P U B L I C A T I O N S CODON ISSN 1120-1770 online, DOI 10.15586/ijfs.v33iSP1.1995 43 change in the lifestyle of consumers, climate changes, and the accompanying rapid changes in food systems. International trade means that unsafe food can be dis- tributed widely (Pinu, 2016; WHO, 2020). Recently, many food-borne disease outbreaks in the world, the most P U B L I C A T I O N S CODON DNAFoil, a novel technology for the rapid detection of food pathogens: Preliminary validation on Salmonella and Listeria monocytogenes Aly Farag El Sheikha1,2,3,4,5 1College of Bioscience and Bioengineering, Jiangxi Agricultural University, 1101 Zhimin Road, Nanchang 330045, China; 2School of Nutrition Sciences, Faculty of Health Sciences, University of Ottawa, 25 University Private Ottawa, ON K1N 6N5, Canada; 3Bioengineering and Technological Research Centre for Edible and Medicinal Fungi, Jiangxi Agricultural University, 1101 Zhimin Road, Nanchang 330045, China; 4Jiangxi Key Laboratory for Conservation and Utilization of Fungal Resources, Jiangxi Agricultural University, 1101 Zhimin Road, Nanchang 330045, China; 5Department of Food Science and Technology, Faculty of Agriculture, Minufiya University, 32511 Shibin El Kom, Minufiya Government, Egypt *Corresponding Author: Aly Farag El Sheikha, College of Bioscience and Bioengineering, Jiangxi Agricultural University, 1101 Zhimin Road, Nanchang 330045, Jiangxi, China. Email: elsheikha_aly@yahoo.com Received: 1 January 2021; Accepted: 4 April 2021; Published: 19 April 2021 © 2021 Codon Publications OPEN ACCESS ORIGINAL ARTICLE Abstract Over the past decades, several tools have been developed for food pathogen detection, including proteom- ics, metabolomics, immunological, biosensor, and nucleic acid-based approaches. Although these techniques are reliable and precise, they are time-consuming, technically challenging, and costly. Hence, it is necessary to develop rapid techniques for food pathogen detection, which can be performed at the household level. DNAFoil mechanism is a portable, completely self-administered, on-site DNA test that does not need expensive instru- ments or settings to confirm food pathogen detection in as little as 30 min. DNAFoil was used successfully for detecting food contamination and adulteration with pork derivatives (down to 0.1%) and vegetal components (down to 0.01%), respectively. In this study, initial validation experiments of DNAFoil were investigated to detect Listeria monocytogenes and Salmonella contamination. To confirm the specificity of the proposed method toward Salmonella, 18 different Salmonella strains, 6 non-Salmonella bacteria, and 2 fungi were investigated; also, in the case of Listeria monocytogenes, five bacterial strains, two fungi, and Listeria monocytogenes were investigated. The results stated that the Swiss Decode Salmonella and L. monocytogenes solutions can detect as few as 1 and 10 copies of DNA per microliter, respectively. The results also showed that the accuracy of our method ranges between 92 and 100%, while the precision value ranged between 88 and 100%. In terms of quality control appli- cability, DNAFoil Salmonella and Listeria monocytogenes reactions could be visually detected with the naked eye using a lateral flow strip, which could be used for in-place quality control during manufacturing and also can be used for more lab tests. In terms of cost, DNAFoil is usually much cheaper than the traditional detection methods. Therefore, DNAFoil is proposed as a promising and universal detection technology for food pathogens. Keywords: DNAFoil technology; food pathogen detection techniques; food safety; foodborne diseases; health and eco- nomics threats Introduction It is well-known that food safety is affected by many factors and variables, including, for example, global- ization of food trade, population increase in the world, Italian Journal of Food Science, 2021; 33 (SP1): 43–54 mailto:elsheikha_aly@yahoo.com 44 Italian Journal of Food Science, 2021; 33 (SP1) El Sheikha AF Salmonella tops the list of foodborne pathogens, with a treatment cost equivalent to $3.6 billion, followed by Listeria monocytogenes, which equals $2.8 billion, and then Escherichia coli with a value of $271 million (USDA ERS, 2014). This is in addition to the cost of recalls of the products as well (Tyco Integrated Security, 2012). Hence, there is an urgent need to develop simple, sen- sitive, specific, robust, reliable, inexpensive, and rapid techniques for food pathogen detection, can perform at the household level and ensure food safety. These requirements comply with new, portable, completely self-administered, on-site DNA test technology called “DNAFoil technology,” which does not need expensive equipment or laboratory settings to get the final results in as little as 30 min (El Sheikha, 2019). Additionally, DNAFoil technique has proven to be effective in: • Detecting food contamination through its ability to detect pork contamination in beef as lower as 0.1% (Meat and Livestock Australia Limited “MLA”, 2018); • Detecting of food adulteration through its ability to detect the adulteration of milk products by vegetal materials as lower as 0.01% (Aronoff et al., 2018). Through five steps (Figure 1), it can be clearly understood as to how this technique works. Sample preparation prominent of which were due to several microbial spe- cies, for example, Listeria monocytogenes, Escherichia coli, Campylobacter jejuni, Salmonella sp., Shigella sp., have proven that food safety is under severe threat from food pathogens (Bintsis, 2017; Chlebicz and Śliżewska, 2018; Faour-Klingbeil and Todd, 2020). Unsafe food con- taining food pathogens, that is, bacteria, viruses, para- sites, or fungi, can cause different diseases ranging from diarrhea to cancers (Dwivedi and Jaykus, 2011; Food Safety Education Program, 2016; WHO, 2020). Centers for Disease Control and Prevention (CDC) esti- mates that each year 48 million people get sick from foodborne diseases, 128,000 are hospitalized, and 3000 die. Foodborne pathogens cause diseases and deaths in all populations, particularly in groups at risk such as infants, children, elderly, and immunocompromised per- sons (CDC, 2020; FDA, 2021; WHO, 2020, 2021). The most common microorganisms responsible for the major foodborne illnesses are shown in Table 1. In addition to the severe health risks caused by foodborne illnesses, they may also threaten international trade and cause significant economic losses. This has been con- firmed by the reports received from the USDA’s Economic Research Service. These reports indicated that foodborne illnesses cost the United States more than $15.6 billion. Table 1. The most common microorganisms responsible for the major foodborne illnesses. Foodborne illness or toxin Associated microorganism Health risks Most population group(s) affected Reference Listeriosis Listeria monocytogenes Meningitis, mild illness in pregnant women, in babies (miscarriage, stillbirth, premature birth, potentially fatal infection after birth) Pregnant women, newborns, the elderly, immuno-compromised individuals Buchanan et al. (2017), Mayo Clinic (2020a) Salmonellosis Salmonella spp. Typhoid fever, inflammatory bowel disease, stomach or bowel disorders All groups Bintsis (2017), Mayo Clinic (2019) Shigellosis Shigella spp. Dehydration, seizures, rectal prolapse, hemolytic uremic syndrome, toxic megacolon, reactive arthritis, bloodstream infections (bacteremia) Malnourished children, immuno-compromised individuals, the elderly NCBI (2017), Mayo Clinic (2020b) Campylobacteriosis Campylobacter spp. Mild to severe diarrhea, bloody diarrhea, stomach pain, cramps, nausea and/or vomiting, fever, muscle pain All groups Bintsis (2017), Ontario Ministry of Health and Long-Term Care (2020) Botulism Clostridium botulinum Breathing problems, trouble swallowing, muscle weakness, slurred speech, headache, nausea All groups Bintsis (2017), Rasetti- Escargueil (2020) Toxoplasmosis Toxoplasma gondii Headache, seizures, lung problems, severe eye infections, e.g., inflammation of retina, enlarged liver and spleen All groups, especially babies, immuno- compromised individuals EFSA and ECDC (2016), Mayo Clinic (2020c) Yersiniosis Yersinia spp. Fever, abdominal pain, diarrhea (which is often bloody) All groups, especially children, adults EFSA and ECDC (2016), Ontario Ministry of Health and Long-Term Care (2018) Amoebiasis Entamoeba histolytica Bowel perforation, gastrointestinal bleeding, stricture formation, intussusception, peritonitis, empyema All groups NCBI (2016), Park (2015) Italian Journal of Food Science, 2021; 33 (SP1) 45 DNAFoil is a promising detection technology anatum, Salmonella Newport, Salmonella thyph- imurium, Salmonella arizonae IIIb, Salmonella saint- paul, Salmonella hadar, Salmonella enteritidis abony); (ii) For exclusivity (Listeria monocytogenes, Bacillus sub- tilis, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Aspergillus brasilien- sis, Candida albicans). Secondly, the strains tested for inclusivity and exclusivity toward Listeria monocytogenes: (i) For inclusivity (Listeria monocytogenes); (ii) For exclu- sivity (Bacillus subtilis, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans). All exper- iments involving living strains were conducted under BSL2 conditions at the independent microbiology lab in Couternon, France. Sample preparation, DNA extraction, and amplification stages DNAFoil mechanism is depicted in Figure 1. Steps 1 and 2 show the DNA extraction, lysing, neutralizing, and stabilizing processes of 200 μL of culture using a barrel without the need to use spin-columns and centrifuges. Briefly, bacteria cells are broken by an alkaline solution that contains chaotropic salts. This allows DNA to be released in the solution. The alkaline pH of the solution is not compatible with downstream DNA amplification; therefore, a neutralization/stabilization step is added, and it consists of buffering the pH with a second solution, which also provides monovalent salts that facilitate the DNA amplification reaction. Amplification stage of DNA target is started via step 3; one drop of extracted DNA is transferred to the reaction tube and is incubated in a water bath at 65°C. Then, using the specific primers and enzymes, the DNA targets will amplify and make multi- ple copies without using thermos-cycling and cold chain. End-point assays After 30 min of incubation at 65°C, DNAFoil strips were dipped into the reaction tube (detection step; 4th step). Migration by lateral-flow caused a positive band’s appearance due to colloidal gold concentrating on the DNA capture line without using electrophoresis and staining (detection step; 5th step). Real-time confirmatory assays For real-time assays, 1 μL of extract was combined with 24 μL of Salmonella and Listeria monocytogenes reac- tions mix and incubated at 65°C. Fluorescence (FAM channel) was monitored every 20 s, and the fluorescence signal was plotted over time. without the need to be pre-enriched before analysis is considered the main obstacle in most methods, but the enrichment remains essential for the revival of stressed or injured cells (Cossarizza et al., 2017; Valderrama et al., 2016). But, through the DNAFoil mechanism, the sample preparation and DNA extraction stages were completed in a single step without the need to use spin-columns and centrifuges. For the amplification stage, cross- contamination is one of the difficulties faced by the com- mercially available kits used to detect food pathogens, that is, Salmonella and Listeria monocytogenes (Baraketi et al., 2018). In contrast to what happened using the DNAFoil technique, it is obvious that the DNA target amplification is done in one pot master mix without requiring trained staff, using thermos-cycling and cold chain. For the final stage (DNA detection stage), there are several problems generated from DNA electrophoresis and staining such as it being time-consuming, gel prepa- ration, smearing, mutagenicity, toxicity, lower efficiency, etc. (Drabik et al., 2016; Hall, 2020). DNAFoil as a detec- tion method during the final stage provides the test strip material, which allows for transport by a capillary force of the target DNA through the detection surface, allowing the target to hybridize specifically to their complemen- tary capture sequences (target DNA fragments are cap- tured in a band). Conjugation of micrometer-sized beads to DNA permits the results to be visualized by the naked eye (visible color reaction), enabling immediate, simple to interpret, cost-efficient, and on-site detection, while eliminating the need for advanced expensive instrumen- tation (El Sheikha, 2019). Hence, the aim of this study was to investigate initial validation experiments of the DNAFoil technique to detect food pathogens, that is, Salmonella and Listeria monocytogenes. Materials and Methods Reference materials Crude bacterial DNA extracts were purchased from the Culture Collection of Switzerland (CCOS), and exper- iments were conducted at the Swiss Decode labs in Renens, Switzerland. Live strains tested were procured from the Pasteur Institute (France). The strains tested were divided into two groups one for inclusivity and the other for exclusivity toward Salmonella and Listeria monocytogenes as follows. Firstly, the strains were tested for inclusivity and exclusivity toward Salmonella: (i)  For inclusivity (Salmonella Montevideo, Salmonella Heidelberg, Salmonella mbandaka, Salmonella enterit- idis, Salmonella agona, Salmonella Indiana, Salmonella infantis, Salmonella arizonae IIIa, Salmonella senfent- berg, Salmonella cerro, Salmonella Virchow, Salmonella 46 Italian Journal of Food Science, 2021; 33 (SP1) El Sheikha AF DNA Extraction Stage “Without the need to use spin-columns and centrifuges” Amplification Stage “Without using thermos-cycling & cold chain” Detection Stage “Without using electrophoresis & staining” As 1st step of DNA extraction: Get 200 µL of culture you want to test for DNA extraction using barrel Transfer one drop of extracted DNA to the reaction tube and incubate it for 30 min in a water bath at 65°C, then use specific primers & enzymes After 30 min of incubation, DNAFoil strip was dipped into reaction tube & migration by lateral-flow was started Positive band means presence of Solmenella’s DNA or Listeria monocytogenes’s DNA As 2nd step of DNA Extraction: Mix hot and cold water in the container provided with the test kit Postive Negative Figure 1. Procedural diagram for the mechanism of food pathogen detection using DNAFoil technology. Source: Adapted from El Sheikha (2019). Reproduced with permission of Elsevier. Statistical analysis All data were presented as the mean value ± standard devi- ation (SD) of independent experiments on various days. Performance metrics Accuracy (%) Results from the experimental specificity (Figure 3A and B) were used to calculate the method accuracy using the following equations: ( ) ( ) Accuracy (%) at 10 min TP TN 100 TP TN FP FN × + + = + + (1) ( ) ( ) ( ) TP TN Accuracy % at 15 min 100 TP TN FP FN + = × + + + (2) ( ) ( ) ( ) TP TN Accuracy % at 20 min 100 TP TN FP FN + = × + + + (3) where TP, FN, FP, and TN represent the number of true positives, false negatives, false positives, and true nega- tives, respectively. Precision (%) Results from the experimental specificity (Figure 3A and B) were used to calculate the method accuracy using the following equations: ( ) ( ) TP Precision % at 10 min 100 TP FP = × + (4) ( ) TPPrecision % at 15 min 100 (TP FP) = × + (5) ( ) TPPrecision % at 20 min 100 (TP FP) = × + (6) where TP, FN, FP, and TN represent the number of true positives, false negatives, false positives, and true nega- tives, respectively. Results Inclusivity and exclusivity To confirm the specificity of our method toward Salmonella, 18 different Salmonella strains, 6 non- Salmonella bacteria, and 2 fungi were investigated; also, in the case of Listeria monocytogenes, five bacterial strains, two fungi, and Listeria monocytogenes were investigated. Independent microbiology lab prepared cultures con- taining 108 CFU/mL. Swiss Decode analyzed the broth media in a blinded manner. The amplification time for each strain is reported in Figure 2A and 2B. The standard amplification time used for our DNAFoil kit is 30 min. Samples for which the amplification signal was not detected after 30 min were considered as negative (no presence of Salmonella or Listeria monocytogenes). Swiss Decode Salmonella solution positively identified the 18 Salmonella strains after 10 to 13 min (Figure 2A). Italian Journal of Food Science, 2021; 33 (SP1) 47 DNAFoil is a promising detection technology S. m on te vi de o S. h ei de lb er g S. m ba nd ak a S. e nt er iti di s S. a go na S. in di an a S. in fa nt is S. a riz on ae Il la S. s en fe nt be rg S. c er ro S. v irc ho w S. a na tu m S. n ew po rt S. th yp hi m ur iu m S. a riz on ae Il lb S. s t P au l S. h ad ar S. e nt er iti di s ab on y Li st er ia m on oc yt og en si s Ba ci llu s su bt ilis En te ro co cc us fa ec al is Es ch er ic hi a co li Ps eu do m on as a er ug in os a St ap hy lo co cc us a ur eu s As pe rg illu s br as ilie ns is C an di da a lb ic an s 0 5 10 A m pl ifi ca tio n Ti m e (m in ) 15 20 25 30 Not Detected Strains Li st er ia M on oc yt og en es Ba ci llu s su bt ilis En te ro co cc us fa ec al is Es ch er ic hi a co li Ps eu do m on as a er ug in os St ap hy lo co cc us a ur eu s As pe rg illu s br as ilie ns is C an di da a lb ic an s0 5 10 15 20 25 30 Not Detected Strains A m pl ifi ca tio n Ti m e (m in ) (A) (B) Figure 2. Amplification time—selectivity experiment. (A) Different cultures of Salmonella (in white), non-Salmonella bacteria (in black), and fungus (in gray) were prepared at an independent microbiology lab. (B) Listeria monocytogenes (in white), dif- ferent bacterial cultures (in black), and fungus (in gray) were prepared at an independent microbiology lab. Broth media (200 μL) was withdrawn from cultures containing 108 CFU/mL. Bacteria were lysed according to the standard DNAFoil method. DNA detection was performed with a real-time assay according to the Swiss Decode protocol. Data represent mean ± SD, n = 3. 48 Italian Journal of Food Science, 2021; 33 (SP1) El Sheikha AF 0 0 500 H2O H2O 1/10 1/10 1/100 1/100 1/1000 1/1000 1/10000 1/10000 1/100000 1/100000 1000 1500 2000 2500 3000 3500 4000 4500 5000 3 6 9 12 15 Time (min) Fl uo re sc en ce 18 21 24 27 30 0 0 500 H2O H2O 1/10 1/10 1/100 1/100 1/1000 1/1000 1/10000 1/10000 1000 1500 2000 2500 3000 3500 4000 4500 5000 3 6 9 12 15 Time (min) Fl uo re sc en ce 18 21 24 27 30 Figure 3. Amplification time-sensitivity experiment. Ten times serial dilution of crude DNA extracts from Salmonella enterica and Listeria monocytogenes were diluted in 10 mM TRIS pH 8.0 or DNAFoil lysis buffer. One microliter of the lysis solution was taken to run a real-time assay. (A) We can observe that the time needed for the detection of Salmonella increases as the amount of DNA present in the sample decreases. However, the detection time is well below the 30 min used as our standard amplifica- tion time. (B) We can observe that the time needed for the detection of Listeria monocytogenes increases as the amount of DNA present in the sample decreases. However, the detection time is well below the 30 min used as our standard amplification time. (A) (B) Italian Journal of Food Science, 2021; 33 (SP1) 49 DNAFoil is a promising detection technology enrichment) followed by biochemical tests (metabolic fingerprinting), molecular tests (typically PCR [poly- merase chain reaction]), or mass spectrometry (Adzitey et al., 2011; Corry et al., 2003; Ellis et al., 2019) to con- firm that the isolate is indeed the pathogen of interest. The gold-standard-based methods have the advantages of being inexpensive, detecting only viable pathogens, and yielding isolates that can further be studied (Adzitey and Huda, 2011; Engberg et al., 2000). However, they are cumbersome, relatively slow, and less efficient (Foddai and Grant, 2020; Jasson et al., 2010; Keramas et al., 2004; Li and Zhu, 2017; Myint et al., 2006). Regarding the bio- chemical and mass spectrometry methods, they are rapid, sensitive, and accurate techniques that involve the analy- sis of entire microbial cells or their extracts (Beale et al., 2014; Cevallos-Cevallos et al., 2011; Singh et al., 2011; Singhal et al., 2015; Toscano et al., 2018; Wu et al., 2016; Yang et al., 2015). However, they are labor- intensive, costly, and the reliance on existing spectral databases of the mass fingerprints of known microbes makes mass spectrometry techniques incapable of identifying new species (Anderson et al., 2012; El Sheikha and Hu, 2020; Jadhav et al., 2018; Mirmajlessi et al., 2015; Reta et al., 2020). Molecular techniques have the advantage of being Other non-Salmonella strain samples were still negative after 30 min. Our method also allowed Salmonella’s iden- tification in broth media samples where Salmonella was mixed with several other non-Salmonella strains (data not shown). Regarding the Listeria monocytogenes, Swiss Decode L.  monocytogenes solution positively identified the Listeria monocytogenes strains after 11 min (Figure 2B). The other strain samples were still negative after 30 min. Limit of detection (LOD) with serial dilutions To determine the sensitivity of our method, a serial dilution of crude bacterial DNA extracts was analyzed. Crude extracts for Salmonella Enterica subspc. enter- ica and Listeria monocytogenes were obtained from CCOS. Crude extracts containing 105 CFU/μL were serially diluted 1:10 either in 10 mM TRIS pH 8.0 or in our DNAFoil lysis buffer. The reactions were analyzed in duplicate by real-time assays (Figure 3A and 3B). The results were similar for both dilution methods (data not shown). Accuracy and precision (%) Accuracy and precision (%) are calculated to measure the performance of our method to identify Salmonella and Listeria monocytogenes. These results are shown in Figure 3A and 3B. The results also showed that the accuracy of our method ranges between 92 and 100%, while the pre- cision value ranged between 88 and 100%. Point-of-need detection with lateral flow As real-time thermocyclers may not be present at the point-of-need (i.e., factory), we verified if the DNAFoil Salmonella and Listeria monocytogenes reactions could be visually detected with the naked eye using a lateral flow strip. Serial dilutions of Salmonella enterica and Listeria monocytogenes extracts were prepared and amplified as before. After 30 min of amplification at 65°C, the results were confirmed with DNAFoil strips (Figure 4A and 4B). Discussion The detection of foodborne pathogens has historically been culture-, or conventional-, or cultural-, or gold- standard-based methods, which were used since the inception of microbiological sampling (Adzitey and Huda, 2010, 2011; Bhunia, 2014). These methods mainly involve enrichment (pre-enrichment and/or selective S al m on el la s am pl e (1 /1 00 00 0) Water Water Listeria monocytogenes (1/10000) (B) (A) Figure 4. Strips results. (A) Amplified samples from Salmo- nella dilution 1/100,000 were applied on DNAFoil strips (trip- licate). A few minutes later, we could see bands appearing. The band on the left confirmed the presence of Salmonella, whereas the band on the right is the positive control of the strips. (B) Amplified samples from Listeria monocytogenes dilution 1/10,000 were applied on DNAFoil strips. Four min- utes later, we could see bands appearing. The band on the left confirmed the presence of Listeria monocytogenes, whereas the band on the right is the positive control of the strips. 50 Italian Journal of Food Science, 2021; 33 (SP1) El Sheikha AF purity in 30 min, without lab equipment, technicians, or scientists. The final report provided by Meat and Livestock Australia Limited (MLA) (2018) illustrated that the DNAFoil kit is able to detect pork contamination in beef as lower as 0.1%. The present study illustrated that the DNAFoil is a fast-detection technique of Salmonella and Listeria monocytogenes that can get the final results in as little as 30 min. In addition, the results of this study stated that the Swiss Decode Salmonella and L. monocytogenes solu- tions can detect as few as 1 and 10 copies of DNA per micro liter, respectively. However, the commercially avail- able kits used to detect food pathogens, that is Salmonella and Listeria monocytogenes, which are based on nucleic acid for detection, are characterized by reliability, high specificity, and sensitivity; they are limited by the diffi- culties of: • differentiating the viable cells from nonculturable cells; • the primers’ design. Moreover, these kits require trained staff to avoid cross-contamination (Baraketi et al., 2018). Table 2 eval- uates the commercial kits used to detect Salmonella and Listeria monocytogenes. In terms of performance metrics (accuracy, precision %), the results show promising per- formance, to be used for Salmonella and Listeria mono- cytogenes detection. The DNAFoil technology is efficient in terms of cost and quality control applicability In terms of cost. DNAFoil is available to the partners (academic and industry) that accede to the Early Access Program. The fee to enter the Early Access Program is rapid, less laborious, more sensitive, specific, and effi- cient, compared to the conventional method (Keramas et al., 2004; Magistrado et al., 2001). Nonetheless, certain components/compounds in foods such as fats, lipids, and salts, enrichment media, or DNA extraction solution can inhibit the sensitivity of PCR-based methods (El Sheikha, 2010; Rossen et al., 1992; Wilson, 1997). To overcome the limitations of traditional methods used for pathogen detection from the side and from the other side to meet industrial and commercial food needs, there is an urgent need for fast, sensitive, accurate, and more efficient detection methods in terms of saving time, labor, and preventing human errors (Baraketi et al., 2018; Law et al., 2015; Mandal et al., 2011; Rajapaksha et al., 2019). Hence, the importance of answering the principal ques- tion, namely, why is DNAFoil technology proposed to detect food pathogens? This question is the hypothesis on which the research idea was based, and which the results of this study approved and provided the answers as follows: DNAFoil is a fast, accurate, precise, sensitive, and reliable technique As a new technology that needs assessment, the “real- time” amplification technology (real-time PCR) is used to evaluate the efficacy and accuracy of DNAFoil technology (El Sheikha, 2019). Aronoff et al. (2018) reported that the efficiency of DNAFoil kit used to detect the vegetal mate- rial in milk products (DNAFoil UniPlant) was confirmed using real-time PCR assays. The same authors concluded that the DNAFoil UniPlant kit provides a quick and reli- able method to validate product content with less than 1% adulteration of any product, confirming identity and Table 2. Commercially available kits based on nucleic acid methods for the detection of foodborne pathogens.* Pathogen Commercially available kits Sensitivity Sample matrix Company Salmonella sp. BAX® System Standard PCR assays for Salmonella 104 CFU/mL, after enrichment Poultry, dairy, fruits, vegetables, bakery products, pet food, environmental samples HYGIENA BAX® System Real-time PCR assay for Salmonella 104 CFU/mL, after enrichment Meat, poultry, dairy, fruits, vegetables, bakery products, pet food, environmental samples HYGIENA GeneQuence® for Salmonella 1–5 CFU/25 g Food and environmental samples HYGIENA Listeria monocytogenes BAX® System PCR Assay for L. monocytogenes 105 CFU/mL, after enrichment Variety of food types HYGIENA BAX® System PCR Assay for L. monocytogenes 24E 104 CFU/mL, after enrichment Dairy, meat, fish, vegetables, environmentals HYGIENA BAX® System Real-Time PCR Assay for L. monocytogenes 104 CFU/mL, after enrichment Dairy, ready-to-eat meat, seafood, vegetables, environmental samples HYGIENA GeneQuence® for L. monocytogenes 1–5 CFU/26 g Food and environmental samples NEOGEN *Source: Baraketi et al. (2018). Licensed under Creative Commons Attribution 3.0. PCR, polymerase chain reaction. Italian Journal of Food Science, 2021; 33 (SP1) 51 DNAFoil is a promising detection technology Although DNA-based techniques have proven to be the best detection tools in food pathogen detection, at the industrial level its practical application has to go a long way (El Sheikha et al., 2018; Zhao et al., 2014). Hence, the demand for a novel, rapid, easy, potent, and universal technology for food pathogen detection is still an urgent need. Therefore, it is hoped that the DNAFoil technology could be a powerful tool that meets all of the require- ments for food pathogen detection and its applications either at the household or industrial level. As a future trend, more applications are recommended for DNAFoil technology as a food pathogen detection tool on different food matrices. Moreover, the DNAFoil test kits for Salmonella and Listeria monocytogenes are qualified for further validation using ISO16140. Compliance with Ethical Standards Acknowledgments This research was supported by the Research Start-Up Fund from Jiangxi Agricultural University, China that granted to Prof. Aly El Sheikah (Fund N° 9232307245). Conflict of Interest The author declares that there is no conflict of interest. References Adzitey, F. and Huda, N., 2010. Listeria monocytogenes in foods: incidences and possible control measures. African Journal of Microbiology Research 4: 2848–2855. Available at: http://www. academicjournals.org/ajmr Adzitey, F. and Huda, N., 2011. Campylobacter in poultry: incidences and possible control measures. Research Journal of Microbiology 6: 182–192. https://doi.org/10.3923/jm.2011.182.192 Adzitey, F., Huda, N. and Gulam, R., 2011. Comparison of media for the isolation of Salmonella (XLD and Rambach) and Listeria species (ALOA and Palcam) in naturally contaminated duck samples. Internet Journal of Food Safety 13: 20–25. Anderson, N.W., Buchan, B.W., Riebe, K.M., Parsons, L.N., Gnacinski, S. and Ledeboer, N.A., 2012. Effects of solid- medium type on routine identification of bacterial isolates by use of matrix-assisted laser desorption ionization-time of flight mass spectrometry. Journal of Clinical Microbiology 50: 1008–1013. https://doi.org/10.1128/JCM.05209-11 Aronoff, R., Vernez, I., Rotman, N. and Rando, G., 2018. Detection of milk adulteration using DNAFoil UniPlant. Application Note: v0.3 Available at: https://www.dropbox.com/s/ho5ich3fpzy5k0s/ application_note_UniPlant_1dec17.docx?dl=0. Accessed 27 January 2021. €  990 and the program makes access free of charge for a kit of 5 tests. The final cost per test is negotiable, and it is usually much cheaper than the traditional detection methods (Aronoff et al., 2018; Lüdin et al., 2018). In terms of quality control applicability. The DNAFoil output gives a (±) answer, for example, while using DNAFoil Pork Test Kit, a positive result indicates the presence of porcine DNA in the sample tested. This is enough for an inspector to take instant action. The strips with the results can be stored as evidence to prove that adequate controls are in place during manufacturing. In the case of litigations, the Swiss Federal Lab concluded that DNA can be easily extracted from the strip, and such DNA can be used for more lab tests (Lüdin et al., 2018). Conclusions, Remarks, and Future Trends In this study, the developed DNAFoil Salmonella and Listeria monocytogenes reactions correctly identify a wide range of Salmonella strains and also Listeria mono- cytogenes, among other bacterial strains. These reactions are specific and sensitive, with a virtual limit of 1 and 10 CFU detection per reaction for Salmonella and Listeria monocytogenes, respectively. In terms of accuracy and precision, the results show promising performance in which the accuracy ranged between 92 and 100% and the precision ranged between 88 and 100%. Real-time PCR may not be present at the point-of-need (i.e., factory); therefore, the DNAFoil Salmonella and Listeria mono- cytogenes reactions could be visually detected with the naked eye using a lateral flow strip. The strips with the results can be stored for quality control in place during manufacturing and also can be used for more lab tests. The results of the present study revealed and confirmed several advantages that considered DNAFoil fact sheet as follows: the DNAFoil is easy to use and does not require specialized skills to perform testing; the DNAFoil is standalone and does not require laboratory equipment other than a source of boiling water. In the absence of boiling water, the kit is compatible with a standard ther- moblock; the DNAFoil detects specifically each target without any cross-reaction; the final results can be inter- preted with the naked eye with a lateral flow dipstick without the need for thermocyclers, electrophoresis, and staining. Food pathogen detection approaches have become ever more substantial for all of the food chain components, that is, consumers, producers, and legislators (El Sheikha, 2015; Hoffmann and Scallan, 2017). 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