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Clinical Chemistry 53: 1741-1748, 2007. First published August 10, 2007; 10.1373/clinchem.2007.087502
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(Clinical Chemistry. 2007;53:1741-1748.)
© 2007 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Identification of 8 Foodborne Pathogens by Multicolor Combinational Probe Coding Technology in a Single Real-Time PCR

Qiuying Huang1, Qinghua Hu2 and Qingge Li1,a

1 Molecular Diagnostics Laboratory, Department of Biomedical Sciences and the Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.
2 Shenzhen Center of Disease Control and Prevention, Shenzhen, China.

aAddress correspondence to this author at: Department of Biomedical Sciences, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China. Fax 86-592-2187363; e-mail qgli{at}xmu.edu.cn.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Real-time PCR assays have been widely used for detecting foodborne pathogens but have been much less frequently applied in species identification, mainly because of the low number of species they can distinguish in 1 reaction. The present study used a new probe coding/labeling strategy, termed multicolor combinational probe coding (MCPC), to increase the number of targets that can be distinguished in a single real-time PCR for rapid and reliable species identification.

Methods: With MCPC, 8 pairs of species-specific tagged primers, 1 pair of universal primers, and 8 unilabeled or mix-labeled molecular beacon probes were included in a single reaction tube. Real-time PCR was performed, and the identity of each of the 8 pathogens was determined by amplification profile comparison. The method was validated via blind assessment of 118 bacterial strains, including clinical isolates and isolates from food products.

Results: The blind test with 118 samples gave no false-positive or -negative results for the target genes. The template DNA suitable for MCPC analysis was simply prepared by heating lysis, and the total PCR analysis was finished within 2.5 h, excluding template preparation.

Conclusions: MCPC is suitable for rapid and reliable identification of foodborne pathogens at the species level.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Rapid identification of the causative organism is critical in the control and prevention of foodborne diseases (1). Unlike species-specific detection that often requires both qualitative and quantitative information, species identification is qualitative in nature, i.e., only a straightforward yes-or-no answer is needed regarding the absence/presence of a suspected pathogen (2). In clinical microbiology laboratories, a pre-enrichment/colony purification process is often routinely conducted before species identification, making identification accuracy rather than quantification sensitivity the crucial issue. Because suspected species of microbial pathogens capable of invoking foodborne diseases are often numerous, it would be very cost-effective if all suspected pathogens involved in a similar clinical manifestation could be accurately identified in a single assay (or a few assays). Currently, the predominant method for foodborne pathogen identification is still cultivation-based, in which comprehensive determinations of phenotypic profiles are carried out through several days of labor-intensive work (3)(4). Even so, unequivocal identification often requires other complementary methods, such as antibody testing. Many nucleic acid–based methods with improved identification resolution have been developed. These methods, such as pulsed-field gel electrophoresis (5), multilocus sequence typing (6)(7), variable number of tandem repeat/multiple-locus analysis (8)(9), and pyrosequencing (10)(11), are more suitable for subspecies analysis and tracking down the dissemination of certain pathogens. But these methods become too complex for routine species identification that involves a large number of clinical samples.

The introduction of real-time PCR technology has revolutionized pathogen detection in clinical microbiology (12)(13). With its simple manipulation, high throughput, minimal risk of contamination, and excellent reproducibility, real-time PCR is becoming the gold standard for detection and quantification of DNA/RNA in diagnostics laboratories (14)(15). So far, however, real-time PCR has found limited use in species identification mainly because of the inadequate number of targets that can be covered in 1 reaction. For example, a uniplex real-time PCR can determine the presence of only 1 target, and multiplex real-time PCR can detect no more than 4 to 5 targets depending on available instruments (16)(17)(18). To determine the existence of a specific species among a large number of suspected pathogens, multiple uniplex or multiplex real-time PCR reactions have to be performed. To be more practical for species identification, a real-time PCR platform must cover more targets that can be distinguished in a single reaction.

In the present study, we report a strategy that significantly increases the number of targets identifiable in a single real-time PCR. This strategy, multicolor combinational probe coding (MCPC), 1 uses fluorophore combinations in addition to single fluorophores to label probes. Similar to the so-called chromosome painting technology (19), the combination rule allows n types of fluorophores to label N = Cn1 + Cn2 + ... + Cnn = 2n – 1 different probes in a combinatorial manner. Thus, up to 15 probes can be labeled using 4 different fluorophores, and 15 targets can be detected on a 4-color real-time PCR machine. Using MCPC, 8 foodborne pathogens can be accurately identified at the species level in a single real-time PCR.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
standard bacterial strains and dna template preparation
Standard strains of 8 foodborne pathogens (Staphylococcus aureus, Listeria monocytogenes, Salmonella typhi, Shigella spp., Escherichia coli O157:H7, Vibrio cholerae, Vibrio parahaemolyticus, and Streptococcus pyogenes) were obtained from the Shenzhen Center of Disease Control and Prevention (Shenzhen CDC, Shenzhen, China). For DNA purification, S. aureus, L. monocytogenes, S. typhi, Shigella spp., E. coli O157:H7, V. cholerae, and V. parahaemolyticus were grown in nutrient broth. S. pyogenes was inoculated in dextrose meat infusion broth. After incubation at 37 °C (225 rpm shaking) overnight, DNA was extracted from each culture using AxyPrepTM Bacterial Genomic DNA Miniprep Kit (Axygen Biosciences). A simple heating lysis procedure was also used for DNA extraction: 1 mL of each culture was centrifuged at 10 000g for 5 min, and the pellet was collected and resuspended in 100 µL distilled water. Bacteria were then lysed by heating at 100 °C for 10 min and snap-cooled on ice. After centrifugation (10 000g for 5 min), the supernatant was collected and diluted 50-fold with water before being used as DNA template for PCR.

primers and probes
The MCPC assay developed in this study was based on real-time PCR detection sets previously developed for the 8 foodborne pathogens. These sets were designed to target 1 specific gene of each causative pathogen with 1 specific primer pair and 1 modified molecular beacon probe (20). An important feature of these sets is that they all work under the same thermocycling conditions, and any combinations of them are compatible in multiplex PCR (up to quadruplex). In the present work, the same primer and probe sequences were used except that tag sequences were added to the 5' end of the primers and different fluorophore/fluorophore combinations instead of only individual fluorophores that were used for probe labeling (see Table S1 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol53/issue10). Among these probes, 4 were each labeled with a single fluorophore, and another 4 were each mix-labeled with various combinations of 2 fluorophores (i.e., mixtures of 2 probes having the same oligonucleotide sequence, but each labeled with a different fluorophore). All primers and modified molecular beacons probes were synthesized and PAGE-purified by Sangon.

uniplex real-time pcr
Uniplex real-time PCR was performed as follows unless otherwise noted. The 25-µL reaction contained 1x PCR AmpliTaq Gold buffer, 1 unit AmpliTaq GoldTM, 2.0 mmol/L MgCl2, 200 µmol/L dNTPs, 0.02 µmol/L tagged primer pair, 0.2 µmol/L universal primer pair, 0.2 µmol/L probe, and 5 µL DNA template. The cycling was initiated by heating to 95 °C for 10 min, continued by 10 cycles of 95 °C for 15 s, 55 °C for 120 s, and 72 °C for 30 s, and followed by another 40 cycles of 95 °C for 15 s, 50 °C for 20 s, and 72 °C for 20 s. Fluorescence was recorded at the annealing steps during the 2nd 40 cycles on an Mx3000P detection system (Stratagene).

mcpc real-time pcr assay
The same conditions were used as in uniplex PCR except that 3.0 mmol/L MgCl2, 1.2 units AmpliTaq Gold, 250 µmol/L dNTPs, 10 nmol/L tagged primers for each of S. aureus, S. typhi, and E. coli O157:H7, 7.5 nmol/L tagged primers for S. pyogenes, 5.0 nmol/L tagged primers for each of L. monocytogenes, Shigella spp., V. cholerae, and V. parahaemolyticus, and 0.2 µmol/L of each differently labeled probe for each of the 8 pathogens were used.

validation of the mcpc assay
A blind test of 118 bacterial samples including the 8 foodborne pathogens and 4 other pathogens (Yersinia enterocolitica, Proteus vulgaris, enteropathogenic E. coli, and enterohemorrhagic E. coli) was performed to validate the MCPC assay. These samples are clinical isolates except that 20 strains were isolated from food products by Shenzhen CDC. All bacteria were previously identified by colony morphology, biochemical properties, and immune agglutination. DNA templates prepared using the heating lysis method were blindly tested by MCPC assay.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
design of mcpc assay
In designing an MCPC assay for 8 pathogens using a 4-color real-time PCR machine, 8 species-specific molecular beacon probes were labeled with 8 different fluorophores or fluorophore combinations, and 8 specific primer pairs were tagged at their 5' ends. Consequently, after initial amplification, all amplicons would have the same universal ends that could be recognized and amplified by a single pair of universal primers (21). The tagged primer pairs were present at relatively low concentrations to facilitate only the initial amplification, whereas the universal primer pair was present at standard concentrations for the subsequent amplification of the initial amplicons. Thus, the MCPC analysis for identification of 8 species involved 8 molecular beacon probes in total, each labeled with a different fluorophore or a mixture (combination) of 2 fluorophores, 8 tagged primer pairs, and 1 universal primer pair in a single tube.

As shown in Fig. 1 , in the presence of a certain target DNA, the preliminary amplification would be initiated by the tagged primer pair specific for this target, followed by a 2nd amplification by the universal primer pair targeting the tag sequences. The presence of a specific target was revealed by the real-time PCR profile characteristic to each uniquely labeled probe. For example, when E. coli O157:H7 was present, only hexachloro-6-carboxyfluorescein (HEX) fluorescence intensity increased in the real-time PCR profile because the probe for E. coli O157:H7 was labeled with HEX alone. With S. pyogenes, both 6-carboxyfluorescein (FAM) and 6-carboxy-X-rhodamine (ROX) fluorescence increased in the real-time PCR profile, because the probe for S. pyogenes was labeled with a mixture of FAM and ROX fluorophores. In total, 8 different types of real-time PCR profiles were generated corresponding to 8 types of pathogens.


Figure 1
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  		Figure 1. Illustration of the principle of MCPC for pathogen identification.

(A), flow chart of MCPC. Genomic DNA is amplified initially by the matched tagged primer pair. Further amplification is mediated by the universal primer pair. The accumulation of the amplicon is detected by the matched molecular beacons probes, producing a corresponding real-time PCR profile. (B), eight possible PCR profiles corresponding with the 8 pathogens according to the MCPC design in this study. Each unique PCR profile is indicated by the appearance of the characteristic fluorescence increase. The color of the curves stands for the different fluorophore labels: FAM (orange), HEX (green), ROX (red), and Cy5 (blue).

establishment of mcpc for identification of 8 foodborne pathogens
The MCPC assay for identification of 8 foodborne pathogens was developed based on the real-time PCR detection sets previously developed. To test whether the same thermocycling conditions could be used for amplification of all targets and whether the specificity remained in the detection of the 8 species in MCPC as in single PCRs, uniplex real-time PCR using tagged primers and universal primers was performed using the original single-fluorophore–labeled (all FAM-labeled) probes. The uniplex PCR thermocycling conditions were directly adopted from the set protocol except that 10 cycles of initial amplification with tagged primers were added. Because the tagged primers were present at low concentrations, relatively long annealing time was allowed for sufficient primer binding. The results showed that all 8 uniplex real-time PCRs could run under the same thermocycling conditions. When tested with 8 different templates (purified genomic DNA), only the matched template produced an amplification signal, whereas the other 7 showed no fluorescence increase (see Supplemental Data Fig. S1). Slight differences in threshold cycle (CT) values were observed among some uniplex PCRs, possibly because of DNA template concentration variations. Thus, after substitution of the original primer pairs with the tagged primer pairs and a universal primer pair, the real-time PCR could be run under the same conditions without losing specificity. These results form the basis for the development of the MCPC assay in which all primers and probes were included in 1 reaction.

Another feature of MCPC is the use of fluorophore combinations rather than individual fluorophores to label probes. In this study, 4 single-fluorophore–labeled probes and 4 dual-fluorophore, mix-labeled probes were used. To test whether the 2 fluorophores of the dual-fluorophore–labeled probes could identically indicate amplicon increase in real-time PCR detection, 3 separate uniplex PCRs, each containing the same specific primer pair, and a probe having the same nucleotide sequence but labeled with different fluorophores or fluorophore combinations were performed and compared. Using V. parahaemolyticus as an example, the 1st PCR used FAM-labeled probe, the 2nd PCR used Cy5-labeled probe, and the 3rd PCR used both FAM- and Cy5-labeled probes. No difference in CT values among these 3 uniplex PCRs was observed in the entire DNA concentration range studied (from 10 ng to 0.1 pg) (Fig. 2 ). Similar results were obtained with 3 other sets of species tested (L. monocytogenes, V. cholerae, and S. pyogenes). Thus, with the dual-fluorophore combination-labeled probe, both fluorophores function equally to indicate PCR amplification. Slightly lowered fluorescence intensity occurred in the reaction using the dual-fluorophore mix-labeled probes. Such fluorescence intensity reduction, however, did not influence the CT value, a parameter reflecting the amplification efficiency. We also observed that, with the dual-fluorophore mix-labeled probes, the relative fluorescence intensity of each fluorophore was determined by the relative concentration of the 2 individual single-fluorophore–labeled probes. Because the relative fluorescence intensity had no influence on the interpretation of the PCR profile, to keep things simple, the dual-fluorophore combination was simply prepared by mixing equal molar amounts of 2 single-fluorophore–labeled probes.


Figure 2
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  		Figure 2. Comparison of single-fluorophore unilabeled and dual-fluorophore mix-labeled probes in uniplex real-time PCR.

Three separate uniplex real-time PCRs for V. parahaemolyticus were performed using FAM-labeled probe (0.12 µmol/L), Cy5-labeled probe (0.25 µmol/L), and both FAM (0.12 µmol/L) and Cy5 (0.25 µmol/L) mix-labeled probes. Purified DNA templates were 10-fold serially diluted from 10 ng to 0.1 pg and water was used as negative control. Note that, among the entire range of template concentrations, no obvious difference in CT values among the 3 reactions was observed, but slightly lowered fluorescence intensity occurred with the dual-color probe reaction.

The MCPC assay contained 8 pairs of tagged primers in 1 reaction. To reduce the complexity, the tagged primers were present at relatively low concentrations. However, if the concentrations were too low, the initial amplification would become inefficient. Thus, we evaluated the effect of the tagged primer concentrations on the amplification efficiency. The tagged primer pairs were serially 2-fold diluted from 40 nmol/L to 1.25 nmol/L and tested in the uniplex PCR. A typical result using L. monocytogenes showed that, as the concentration of the tagged primer pair increased, uniplex PCR became more efficient as indicated by continuous decrease of CT values (see Supplemental Data Fig. S2). Interestingly, a linear relationship between the logarithm of the tagged primer concentrations and the CT was observed. We attributed this relationship to the fact that the template for the 2nd-round amplification is produced by tagged primers, and higher concentration of tagged primer will thus produce more amplicon. This linear relationship is helpful in the optimization of the relative concentrations of tagged primer pairs in the MCPC assay.

Finally, MCPC was established by stepwise combination of 4, 6, and 8 individual uniplex real-time PCRs into 1 reaction. Compared with thermocycling conditions for uniplex PCR, higher concentrations of Mg2+, dNTPs, and AmpliTaq Gold were found to be helpful for achieving better signals in MCPC. In addition, similar amplification efficiency for all 8 pathogens was achieved by adjustment of the tagged primer pair concentration without changing probe concentrations. With the use of DNA templates purified with the commercial set, 8 standard pathogens were successfully identified by MCPC analysis as indicated by real-time PCR profiles corresponding to each pathogen (Fig. 3 ).


Figure 3
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  		Figure 3. Identification of 8 bacterial species with a single MCPC assay.

According to the designs in this study, the probes for S. aureus, E. coli O157:H7, S. typhi, Shigella spp., L. monocytogenes, V. parahaemolyticus, S. pyogenes, and V. cholerae were labeled with FAM, HEX, ROX, Cy5, FAM + HEX, FAM + Cy5, FAM + ROX, and HEX + ROX, respectively. For each of the 8 pathogens, FAM fluorescence (orange), HEX fluorescence (green), ROX fluorescence (red), Cy5 fluorescence (blue), both FAM and HEX fluorescence, both FAM and Cy5 fluorescence, both FAM and ROX fluorescence, and both HEX and ROX fluorescence were increased correspondingly.

The limit of detection (LOD) of MCPC was also estimated by serial dilution of purified DNA templates. Reproducible results from 100 ng to 0.1 ng DNA templates were observed. When converted into copy numbers, the estimated LODs varied from 2.12 x 10 1 copies (V. cholerae) to 3.24 x 104 copies (S. aureus) per reaction, depending on the type of the pathogen. These LOD values were approximately 3–4 orders of magnitude higher than those observed with corresponding uniplex PCRs but were low enough for species identification with pre-enriched samples.

To be useful for routine use, MCPC was also tested with coarsely prepared DNA templates. DNA templates extracted by commercial set and by a simple heating lysis step were compared. No obvious difference was observed between DNA templates prepared by the 2 methods. For routine use, the supernatant collected from heating lysis was diluted 50-fold with water and directly used as PCR templates. Stability studies showed that heat-lysed DNA was still suitable for MCPC assay after 3-month storage at –20 °C.

validation of mcpc assay with a blind test of 118 samples
The blind test of 118 samples by MCPC showed that 101 samples were correctly identified. Eight unrelated bacteria were not detected, and 3 strains of V. cholerae and 6 strains of V. parahaemolyticus isolated from food products were not identified (Table 1 ). To confirm these results, all samples were retested using the corresponding uniplex (single) real-time PCR sets, which showed complete agreement with MCPC analysis. Thus, we reasoned that strains not identified by MCPC may actually not contain the toxigenic target genes, i.e., ctxA for V. cholerae and tdh for V. parahaemolyticus, in their genetic material. These data indicate that the MCPC assay is as specific and sensitive as the uniplex real-time PCR assays but can identify many more species in a single reaction.


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  		Table 1. Comparison of bacterial species identified by MCPC and by classic uniplex real-time PCR analysis.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A new real-time PCR strategy for foodborne pathogen identification has been proposed and experimentally validated. By using a variety of fluorophores and fluorophore combinations to label species-specific probes, 8 probes can be included in 1 tube to target the species-specific genes of 8 foodborne pathogens in a single real-time PCR. As demonstrated by both proof-of-principle experiments and a large-scale validation, all 8 foodborne pathogens targeted are accurately identified and distinguished from other pathogens using the MCPC strategy.

The MCPC assay fills the gap between the current, nucleic acid–based single species-specific detection and high-resolution (sub)species identification methods. Various species-specific PCR detection methods have been developed, but these methods, because of the 1 color/1 target strategy used, have limited applicability in identification of 1 species from a number of possible candidates. On the other hand, many high-resolution identification methods, such as pulsed-field gel electrophoresis (5) and multilocus sequence typing (6), as well as high-throughput techniques, such as denaturing HPLC (22), MALDI-TOF (23), and microarray analysis (24), offer comprehensive sequence information. However, these methods require separate DNA amplification procedures and sophisticated equipment that are not always available on a routine basis. Recently, real-time PCR combined with high-resolution melting analysis has also been explored for rapid bacteria identification (25). In all, 25 clinically important bacterial species could be identified after LightCycler real-time PCR amplification of the 16S rRNA gene in the presence of LCGreen I fluorescent dye followed by high-resolution melting analysis of the PCR products with the HR-1 instrument. However, due to the limitation of 16S rRNA-based typing (e.g., existence of nucleotide polymorphisms among the same species and identical sequences shared by different species) and technical challenges (different sequences may have identical melting curves), further analysis, either by a 2nd PCR or by heteroduplex melting-curve assays, was needed for identification of more than half of the bacterial species. Compared with existing methods, MCPC substantially increases the number of targets that can be identified in a single reaction and significantly decreases the workload, time to result, and the need for sophisticated equipment often involved in high-resolution or high-throughput methods. We speculate that any real-time PCR machines with 4 or more fluorescence detection channels (e.g., ABI Prism® 7500, LightCycler® 2, LightCycler® 480, Mx3005P, Rotor-Gene 6000) are suitable for MCPC analysis. Based on the combination rule, with the advent of 5- or 6-color channel real-time thermocyclers, up to 31–63 targets could be covered by a single-tube MCPC assay.

At first glance, the MCPC assay involves a large number of primer pairs and probes in 1 reaction and seems difficult to establish. In fact, the setup is much easier than conventional multiplex PCR. In the current implementation, only 1 of 8 target sequences in the MCPC reaction is amplified and thus there is no concern about balanced coamplification of different targets. Also, the tagged primer pairs are present at low concentrations and function mainly in the initial rounds of amplification, which allows easy tuning of their concentrations by considering their nearly linear relationship with the observed CT value. Moreover, dual-fluorophore mix-labeled probes function similarly as single-fluorophore unilabeled probes in indicating the fluorescence signals because they are simply the equal molar mixture of the same DNA oligonucleotide labeled with 2 different fluorophores. As embodied in the present study, the primers and probes used in MCPC were directly adopted from the 8 single (uniplex) real-time PCR sets. Optimization involved only slightly adjusting the concentration of primer, dNTP, and DNA polymerase. We are aware that, in the current MCPC setting, any primer dimers, once formed among the tagged primers (even present in small amounts), can be amplified by the universal primer pairs. Although these primer dimers may not cause any false signal in MCPC in the presence of the specific probes, they definitely decrease the overall amplification efficiency. Indeed, higher estimated LOD values were observed with MCPC than with the corresponding uniplex PCR setting. However, such an observation does not compromise the overall performance of MCPC, because in the present setting pre-enriched samples provide much more DNA template than needed for each analysis. The problem may be partially solved by using single-tail strategy (26) to eliminate primer dimer formation and/or by extensive optimization of reaction conditions to reduce nonspecific amplification. Modified molecular beacons were used as probes in the present work because of their convenient availability. Any types of probes used for real-time PCR detection can be adapted to MCPC. However, considering the coexistence of a large number of probes, the so-called dark quencher–labeled probes [such as molecular beacons (27), Scorpion primers (28), displacing probes (29), and MGB EclipseTM probes (Epoch Biosciences)] may be especially useful owing to their relatively low background.

At the current stage, MCPC is limited to identifying only 1 single pathogen among a number of suspected candidates, and its performance may be compromised by sample contamination or coinfections, if single-colony prepurification, a classic laboratory procedure, is not performed before the identification step. Whether an MCPC profile could provide additional information for differentiating single vs multiple infections in clinical specimens without the need of sample prepurification needs further investigation. We speculate that CT value differences could be used for further distinguishing each target signal, especially when the concentrations of each target are different. If combined with melting analysis (30)(31), even multiple targets might be simultaneously identified in 1 MCPC reaction. It should be noted that multiple infections of foodborne pathogens are relatively rare and that DNA templates are often, if not always, prepared from pure single-colony cultures. Thus, we expect that MCPC should find valuable clinical applications in rapid and accurate identification of suspected causative pathogens.


   Acknowledgments
 
Grant/funding support: The work was partially supported by National Science Foundation of China (Grant 30300281), Natural Science Foundation of Fujian Province (Grant 2003Y004), Xiamen Municipal Commission of Science and Technology Key Program, and Xiamen University Action Project.

Financial disclosures: None declared.

Acknowledgments: We thank Drs. Luming Zhou, Mark Poritz, Xilin Zhao, Yongyou Zhang, Jinping Cheng, and Robert Palais for critical reading of the manuscript. We also thank Jianwei Huang for providing bacterial cultures and technical assistance.


   Footnotes
 
1 Nonstandard abbreviations: MCPC, multicolor combinational probe coding; HEX, hexachloro-6-carboxyfluorescein; FAM, 6-carboxyfluorescein; ROX, 6-carboxy-X-rhodamine; CT, threshold cycle; LOD, limit of detection.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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