Validation and Application of FBPP: Software to Design Foodborne Pathogen PCR Primers And Probes

The nucleic acid base method is a widely used method in foodborne pathogen detection as a rapid method and confirmation of traditional methods. This method is simply hybridizing the target region sequence of a foodborne pathogen with a synthetic oligonucleotide (probes or primers), and the prediction of this primer or probe is a critical step that needs more care. Hence Proper validation of the application used for this prediction is important. In this paper, we validate FBPP application by the evaluation of the predicted primers takes place at the three levels: genus level by using the Inv A gene for all salmonella species; species level by using the HipO gene for only Campylobacter jejuni species; and serotype level through the O antigen flippase gene for Escherichia coli O157 : H7 . It includes measures of inclusivity (detection of the target microorganism) and exclusivity (non-detection of non-target microorganisms) as recommended for the performance characteristics of the PCR-based method (ISO 22118). Furthermore, the sequence of the PCR product has been verified to confirm the correct PCR product has been amplified. The results demonstrate the ability of FBPP to create useful primers for foodborne pathogens under the three levels of evaluation.


INTRODUCTION
Foodborne diseases are an important cause of morbidity and mortality, and a significant impediment to socioeconomic development worldwide (Organization, 2017).One strategy to lower the incidence and the costs of food borne illnesses is the detection of foodborne pathogens to provide safe food supply and to prevent foodborne diseases.
The nucleic acid base method is one of the most widely used method in food applications.It is considered a rapid method used in the detection of foodborne pathogens; moreover, recently it has been part of the conformation test of conventional methods, for instance, ISO method for Bacillus cereus group (ISO 7932 :2004/Amd1:2020) (Standardization, 2020) and Vibrio parahaemolyticus (ISO 21872-1:2017) (Standardization, 2017).it depends on the hybridizing of the target nucleic acid sequence with a synthetic oligonucleotide (probes or primers) that is complementary to the target sequence (Zhao, Lin, Wang, & Oh, 2014).
The process of predicting Primers and Probes Design is essentially important in this method, especially for detection purposes, and it is a preliminary and critical step (Soliman, Azab, Hussein, Roushdy, & Abu el-naga, 2024).This task can be challenging because it requires the identification of highly unique conserved regions of target sequences and the ability to display highly sensitive (i.e., able to amplify its intended target), and specific (i.e., able not to amplify any non-target) (Sachse & Frey, 2003).In addition to computing the biological parameters of the primers, like GC content, melting temperature, and the formation of secondary structures, which include self-dimers, hairpins, and crossdimers, they are essential for evaluating the efficient amplification of a target sequence (Yuryev, 2008).
There are a number of public software tools available to predict primers / probe design (Abd-Elsalam, 2003).However, these tools often present limitations in their capabilities to define the target region.Also, the users need to evaluate the primer specificity by using additional tools that are not sensitive enough to detect targets that have a significant number of mismatches and consequently not suitable to apply in the nucleic acid detection method.There are several simulation software programs that do not have the ability of primers design but rather determine the amplification targets of user-supplied primer pairs (Ye et al., 2012), and some of them are not easily usable without skill in bioinformatics (Sobhy & Colson, 2012).
FBPP, an open-source Python-based application, could: (i) design primers /probes; (ii) perform PCR and gel electrophoreses Photo simulation.(iii) checks the specificity of primers / probes, to overcome many of the earlier limitations (Soliman et al., 2024).However, it requires careful validation and evaluation before it can be adopted as a primer / probe design tool.
The aim of this paper is to validate and apply the FBPP application as a useful tool for predicting primers and probes in foodborne pathogen nucleic acid base detection methods.
MATERIALS AND METHODS 1-Design of Specific Oligonucleotide Primer: Three primers were designed using the FBPP application (Soliman et al., 2024), and the common published primer (16s rRNA) (Lane, 1991) were synthesized by Invitrogen (Thermo Fisher Scientific), then reconstituted as the below equation to prepare master stock 100µM.100µ =   ℎ  + ( × 10 µ   2) Primers were designed for Salmonella species (spp.) based on the InvA gene (Sachse & Frey, 2003), Campylobacter jejuni based on the hipO gene (Wu, Hu, & Li, 2022), Escherichia coli O157:H7 based on the O antigen Flippase (Liu et al., 2020), and the 16s rRNA gene.The primer sequences for each gene are illustrated in Table 1.2) were used for the validation of the program.The KWIK-STIK strains are lyophilized microorganism pellets with ampoules of hydrating fluid and inoculating swabs.By cracking ampoules, the fluid flows into a lyophilized bacterial pellet, where the specific bacterial strain is dissolved and ready for transfer to the appropriate agar medium and incubation at a suitable temperature and time (Table 2).Primer, template concentration, and annealing temperature were determined by applying different primer and template volumes, various annealing temperatures and SYBR™ Green PCR Master Mix (Applied Biosystems™) (Table 3) to PCR plate and placing it in QuantStudio™5 (Applied Biosystems™).Then compile the result for Rn and Ct.

4.3-PCR Reaction:
PCR was performed using a thermal cycler (SimpliAmp "Applied Biosystems™").The genomic DNA from 45 strains representing major foodborne pathogens and closely related strains (Table 2) was used as a templet with four different primers (Table 1) for validation.The PCR reaction mixture performed in 50μL consisted of 5μL 10X Dream Taq Green Buffer (ThermoScientific), 5μL dNTP Mix (2mM), 1μL of MgCl2, optimized value of each primer set (10μM), optimize volume of template DNA, Dream Taq DNA Polymerase (1.25U), and water nucleasefree (filled to a final volume of 50μL).
The PCR program was performed with the following parameters: initial denaturation step at 95°C for 3 min, followed by 30 cycles of denaturation at 95°C for 30 s, annealing for 30 s at different grading temperatures according to primers (table 1) , extension at 72°C for 1 min, and a final extension at 72°C for 10 min.The PCR products were purified using the Thermo Scientific GeneJET PCR Purification Kit, quantified by electrophoresis in different concentration s of agarose gel along with the expected amplicon yield, and visualized by ethidium bromide (EB) with the quantitative GeneRuler 100 bp DNA Ladder (ThermoScientific) under a UV transilluminator.

5-DNA Sequencing and Alignment:
Amplicons from the expect band were purified using GeneJET Gel Extraction Kit, and sequencing was performed at Bioneer Company using automated sequence analyzer ABI 3730xl.In order to validate the gene from predicted primers, sequences were input as a query against the NCBI databases and showed similarity by suing clusatlX.

RESULTS AND DISCUSSION
The DNA from all 45 strains was extracted and examined by electrophoresis (Fig. 1.A).high-molecular -sharp bands with a minimum of fuzzy were noted for all strains.Moreover, the concentration of the extracted DNA was determined.The extracted gDNA was used as a template to evaluate the predicted primers designed by FBPP software.
The three predicted primers designed by FBPP were evaluated theoretically by performing a sequence similarity search against the NCBI GenBank database (Primer-BLAST), and the result confirmed that the complete identity of each primer only matches the expected sequence of the target organism.
The PCR conditions were optimized by establishing the primer, template concentration, and annealing temperature for the three predicted primers, as shown in Table 3.Then, the minimum forward and reverse primer concentrations that yielded the maximum ΔRn values and low Ct values were selected.The optimum conditions for the three primers InvA, hipO, and O antigen were 57.4°C for the annealing temperature, 50 ng of gDNA for the template per reaction and 500/500, 500/800, and 500/500 nM for the forward/reverse primer concentrations, respectively.Four PCR experiments were performed using the optimized conditions for each primer to validate the design.Each primer was evaluated against the 45 extracted DNA templates, which contain sequence complementary to target primers and DNA from closely related species and other foodborne pathogen species as non-target templates.
In a way to verify the PCR amplicon, one band from each primer was purified and sequenced.The sequence was evaluated by performing a sequence similarity search against the NCBI GenBank database (BLAST), and the results showed a high similarity of each amplicon with the desired organism; moreover, the sequence similarity with the target gene was determined by a cluster tool and highly showed similarity with the desired gene.
The nucleic acid base method is currently widely applied for the detection of foodborne microorganisms, in addition to being part of conformation of the traditional method (ISO 7932:2004/Amd 1:2020) (ISO 21872-1:2017).(Zhao et al., 2014).The design of Primer and probe in this method is considered a fundamental and critical step (Sachse & Frey, 2003).Hence Proper validation of the tools used for design is a preliminary step (Soliman et al., 2024).Therefore, we have validated and applied primers predicted by using the FBPP tool for detecting the foodborne pathogen to careful validation before it can be adopted as an accepted application.
The evaluation of the predicted primers took place at three levels: genus level by using the primer Inv A gene for all salmonella species; species level by using the HipO gene for only Campylobacter jejuni species; and serotype level through the O antigen flippase gene for E. coli O157.It includes measures of inclusivity (detection of the target microorganism) and exclusivity (non-detection of non-target microorganisms) as recommended for the performance characteristics of the PCR-based method (ISO 22118).Furthermore, the sequence of the PCR product has been verified to confirm the correct PCR product has been amplified.The results demonstrate the ability of FBPP to create useful primers for foodborne pathogens under the three levels of evaluation.Proper validation of the application took place through apply of the ISO standard method (ISO 22118:2011) for the performance characteristics for the detection and quantification of food-borne pathogens by using PCR (Standardization, 2011).According to the last report of the global burden of foodborne diseases from the World Health Organisation (WHO), The most frequent causes of foodborne illness were diarrheal disease agents, particularly norovirus, Campylobacter spp,, nontyphoidal Salmonella enterica, and enteropathogenic Escherichia coli 2017)(Organization, 2017)(Organization, 2017) [14] .So, the evolution of application is applied to these organisms.The genes nominated for three levels, in the first level under the genus level by selecting the InvA gene, this gene is widely used in PCR method detection and use for the detection of all Salmonella spp.(Sachse & Frey, 2003), because it is found in both Salmonella species (Salmonella bongori and Salmonella enterica) (Microorganisms & Toxins, 2005).At the species level, the hippuricase gene (hipO) was used for the identification of C. jejun.The hipO gene is considered the key gene for Campylobacter jejuni other than other campylobacter species (Wu et al., 2022).Finally, for the identification of serotype strains of E. coli primers based on the O antigen flippase gene were designed.Several E. coli O157 were examined by using these gene in PCR reactions (Liu et al., 2020).
Many primers and probes designed by the FBPP program for foodborne pathogens such as Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, and Vibrio parahaemolyticus have been tested and validated in numerous laboratories; furthermore, some of this work is under publication.

Conclusions:
In this paper, we validate and apply FBPP as a specific-purpose tool for predicting foodborne pathogen primers and probes that use the nucleic acid-based detection method.FBPP has been confirmed on the three levels of foodborne pathogen bacteria: genius, specie, and serotype, by using remarkably similar strains of foodborne pathogen, and it was able to create highly selective primers for each of them.This result proves the ability of the FBPP application to predict and probe for the detection of foodborne pathogens.Further development could be applied to the software that can be used to predict multiplex primers.Declarations: Ethical Approval: This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of interests:
The authors have no relevant financial or non-financial interests to disclose.Authors Contributions: All authors contributed to the study conception and design.Material preparation, data collection and analysis were performed Mohamed A. Soliman, Mohamed S. Azab, Hala A. Hussein, Mohamed N. Abu el-naga.The first draft of the manuscript was written by Mohamed A. Soliman and all authors commented on previous versions of the manuscript.All authors read and approved the final manuscript.Funding: The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.
Extraction:By using loop 10 µl, a colony from bacteria strain plates were picked up and resuspended in 500 µl of Saline Solution (0.85%) in 1.5 mL microcentrifuge tube, then centrifugated for 3 min at 10000 X g.Discard the supernatant.Total bacterial genomic DNA was extracted by means of the Thermo GeneJET Genomic DNA Purification Kit (ThermoScientific).According to the manufacturer's instructions.The purified DNA was detected by electrophoresis in 1% agarose gel and visualized by ethidium bromide (EB) with the quantitative GeneRuler 1 kb DNA Ladder (ThermoScientific) under a UV transilluminator, and concentration was measured by using a Qubit 4 fluorometer (Invitrogen), then stored at -20 °C until further use.

Table 1 :
The predicted primer used in validation.

-3` Bacterial species Target gene description Gene ID
2-Theoretical Evaluation of theDesign primer:The three primers predicted by FBPP were evaluated by performing a sequence similarity search against the NCBI GenBank database.3-Bacterial Strains: 45 bacterial strains bought from Microbiologics (KWIK-STIK), and the Microbiological Resources Centre (Cairo MIRCEN) (slant) and collected from King Abdulaziz University, Al Azhar university, and Egypt Atomic Energy Authority listed in Table (

Table 2 :
List of Reference strains used for the validation of program .

Table 3 :
List of PCR components concentration with different annealing Temperature.