetii,22 Salmonella species, Shigella species, Escherichia coli O157:H7, and Vibrio cholera.23−27 The SSMP-SBA used a combination of two-step multiplex PCR, T7 exonuclease-mediated DNA hydrolysis, and SBA enhanced highly sensitive, specific identification and quantification of the infectious agents within 6 h after nucleic acid extraction. Combining multiplex and high-throughput capability for multiple pathogens detection, this novel method can largely decrease the labor, processing time, costs, sample and reagent consumption as well as acquire more information in a short period of time.

Table 1. Assignment of Pathogen Species and Target Genes to Individual Bead Sets and the Reference Strains Validated by SSMP-SBA no.

bead name

B. anthracis

sap

BANsap

2

B. anthracis

pag (on pXO1)

3

B. anthracis

cap (on pXO2)

4

Y. pestis

5

Y. pestis

putative methyltransferase pla (on pP1a)

6

F. tularensis

ISFtu2

BANpag BANcap YPEmet YPEpla FTU

7

Brucella species B. mallei

alkB

BRU

IS407A-f liP

BMA

B. psudomallei C. burnetii

orf11

BPS

IS1111

CBU

rfb

ECO

ttrC, ttrA

SAL

ipaH1.4

SHI

recA

VCH

9 10

14

E. coli O157:H7 Salmonella species Shigella species V. cholerae

15

internal control

11 12 13

MATERIALS AND METHODS Bacterial Strains, Media, and Cultures. The lists of reference strains used in this study can be found in Table 1 and the Supporting Information. In accordance with the biosafety guidelines from Centers for Disease Control (CDC), all culture work involving biosafety level (BSL)-3 pathogens was permitted and performed in a BSL-3 facility. DNA Extraction. Genomic DNA was extracted with the QIAamp DNA-mini kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Finally, DNA was eluted in 100 μL of elution buffer (10 mM Tris−Cl, pH 8.0) and stored at −20 °C until use. The concentration of DNA was determined with a Qubit dsDNA HS assay kit with the Qubit 2.0 fluorometer (Invitrogen, Ltd., U.K.) according to the manufacturer’s protocol. Primers and Probes. The primers and probes were designed using the Primer Select module in the Lasergene 7 software (DNASTAR) and PrimerPlex software. The specificity of the primers was assessed with GenBank BLAST. The melting temperature (Tm) (°C) of each specific primer was within 72− 75. The primer specificity was examined by conventional monoplex and multiplex PCR to make sure only a single band with the predicted size was appeared. To select the probe sequence, the Tm of the probe was defined at 55 ± 2 °C, and the specificity of each probe was assessed with BLAST. Then the probe specificity was further examined by SSMP-SBA with DNA template from reference strains. All primers and probes were manufactured by Eurofins MWG Operon (Ebersberg, Germany). The target genes, primers, and probes are listed

target gene

1

8

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bacteria

IC

reference strains Ban1, B. anthracis ATCC 4229 Ban2, B. anthracis ATCC 14186 Ban2, B. anthracis ATCC 14186 Ban1, B. anthracis ATCC 4229 Ype, Y. pestis ATCC 19428 Ype, Y. pestis ATCC 19428 Ftu, F. tularensis ATCC 15482 Bru, B. abortus ATCC 4315 Bma, B. mallei ATCC 15310 Bps, B. pseudomallei ATCC 11668 Cbu, C. burnetii QNM strain Eco, E. coli O157:H7 ATCC 43895 Sal, S. typhi ATCC 167 Shi, S. dysenteriae ATCC 11835 Vch, V. cholera ATCC 9458 ICO, artificial sequences with forward and reverse unique tails

(Table S-1, Supporting Information). The sequences of primers and probes are patent pending. The reverse unique primer was modified at the 5′-end with biotin and four phosphorothioate linkage between the five terminal bases. Each probe was labeled with amine at its 5′-end with 12-C spacer between the reactive group and 5′-end of the oligonucleotide for bead coupling use. Multiplex PCR Amplification. Multiplex PCR amplification was performed in a 25 μL reaction containing the following: 4 μL of template DNA, 60−80 nM of each targetspecific primer, 1.0 mM of unique primers, 5 pM of internal control oligonucleotide (ICO), and 1× AmpliTaq Gold PCR master mix (Applied Biosystems, U.S.A.). PCR was performed on an ABI 9700 thermal cycler by using the cycling program: 95 °C for 15 min for enzyme activation: first PCR, 25 cycles of 94 °C for 30 s, 65 °C for 1 min, and 72 °C for 30 s; second PCR, 30 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 30 s, and 1 cycle of 72 °C for 5 min. T7 Exonuclease Hydrolysis. SSMP products were prepared by adding 21 U of T7 exonuclease (New England Biolabs Inc., Ipswich, MA) to a mix of 18 μL of multiplex PCR product, water, and 1× buffer supplied with the enzyme in a final volume of 36 μL according to ref 28. The mixture was incubated at 25 °C for 40 min. Suspension Bead Array. (1) Preparation of probe-coupled microbeads: 15 carboxylated microbead sets (Luminex, Austin, TX) with different fluorescent dye mixes were chosen, and each microbead set was coupled to a 5′-amine-C12 modified probe according to the carbodiimide coupling method.29 Detailed B

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Figure 1. Schematic of SSMP-SBA procedure. (A) For simplicity, ICO and two target genes denominate as A and B are presented. After the first PCR, each target gene except ICO was amplified by its corresponding specific primer pair. In the second PCR, amplicons of A and B including ICO were amplified with unique primers and all of the amplified reverse strands were labeled with biotin and modified with phosphorothioate. (B) After T7 exonuclease hydrolysis, these biotin-labeled reverse strands (SSMP products) were conserved under the protection of phosphorothioate bonds and captured by the probes coupled to the appointed bead set. Finally they were detected by streptavidin-R-phycoerythrin (SAPE) and analyzed using flow cytometry.

95 °C for 10 min, 40 cycles of denaturation (94 °C for 0 s), annealing and extension (60 °C for 20 s), and cooling at 40 °C for 30 s. Statistics. Data were expressed as mean, mean + SD, or mean ± SD, and statistical significance was assessed by paired t test.

procedures can be found in the Supporting Information. (2) Hybridization: an amount of 17 μL of the ss-PCR product was analyzed in a 50 μL hybridization assay mixture containing 2500 probe-coupled microbeads from each set (see the Supporting Information). (3) Flow cytometry analysis: the reactions were analyzed with Bio-Plex 200 system (Bio-Rad) using high RP1 setting and Bio-Plex Manager (version 5.0) according to the manufacturer’s instructions. For each sample, at least 100 beads were analyzed from each bead set and the results were reported as the median fluorescence intensity (MFI). Analytical Sensitivity and Quantification. The extracted DNA from each reference strain was diluted with 10 mM Tris, pH 8.0. Solutions of 10-fold serial dilutions ranging from 106 to 1 and 500, 250, 50, 25, 5 genome equivalents (GE) per reaction mixture were tested in triplicate with SSMP-SBA or in duplicate with real-time PCR. The limit of detection (LOD) was the one with the fewest GE of each reference strain that gave a positive signal. Real-Time PCR Assays. Fourteen in-house real-time PCR assays that targeted the same genes as applied in SSMP-SBA were performed using a LightCycler 2.0 system (Roche Diagnostics, U.S.A.). In brief, 20 μL of reaction mixture containing 4 μL of extracted DNA, 0.5 μM of each primer, and 0.25 μM TaqMan probe (Applied Biosystems, U.S.A.) was mixed with 1× Master Mix of LightCycler FastStart DNA Masterplus HybProbe (Roche Diagnostics, U.S.A.). Probes were labeled with FAM at the 5′-end and a minor groove binder (MGB) quencher at the 3′-end (Applied Biosystems, U.S.A.). The reaction was performed as follows: initial denaturation at

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RESULTS AND DISCUSSION

Universal genes such as 16S rDNA or 23S rDNA have been widely applied in molecular diagnostics of microorganisms. However, the main disadvantages include lack of diversity and consistent differentiation between species as well as being short of identification of bacterial virulence and pathogenesis. Besides, the analytical efficacy is more likely to be compromised if the specimen is contaminated with other environmental microorganisms. Therefore, we selected genus-specific, speciesspecific, and pathogenic genes as identification targets and established a novel assay of SSMP-SBA in this study. Experimental Design of SSMP-SBA. A schematic illustration of the strategy is shown in Figure 1. The first part of the assay was a two-step multiplex PCR using 14 pairs of gene-specific primers and one pair of unique primer (Figure 1A). Each specific primer pair was composed of sequences specific to the selected target gene and one forward/reverse unique tail sequences on the 5′-end, respectively. The unique primer pair consisted of forward/reverse unique tail sequences, and the reverse primer was modified by biotin and phosphorothioate. The unique tails were artificial sequences not relevant to any gene but for amplification. C

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Table 2. Representative Data from SSMP-SBA on Positive Controls

1

DNA with 105 GE from each reference strain was used as template. Each sample was tested in triplicate, and the mean MFI from each bead set is presented. The cutoff values were determined as the sum of the mean plus 4 times the standard deviations of the no-template controls (NTCs) and unrelated bacterial species analyzed during the study. Any signal that was greater than the cutoff value for a given microbead set was considered as positive and is presented in bold type on a gray background.

The multiplex PCR is a challenging technology because primer dimers may form and nonspecific binding of primers may be induced as a result of too many primers mixed in one reaction. To reduce the formation of primer dimers and nonspecific binding, we lowered the concentration of each specific primer to 1/5 to 1/10 of the amount applied in realtime PCR. In the first step of PCR, each target gene was amplified by its specific primer pair under the annealing condition of 65 °C. In the second step, these amplicons were then reamplified with the unique primer pair under the annealing temperature of 50 °C and each amplified reverse strand was tagged with biotin and phosphorothioate modification. The second part is the generation of SSMP products (Figure 1B). Following T7 exonuclease-mediated hydrolysis, all of the biotin-labeled reverse strands were conserved because the phosphorothioate bonds protected them from 5′-to-3′ hydrolysis.28 The third part is suspension bead array (Figure 1B). For multiplex analysis, each 5′-amine-C12 modified specific probe with sequences complementary to the target gene was coupled to an appointed microbead set. The biotinlabeled SSMP products were hybridized to the probes on a particular bead set and subsequently marked with streptavidinR-phycoerythrin (SAPE). Finally, the beads were analyzed by the flow cytometry, and the median fluorescence intensity (MFI) values from each bead set were calculated.

In this study, 15 bead sets were used in SBA. Among them, 14 bead sets were specific for pathogen detection related to 14 selected target genes and one bead set was for internal control (Table 1). To verify the analysis, a constant amount of internal control oligonucleotide (ICO) was included in each reaction. ICO was 80-mer long with artificial sequences unrelated to any known gene but contained the forward and complementary reverse unique tail sequences (Table S-1, Supporting Information). It was amplified in the second step of multiplex PCR, and its generated SSMP products were captured by internal control (IC) beads in the SBA (Figure 1B). Therefore, the reactions were regarded as valid if positive signals were detected in the IC bead set. Optimization of SSMP-SBA. The target genes selected for the 11 pathogens and the assignment of the individual bead set to the pathogen and target gene are listed (Table 1). Because of the high degree of genomic homology, it is necessary to differentiate pathogenic B. anthracis from avirulent strains and the closely related spore-forming Bacillus species. In this study, sap was a chromosomal marker specific for B. anthracis; pag (on plasmid pXO1) and cap (on plasmid pXO2) were applied to differentiate the full virulent strains, which contained both pXO1 and pXO2, or avirulent strains, which contained only pXO1 or pXO2.30 In addition, we selected a putative methyltransferase gene as a specific chromosomal marker for Y. pestis. A virulence gene pla (encoding plasminogen activator) D

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Figure 2. Effect of T7 exonuclease treatment in the SSMP-SBA and uniplex vs 15-plex bead assay. (A) Comparison of the MFI values generated from multiplex PCR, with or without T7 exonuclease hydrolysis followed by SBA. Each positive control with 105 GE was tested in triplicate, but only the data (mean + SD) from the specific bead set are presented (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (B) Positive controls with 105 GE were analyzed by generation of SSMP products followed by uniplex or 15-plex bead array. The MFI values (mean + SD) from the specific bead set are presented.

Table 3. Multiple Agents Detection with SSMP-SBA

1

Each sample was tested in triplicate, and the mean MFI greater than the cutoff value for a given microbead set was considered as positive. The templates added in the reaction are presented on a gray background. (Abbreviations: 2-DNA-1 for a mixture of DNA from Bma and Bps; 2-DNA-2 for a mixture of DNA from Sal and Shi; 4-DNA-1 for a mixture of DNA from Ype, Ftu, Bma, and Bps; 4-DNA-2 for a mixture of DNA from Eco, Sal, Shi, and Vch; 6-DNA for a mixture of DNA from Ype, Bma, Bps, Cbu, Sal, and Shi; +, positive; −, negative.)

Both B. anthracis strains including Ban1 and Ban2 showed positive signals of the sap gene. Ban1 containing only pXO2 was also positive for the cap gene, and Ban2 containing only pXO1 produced positive signal for the pag gene. Ype (Y. pestis ATCC 19428), a virulent strain containing three virulence plasmids,31 produced positive signals for YPE-met and YPE-pla in this assay. DNA from each reference strain gave the expected positive results on the corresponding specific bead sets but negative on the other bead sets. All of the reactions including no-template control (NTC) showed positive signals on the IC bead set. To verify the effect of T7 exonuclease hydrolysis, we found the MFI values on the specific bead sets were significantly increased and the signal-to-noise ratio was enhanced after T7 treatment (Figure 2A). It suggests the hybridization efficiency between PCR products and their specific probes can be substantially improved by this strategy.

on pPla essential for virulence of Y. pestis was also included in this assay to detect pathogenic Y. pestis.31 A variety of conditions including polymerase selection, PCR cycle numbers, reaction volume, ICO concentration, concentrations of specific and unique primers for multiplex PCR, T7 exonuclease concentration, and hybridization temperature and duration were optimized to get the efficient hybridization, high sensitivity, and specificity for the analysis (data not shown). As shown in Figure S-1 (Supporting Information), 21 U of T7 exonuclease and denaturation at 95 °C for 10 min and hybridization at 50 °C for 30 min were applied in the SSMPSBA. During the development of this assay, the multiplex PCRamplified products from each reference strain were subjected to gel electrophoresis and the sizes were confirmed as predicted (data not shown). Analytical Performance of SSMP-SBA. To validate this assay, the purified DNA from each reference strain (Table 1) was analyzed as positive control with SSMP-SBA (Table 2). E

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Figure 3. Quantitative analysis of SSMP-SBA. Serial dilutions of DNA from reference strains were tested with SSMP-SBA in triplicate, and the MFI values (mean ± SD) from the specific bead set were plotted against the amounts of added templates: Ban-cap detection (A) and Sal detection (B).

To examine whether the MFI signal would be affected by combining multiple bead sets, each reference strain was investigated with SSMP followed by uniplex or 15-plex SBA. The data show there was no statistical difference in MFI signal when the tests were performed in uniplex or 15-plex SBA (Figure 2B). To demonstrate the feasibility of SSMP-SBA for detection of multiple pathogens in a single reaction, equal amounts of template DNA from two, four, and six different pathogens were mixed and analyzed (Table 3). The positive signals we obtained faithfully indicated the target templates included in the reaction mixture. We achieved a 100% accuracy of identification when the samples were mixed with 2- to 6DNA templates of pathogens. Since multiple infections of more than five pathogens are rare, the results suggest SSMP-SBA can deal with most of the multiple infections and is promising for detecting different targets according to user’s demand. The specificity of SSMP-SBA was further examined by testing 13 different bacteria other than these 11 selected agents (Table S-2, Supporting Information). Three nonanthracis species including B. cereus, B. thuringiensis, and B. circulans were negative for all targets and distinguished from B. anthracis in our system. For these tested bacterial strains, only the IC bead set presented positive signals and all of the specific signals were negative. It indicates that SSMP-SBA can be applied to single or multiple pathogen identification with high specificity. Limit of Detection and Quantification of SSMP-SBA. To determine the analytical sensitivity of SSMP-SBA for each pathogen, serial dilutions of DNA from each reference strain were tested, and the resulting MFI values of the specific target versus the amounts of added templates were plotted (Figure 3 and Supporting Information Figure S-2). The LOD was determined as the one with the fewest GE that gave positive signal to the respective bead set. For all the pathogens tested, the LOD was 5−100 GE per reaction (Table 4). The data produced in SSMP-SBA were quantitative with a good linearity (R2 = 0.9604−0.9959) between the logarithm of input quantity and the MFI values (Figure 3 and Supporting Information Figure S-2). The linear dynamic range for each target was between 3 and 5 logs (Table 4). Furthermore, SSMP-SBA could quantitatively analyze multiple pathogens in one sample under optimized dilution of pathogen DNA (data not shown). Comparison of SSMP-SBA with In-House Real-Time PCR. For a practical demonstration of SSMP-SBA, we compared the detection of each target gene by SSMP-SBA and in-house real-time PCR with the same DNA dilutions. As shown in Table 4, SSMP-SBA achieves similar sensitivity with in-house real-time PCR system but presents superior performance with multiplexing capability.

Table 4. List of Dynamic Range and LOD for Each Pathogen and Target Gene Detection with SSMP-SBA and Real-Time PCR SSMP-SBA

real-time PCR

target gene

dynamic range (GE)

LOD (GE)

LOD (GE)

Ban-sap Ban-cap Ban-pag Ype-met Ype-pla Ftu Bru Bma Bps Cbu Eco Sal Shi Vch

500−106 100−106 50−105 100−106 25−105 250−106 250−106 10−106 25−106 10−106 100−106 10−106 10−105 50−106

50 25 25 25 25 100 50 10 10 5 100 5 10 10

10 5 5 5 1 5 10 5 10 1 5 10 1 1

Blind Test and Clinical Application. We validated this assay in bacterial species identification by performing a blind test with 57 bacterial culture colonies of reference strains from ATCC and one clinical bacterial isolate obtained from the CDC of Taiwan (Table S-3, Supporting Information). In this study, all of the blind samples were correctly identified. In addition, the clinical isolate (CI) from a 54 year old woman who was hospitalized with fever, low-back pain, and disorder of liver function was tested with SSMP-SBA and showed positive for Brucella species. This result was consistent with the other two methods: a rapid lateral flow chromatographic immunoassay and a conventional PCR analysis (Figure S-3, Supporting Information). By using SSMP-SBA, we achieved a 100% (22 of 22) overall accuracy rate for bacterial species identification and no false positive result was observed.

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CONCLUSIONS In this study, we developed a novel detection method based on analysis of SSMP followed by SBA to accurately diagnose multiple infectious agents. By utilizing artificial unique primers and T7 exonuclease in SSMP-SBA, it enhances the assay sensitivity to achieve the LOD similar to real-time PCR and allows broad dynamic range for the microorganism quantification. Without considering the cost of machinery, labor, and DNA extraction, the reagent cost is estimated to be approximately US $10.00 with SSMP-SBA and less than US $1.00 per target. This novel assay has the potential to be applied to other infectious pathogens in the future. F

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(23) Kothary, M. H.; Babu, U. S. J. Food Saf. 2001, 21, 49−68. (24) Musher, D. M.; Musher, B. L. N. Engl. J. Med. 2004, 351, 2417− 2427. (25) Rabbani, G. H.; Greenough, W. B., III. J. Diarrhoeal Dis. Res. 1999, 17, 1−9. (26) Ackers, M.-L.; Mahon, B. E.; Leahy, E.; Goode, B.; Damrow, T.; Hayes, P. S.; Bibb, W. F.; Rice, D. H.; Barrett, T. J.; Hutwagner, L.; Griffin, P. M.; Slutsker, L. J. Infect. Dis. 1998, 177, 1588−1593. (27) Tarr, P. I. Clin. Infect. Dis. 1995, 20, 1−8. (28) Nikiforov, T. T.; Rendle, R. B.; Kotewicz, M. L.; Rogers, Y. H. PCR Methods Appl. 1994, 3, 285−91. (29) Fulton, R. J.; McDade, R. L.; Smith, P. L.; Kienker, L. J.; Kettman, J. R. Clin. Chem. 1997, 43, 1749−1756. (30) Ryu, C.; Lee, K.; Yoo, C.; Seong, W. K.; Oh, H. B. Microbiol. Immunol. 2003, 47, 693−9. (31) Parkhill, J.; Wren, B. W.; Thomson, N. R.; Titball, R. W.; Holden, M. T. G.; Prentice, M. B.; Sebaihia, M.; James, K. D.; Churcher, C.; Mungall, K. L.; Baker, S.; Basham, D.; Bentley, S. D.; Brooks, K.; Cerdeno-Tarraga, A. M.; Chillingworth, T.; Cronin, A.; Davies, R. M.; Davis, P.; Dougan, G.; Feltwell, T.; Hamlin, N.; Holroyd, S.; Jagels, K.; Karlyshev, A. V.; Leather, S.; Moule, S.; Oyston, P. C. F.; Quail, M.; Rutherford, K.; Simmonds, M.; Skelton, J.; Stevens, K.; Whitehead, S.; Barrell, B. G. Nature 2001, 413, 523−527.

ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +886-2-2826-7228 (K.-H.S.); +886-2-8792-3100, ext 18531 (J.-H.K.). Fax: +886-2-2826-4092 (K.-H.S.); +886-226733025 (J.-H.K.). E-mail: [email protected] (K.-H.S.); [email protected] (J.-H.K.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Science Council (101-2321-B-010-005), Ministry of Education (Aim for the Top University Plan), Taipei City Hospital, and National Defense Research Program, Taiwan, Republic of China. The authors thank Professor Shy-Jye Tang, Ching-Ping Tseng, and Wailap Victor Ng for discussion.

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dx.doi.org/10.1021/ac400778b | Anal. Chem. XXXX, XXX, XXX−XXX