Study on rapid detection of seven common foodborne pathogens by gene chip

To develop a rapid, effective, specific, and sensitive method to detect foodborne pathogens, 13 sets of primers were designed to amplify the conservative and specific genes of rfbE, fliC, invA, hilA, ipaH, femA, nuc, hlyA, prfA, tuf, speB, tlh and tdh, respectively. Establishment of foodborne pathogens detection chips was conducted by spotting the target genes on the chips by Nano-PlotterTM NP 1.2 printing system. The DNA of 7 standard pathogenic strains and 147 strains extracts from food samples was amplified and labeled for hybridization. The results demonstrated that enterhemorrhagic Escherichia coli O157:H7, Salmonella enteritidis, Shigella flexner, Staphylococcus aureus, Listeria monocytogenes, β-hemolytic streptococcus, and Vibrio parahaemolyticus could correctly be identified by the designed gene chip at an optimal temperature of 58°C and were proved as a potential method with good stability and sensitivity (5 pg/μl of template DNA).


INTRODUCTION
It is well known that food is the basis of human beings.In recent years, hundreds of outbreaks of foodborne infection cases occur in the world (Keener et al., 2014;Scallan et al., 2015;Centers for Disease Control and Prevention, 2013) and the species of bacteria causing foodborne infections have continuously become more diversified (Van Doren et al., 2013;Crim et al., 2015;Korsak et al., 2015), resulting in serious harmfulness to human's health.Generally, the food-borne pathogens are some micro-organism from food-processing and transporting, which usually are the main murderers making human's diseases.Therefore, analyzing pathogenic bacteria in food is a standard practice to ensure safety and quality of the food.Presently, there are *Corresponding author.E-mail: fjwzhuhai@sina.com.cn or Xiaoyanhu5@aliyun.com.Tel: +86 756 3333061.Fax: +86 756 3333061.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License many methods, such as culture-medium enriching, culturing, isolating, and biochemical identification available to detect foodborne pathogens (Feng et al., 2013;Wang et al., 2014Wang et al., , 2012)).However, these methods are as long as 3-to-7-day duration, laborious, and fewer species detected.Compared with the above methods, though polymerase chain reaction (PCR) is rather rapid, its disadvantages are that only one strain and gene can be detected at a batch sample, which greatly lowers the working effectiveness and prevents the feasibility of detecting high-flux samples.Further studies are needed to develop rapid and objective methods for foodborne pathogen detection.In this study, the multi-gene methods have been successfully applied for the simultaneous detection of common foodborne pathogens in real food samples.The design of primers, the amplification of PCR, and the formation of gene chip matrix of probes are based on the steady virulence gene or non-virulence gene in the enterohemorrhagic Escherichia coli (EHEC) O157:H7, Salmonella enteritis, Shigella, Staphylococcus aureus, Listeria monocytogenes, β-hemolytic streptococcus, and Vibrio parahaemolyticus.Our methods are highly specific, sensitive, time-saving, and effective in the simultaneous detection of foodborne pathogens.The established methods have shown satisfactory results applied to detect the 7 standard strains of foodborne pathogens and 147 isolation strains from food samples.

Seven food isolation strains
Each isolation strain is composed of 21 strains, including 5 negative and 16 positive ones, and all were granted by the Food Safety Laboratory of Technology Center, Zhuhai Entry-Exit Inspection, and Quarantine Bureau (Zhuhai, China).

Design of the primers
The good conservative domains were picked by DNAMAN software from the conservative and specific genes of seven types of foodborne bacteria.The various primers (Shanghai Yingjun Co. Ltd., China) were also designed by Array Designer 2.0 software (Primer Biosoft International, CA).

Design of aligned genes (positive references)
The aligned gene was picked up from one specific DNA gene existing in the lambda bacteriophage, a positive reference, used as quality control (QC) in the experimental program.The results will not be reliable unless the aligned gene was found.The designs of prime and the composition of aligned genes are identical to the aforementioned workflow.

Extraction and preparation of the target and the aligned genes in samples
The extraction and preparation of DNA sample template was processed based on the User's Manual from TIANamp Bacteria DNA Kit.The amplification was processed by using PCR gradient instrument and then the production was recovered by the gel-cut after electrophoresis (the annealing temperature of the primers shown in Table 1.The cut gel was amplified as a template after tenfold dilution and the purification was processed with isopropyl alcohol precipitation method.The amplification gene was sequenced by Shanghai Yingjun Company and Shanghai Biological Engineering Company (Shanghai, China), from which the obtained data was analyzed by Blast search and DNAMAN software.

Preparation of the gene chip of foodborne pathogens
The concentrations of target gene and aligned gene were diluted to 250 to 300 ng/μl prior to spotting on the amido slide by spotting instrument.Three sets of comparative experiments were designed as follows: positive group (aligned gene), negative group (3×SSC), and blank group (Milli-Q water), among which each sample had ten same positions and each slide repeated two arrays.Prior to being fixed at 65°C for an hour in an oven, the prepared samples should first be dried overnight; next hydrated and crosslinked by UV; finally, centrifugated after washing by 0.2% SDS solution and stored at room temperature.

Hybridization, scanning and data analysis for gene chip
Gene chip was hybridized with the introduced probes at 58°C for 10n to 18 h after pre-hybridization at 50°C for an hour.After hybridization, the chip was in turn washed by cleaning solution 1, 2, 3, and Milli-Q water, prior to being centrifuged and dried, respectively.The gene chip signal was obtained by a 532-nm excitation light source with 100% laser power, 600 Photo Multiplier Tube (PMT) Gain, and 10-μm resolution.The final information was taken using GenePix Pro Ver.4.1 software, both mean values of ten-point signals from same target gene and ten-sample signal-tonoise ratio (SNR) were used to characterize the signal value of hybridization for each target gene.

Optimized temperature of hybridization of gene chip
Different tagged probes and prepared chip were hybridized at 52, 56, 58, and 60°C, respectively.The optimized temperature was obtained by analyzing specificity of hybridization of gene chip at different temperature.

Testing of specificity and sensitivity
The testing of specificity has been done based on seven probes of standard foodborned pathogens and detection chip hybridized at an optimized temperature, respectively.The results have shown that there are not distinct interferences in difficult matrix samples.Additionally, DNA template purified by Shigella was diluted to 3000, 300, 30, 10, 5, and 1 pg/μl and then hybridized with relevant detection chip before getting data and verifying sensitivity of chip by fluorescence scanner.

Testing of isolated food strains
The data was obtained by scanning after the isolated food strains were amplified to notate by relevant primers and hybridized with the detection chips, successively.

Design of primers, sequence comparison of fragment and analysis of homology
Fourteen pairs of primers were made among which rfbE, tdh, and λDNA were candidates and the remaining was designed by array designer (design software of gene-chip primer).As shown in Table 1, the target gene fragments with other genus or species strains have more than 88% sequence homologies except tdh gene.It approved that the designed target gene fragments have shown good conservation and are irrelative with homology with nucleoside sequence of other genus and species.Moreover, Less than 43% ratio of homologies was produced between 13 sets of target genes fragments and aligned genes, greatly preventing gene-gene from being hybridized, which theoretically guaranteed the good specificity of detecting chip.Moreover, double gene combined detection supplied to each foodborne pathogen will greatly lower the probability of false positive and improve the specificity in the difficult matrix samples.

Testing of amplification effect and specificity of target gene and aligned gene
In this research, thirteen target genes and one aligned gene were successfully amplified from seven types of foodborne pathogens and λbacteriophage DNA gene, respectively.According to the data obtained by nucleic acid protein spectrophotometer, the concentrations and the purities (A260:A280) of genes were 449.3 to 1917.0 ng/μL and 1.70 to 1.87, respectively.Figure 1 shows that the strips of targeted genes and aligned genes are not distinct interferences and tailing found, meaning that the methods may be reasonable for the successful application of the detection.

Preparation of probes and detection of concentration
Probes were prepared by using enzymatic reaction notation.The experimental data shows that the values of A260:A280 were 1.70 to 1.96 and the concentrations were 307.2 to 846.3 ng/μl, consistent with the experimental requirements (Figure 2).

Experimental results of target gene at different temperature
The gene chip was hybridized at 52, 56, 58 and 60°C, respectively.The experimental data showed that better results can be obtained at 56, and 58°C, where each target gene had a strong signal of hybridization with relevant one on the detection chip and visible interference signals were 'hidden'.Taking into account the signal intensity and stability, 58°C was employed as the optimized temperature.

Experimental results of hybridization of seven pathogens
The tagging probes can well hybridize with the detection chips (Figure 3a to g).The results showed that more than both 500 strong signals and 4.0 SNR were obtained and invisible interference signal appeared.These proved that the prepared chips had good specificity and did not hybridize with irrelevant genes.

Sensitivity of gene-chip detection for Shigella
When the concentrations of template DNA varied from 5 to 3000 pg/μl, SNR was 1.5 above and all signal intensities of hybridization were more than 150, with distinct hybridization spots.Notably, when the concentration was down to 1 pg/μl, SNR was 0.40 and the signal intensities of hybridization were less than 100, without visible signals that appeared.These indicated that 5 pg/μl was the sensitivity of detection in this study.

Specificity, repeatability and sensitivity of the isolation strains for food samples
Using the detecting chips of gene, the detecting results of 147 food isolation strains of seven foodborne pathogens have shown that the probes from 16 positive strains and the targeted genes on the chips had strong hybridization signal, meaning good repeatability; at the same time, there are no distinct interferences between positive strains and targeted genes of other strains, also five negative strains did not show any hybridization signal, bringing high specificity.
In difficult matrix samples, the detecting results of 147 food isolation strains displayed that the limit of detection can be 5 pg/μl.

Analytical applications
Each probe appeared strong signal of hybridization with relevant target gene but did not display similar phenomenon with irrelevant ones.High sensitivity of detection and good specificity had proved that our methods can well be applied to rapid and simultaneous detection of foodborne pathogens in real food samples.

Design of the target gene chip
The selection of the target genes will greatly affect the detection chip, so the target genes must be designed in accordance with conservative and specificity in its genus or species.Using the homology analysis of the detection genes, it can be clearly know whether target gene may be detected.Girke et al. (2000) have reported that the hybridization of crossing can be processed in the case of more than 70 to 80% homologies of gene sequences, whereas the hybridization will least or even do not appear under 75% homologies of sequences (Schena, 2003).In this study, the homology between 13 target genes and 1 aligned gene is less than 43%, indicating a good specificity.The homologies of the fragments are more than 88% in the remaining target genes and different genus or species except 70% homology in tdh, displaying a satisfactory conservative.The remaining bacteria were simultaneously detected by bi-genes except Shigella, thus greatly lowering the ratio of false positive and improving the specificity of detection.

UV-crosslinked and hydration time
To obtain a uniform distribution after inoculation, DNA must be re-hydrated and dried quickly.It is worthy of mentioning that excessively low water temperature and insufficient time will greatly suffer irregular sampling sites which affect the following hybridization and data analysis.By contrast, the sampling sites may be rapidly enlarged under excessively high water temperature and time and thus lead to the pollution of blending of the sampling sites.Generally, the optimized temperature and time is 70 to 80°C and 10 s, respectively.In addition, the chip should be crosslinked from a 10-cm vertical distance by a 15-mJ UV irradiation, which will successfully form a crosslink bond between a fraction of thymine residues in DNA and amino groups on the surface of slide, thus greatly improving the fixed effect.Noticeably, DNA will severely be destroyed for excessive crosslinking time, 20 min were taken as the optimization time.

Treatment parameters of images and choice of signal output mode
The images were scanned to produce the exactly aligned sites of gridding by GenePix Pro Ver.4.1 chip data analysis software and then automatically analyze signal median value, mean value, SNR and standard deviation in each sampling site.Among the mentioned data, the median value is widely used (He and Zhou, 2008;Sarder et al., 2008;Wentzell and Karakach, 2005) owing to its less sensitivity with fragments and dust pollution (Wentzell and Karakach, 2005).In this work, compared with mean value, one is that the median value can better embody hybridization signal in the case of pollution or high background signal; the other is that SNR is relative to signal and background and may well evaluate reliability of the obtained data, bigger SNR value with stronger sampling site signal and weaker background signal.Based on the discussion, median value and SNR were used to characterize the hybridization results.

Detection standard of positive signal
So far, there has still not been a standard model for gene chip adjustment.Al-Khaldi et al. (2004) have reported that the adjustment standard is to contrast the fluorescence signals in the sampling sites and the probe sites.Murray et al. (2001) have suggested that 55% pixels in the sampling sites are above 1.5-fold local background and the intensities of signals are more than the standard deviation of 2-fold mean value of background signal (Murray et al., 2001).Until now, it is an accepted-widely method to find a proper threshold such as background mean value plus 2-fold variance or negative contrast mean signal plus 2-fold variance, in which the value of less than threshold should directly be ignored and the remaining should be kept for further data analysis.It was considered that the reliable adjustment standard of positive signals is based on more than 1.5 SNR, visible hybridization sampling sites in the scanned images and more than 150 median value of fluorescence intensity in the 10 repeated sampling sites of target genes.

Table 1 .
Related information on the primers of target gene.