Development Of Real-time Quantitative PCR Method For Detection Of Several Food-borne Pathogens

BackgroundFoodborne diseases are recognized as one of the most serious public health concerns today, and are the main reasons for human death and property damage. With travel and trade increasing over the years, the risk of dissemination of pathogens grows continuously. Traditional and standardized analysis of food for the presence of bacteria relies on the enrichment and isolation of presumptive colonies on solid media, using approved diagnostic artificial media. This is usually followed by biochemical and/or serological identification. Traditional methods of detection, though reliable and efficient, require several days to weeks before results are obtained, and may prolong definitive diagnoses and treatment of the patient. Furthermore, phenotypic properties by which the bacteria are identified may not always be expressed; and when expressed, they may be difficult to interpret and classify. Another disadvantage of traditional methods is that cells which are viable but otherwise nonculturable cannot be detected. More over, they can only confirm the present or absent of one pathogen and cannot cater to all the needs of public health surveillance, clinical diagnosis and epidemiological investigation. Thus, the availability of reliable, rapid and accepted test systems to detect the presence or absence, or even the degree of contamination of pathogens, becomes increasingly important for the agricultural and food industry.A technological breakthrough in molecular biology came in 1983 with the development of polymerase chain reaction (PCR). In subsequent years, the greatest recent advancement in amplification technology has been the development of systems that allow monitoring of amplification in real-time, which was in 1992 first proposed by the Japanese Higuchi and used ethidium bromide and a mounted CCD camera to monitor PCR amplification in a closed reaction tube. In 1996, the first quantitative PCR equipment was developed by Applied Biosystems, which achieved the leap of PCR from qualitative to quantitative. The advent of real-time quantitative PCR (qPCR) has overcome a number of short-comings of conventional PCR. qPCR readily allows quantitation of DNA over a broad dynamic range and it is a closed-tube format that requires no post PCR handling for identification of amplicons, reducing the potential for sample contamination and making the entire process more amenable to high throughput analysis. As a result, qPCR now provides researchers and diagnostic laboratories with additional tools for disease diagnosis, identification of species, quantifying gene expression, single nucleotide polymorphism (SNP) detection and monitoring infection loads during therapy.qPCR assays may use the intercalating fluorescent dye, SYBR Green, TaqMan, hybridization probes, Molecular Beacons, or Scorpionsi as the means of detecting amplification. Currently the most widely used, the most promising is the TaqMan probe technique because assay design is relatively simple and assays are generally robust. Taqman probe have a 5' fluorescent reporter molecule and a 3' quencher molecule. Taqman probes are typically 20-30 nucleotides in length and the fluorescence of the reporter molecule is quenched at this proximity between reporter and quencher molecule. Hydrolysis of the Taqman probe by the 5' exonuclease activity of Taq DNA polymerase at 60℃separates the reporter and quencher molecule and a signal is emitted by the reporter molecule that is detected by the real-time PCR instrument. As a result of probe degradation, fluorescent signal increases as a function of the number of amplification cycles and allows specific detection and quantitation of the target DNA. Taqman probes do not allow confirmation that the correct fragment has been amplified (other than by running a gel), and as a result it is particularly important to validate the specificity of primer/probe combinations. Multiplexing Taqman assays is achieved using probes labelled with different reporter molecules that have distinct fluorescence properties (namely excitation/emission maxima). The combinations of fluorophores that can be used is dependant on the technical specifications of the qPCR instrument used (an upper limit of six fluorophores can be detected on some instruments).Sensitivities and specificities of these tests, however, may be affected by the specimen type used, food matrix, medium, nucleic acid extraction reagent, and quality of the primers and fluorescent probes used in the tests, which can interfere in the amplification reaction and result in even a false negative. The reaction inhibition can be partially overcome by improvement of sample pretreatment and use of appropriate DNA extraction protocols, but these sometimes are not efficient enough in the total removal of inhibitors. To recognize false negatives due to inhibition of PCR, the only pathway, currently, is addition of an internal amplification control (IAC) at the appropriate concentration in the same reaction system. This IAC is detected using a second TaqMan probe labeled with a different fluorophore, enabling the simultaneous monitoring of the target and IAC signals. Co-amplification of the IAC and target sequence ensures the attainment of at least one amplification product in every PCR reaction if the DNA extracted is of high enough quality to be amplified.qPCR techniques can determine the number of intact stretches of DNA of specified nucleotide sequence in an extremely small sample; however, these assays must be calibrated with DNA quantitation certified reference material (CRM) with well-characterized and stable composition. CRM would contribute to reducing variability in testing from one laboratory to the next.In this study, foodborne pathogens, such as Staphylococcus aureus (S. aureus), Shigella dysenteriae (S. dysenteriae), Salmonella spp., Clostridium botulinum (C. botulinum) type A, B, and E, were applied to the research target. Pathogen-specific genes were selected through bioinformatics and molecular biology analysis. Several primers and corresponding probes based on these specific genes were designed, and mono-/multi-plex qPCR based TaqMan technique was established, which can not only detect pathogen-specific genes, but also synchronously detect the IAC signal for control of false negative.Lambda phage was selected as the candidate to develop CRM with an accurate and precise mass fraction traceable to the SI, and applied to calibrate genome copy number and standardize qPCR assays for foodborne pathogens:S. aureus, S. dysenteriae, Salmonella spp., C. botulinum type A, B, and E. In this way, CRM could help reduce within-and among-laboratory quantitation variability. At the same time, pathogen-specific gene was cloned to the T vector as the working reference material.Objective1. To develop lambda DNA CRM and several foodborne pathogens working standard reference materials to ensure accurate measurements in qPCR assays.2. To develop several highly sensitive and specific, convenient and rapid qPCR assay for quantitative detection of foodborne pathogens with SmartCycler, and to certify on Light Cycler 2.0, ABI 7300 instruments.Methods1. Lambda phage strainλcI857 Sam7 was selected as the candidate to develop CRM in order to calibrate genome DNA (gDNA).2. S. aureus strain ATCC 6538, S. dysenteriae strain ATCC 51197, Salmonella spp. strain ATCC 14028, C. botulinum type A strain 62A, type B strain 621, and type E strain 619 were selected as the candidate to develop working reference material.3. For development of the qPCR assays for the foodborne pathogens above, specific genes of pathogens were selected; nucleotide sequences were retrieved from the GenBank and aligned by using the ClustalW program to localize homologous regions, allowing the design of specific primers and probes with the Beacon Designer 7.5 software. BLAST searches were performed on all primer and probe sequences to ensure the desired specificities.4. qPCR reaction system was optimized to determine the optimal concentrations of primers and probes.5. This I AC was added in the reaction system and detected using a second TaqMan probe labeled with a different fluorophore, enabling the simultaneous monitoring of the target sequence and IAC signals.6. A triplex qPCR assays for detection of C. botulinum neurotoxin (BoNT) gene fragments specific to C. botulinum type A, B, and E, was developed and enables a simultaneous detection of the three serotypes. 7. The specificity of the qPCR assays was further evaluated by empirical tests against DNA extracted from 61 pure bacteria cultures, including various similar genetic, foodborne and environmental species frequently found in food.8. Specific gene fragments obtained by PCR were cloned into pMD18-T vector to construct recombinant plasmids, which were then linearized with restriction enzyme EcoR I. The standard curve was constructed by plotting the Ct values against the log of the rate of change of fluorescence for a 10-fold serial dilutions ranging from 1.0×107 to 1.0×101 copies of linear recombinant plasmid DNA.9. Analytical sensitivity and limit of detection of qPCR assay was determined by amplification of the 10-fold serially diluted gDNA, linear recombinant plasmids DNA, pure culture cells, respectively.10. The performance of qPCR was evaluated using artificially contaminated drinking water and commercial ultrahigh-temperature treated (UTH) whole milk samples spiked with strains ATCC 6538, ATCC 51197, ATCC 14028,62A,621, and 619, respectively.11. Moreover, we investigated whether these assays produce comparable sensitivity and specificity on these rapid cycling instruments:LightCycler, ABI and Smart Cycler Platforms.Results1. Different methods of extractingλDNA were compared; finally, QIAGEN Lambda Maxi Kit was selected to extract and purify lambda DNA (certified reference material). After that, purified lambda DNA was identificated by restriction enzyme digestion, PCR reaction, and sequencing, and the results showed the DNA extracted to be DNA of lambda phage mutant strainλCI857 Sam 7.2. Two different kits for extracting bacterial gDNA were compared; finally, TIANamp Bacteria DNA Kit was selected to prepare pathogenic gDNA (working reference material).3. The specificity of qPCR was further evaluated using 61 reference strains. As a conclusion, the qPCR unequivocally distinguished target strains from nontarget bacteria and was fully selective.4. The slopes of the linear regression curves calculated over a 7-log range were similar to the theoretical optimum value of-3.32 and showed amplification to be very efficient. The quantification was linear (R2≥0.999) with a PCR efficiency of>90%.5. The sensitivities of the qPCR assays for linear recombinant plasmids DNA were 2 copies,10 copies, and 5 copies per reaction for S. aureus, S. dysenteriae, Salmonella spp., and were 5 copies,2 copies, and 5 copies per reaction for C. botulinum type A, B, and E, respectively. The sensitivities determined using 10-fold dilutions of purified gDNA were 10 fg per reaction for all qPCR assays.6. It is capable of detecting 5.0×101 cfu/ml for ATCC 6538,2.74×101 cfu/ml for ATCC 51197,1.41×101 cfu/ml for ATCC 14028,2.11×101 cfu/ml for 62A,1.02×101 cfu/ml for 621,5.38×101 for 619, which was calculated by standard plating technique, assuming 100% efficiency of DNA extracting.7. Using prior centrifugation, the assay was able to consistently and quantifiably detect 5.0×101 cfu for ATCC 6538,2.74×101 cfu for ATCC 51197,1.41×101 cfu for ATCC 14028 in 25 ml artificially contaminated drinking water samples.8. With a simple detergent and enzymatic sample pretreatment procedure ahead of DNA extracting and PCR amplification, the assay was able to consistently detect as few as 5.0×102 cfu for ATCC 6538,2.74×102 cfu for ATCC 51197,1.41×102 cfu for ATCC 14028,2.11 X 102 cfu for 62A,1.02×102 cfu for 621,5.38X 102 for 619 in 25 ml artificially contaminated UTH whole milk samples, which indicated that the performance of the qPCR assay was excellent.9. This qPCR assay established in the Smart cycler, Light cycler 2.0, ABI7300, can get better results, fully applicable to these different qPCR instruments.Conclusion1. It was the first time to develop theλDNA as CRM in measurement traceability of foodborne pathogens in China.2. The qPCR assays for S. aureus, S. dysenteriae, S. spp., with IAC, could not only be applied for the quantitative dectection of foodborne pathogens, but also avoids the false negatives and underestimations of contamination loads due to PCR failure. The assays will be a useful addition to the range of diagnostic tools available for the study of foodborne pathogens.3. These qPCR assays for the detection of S. aureus, S. dysenteriae, Salmonella spp., C. botulinum type A, B, and E were developed, which are rapid, sensitive, specific, and can be used on different types of instruments, with a wide range of applications in public safety, health monitoring, clinical diagnosis, epidemiological investigation and so on.