Digital PCR assay for the specific detection and estimation of Salmonella contamination levels in poultry rinse.

Strains of Salmonella are a frequent cause of foodborne illness and are known to contaminate poultry products. Most Salmonella testing methods can qualitatively detect Salmonella and cannot quantify or estimate the Salmonella load in samples. Therefore, the aim of this study was to standardize and validate a partitioned-based digital PCR (dPCR) assay for the detection and estimation of Salmonella contamination levels in poultry rinses. Pure culture Salmonella strains were cultured, enumerated, cold-stressed for 48 h, and used to inoculate whole carcass chicken rinse (WCCR) at 1-4 log CFU/30 mL and enriched at 37 °C for 5 h. Undiluted DNA samples with primer and probes targeting the Salmonella-specific invA gene were used for the dPCR assay. The dPCR assay was highly specific, with a limit of detection of 0.001 ng/µL and a limit of quantification of 0.01 ng/µL. The dPCR assay further showed no PCR reaction inhibition up to 5 µg of crude DNA extract. The assays accurately detected all cold-stressed Salmonella in inoculated WCCR samples following a 5-h enrichment. Most importantly, when converted to log, the dPCR copies/µL values accurately estimated the inoculated Salmonella levels. The dPCR assay standardized in this study is a robust method for the detection and estimation of Salmonella concentration in contaminated food samples. This approach can allow same-day decision-making for poultry processors attempting to maintain limits and controls on Salmonella contamination.

Seasonal variation of Salmonella enterica prevalence in milk and cottage cheese along the dairy value chain in three regions of Ethiopia.

Seasonal fluctuations influence foodborne illness transmission and affect patterns of microbial contamination of food. Previous investigations on the seasonality of Salmonella enterica prevalence in dairy products in Ethiopia have been minimal. However, such data are needed to inform strategic development of effective interventions to improve food safety, as seasonal differences may affect intervention strategies. This study was conducted to identify differences in the prevalence of Salmonella in milk and cheese samples between wet and dry seasons. A longitudinal study design was utilized with a random sampling occurring during both dry and wet seasons. A total of 448 milk and cottage cheese samples were collected from Oromia, Sidama, and Amhara regions. Samples were tested for Salmonella using the ISO 6579-1: 2008 method, followed by PCR confirmation. A chi-square test was conducted to assess the significance of differences in the prevalence of Salmonella in the samples between the two seasons. Results from this study showed a higher prevalence of Salmonella in all sample types during the dry season (P < 0.05). Moreover, when comparing raw milk, pasteurized milk, and cottage cheese samples, a significant difference was observed in Salmonella prevalence from raw milk samples (27.08%) collected in the Oromia region. Additionally, data showed a significantly higher prevalence of Salmonella in samples collected from raw milk producers (29.17%) during the wet season (P < 0.05). This study indicates that in order to enhance the safety of dairy products in Ethiopia, comprehensive, long-term awareness building on hygienic milk production and handling that consider seasonal influence is warranted. Supplementary Information: The online version contains supplementary material available at 10.1186/s40550-024-00108-4.

Serological investigation and isolation of Salmonella abortus equi in horses in Xinjiang.

BACKGROUND: Salmonella enterica subspecies enterica serovar abortus equi (S. abortus equi) is one of the main pathogens that causes abortion in pregnant horses and donkeys, which was highly infectious and greatly restricts the healthy development of the horse industry. OBJECTIVES: In order to investigate the prevalence and biological characteristics of S. abortus equi in different regions and breeds of horses in Xinjiang. METHODS: This study conducted ELISA detection of S. abortus equi antibodies on serum samples of 971 horses collected from three large-scale horse farms and five free-range horse farms in Yili Prefecture and Bayingol Mongolian Autonomous Prefecture of Xinjiang from 2020 to 2023. On this basis, bacterial isolation, culture, identification, and drug sensitivity tests were conducted on 42 samples of aborted foal tissues and 23 mare vaginal swabs. RESULTS: The results showed that the positive rate of S. abortus equi antibody was as high as 20.91% in 971 horse serum samples. Among them, the positive rate in the Ili region (29.09%) was significantly higher than that in the Bayingole region (11.24%), and the positive rate in mares (22.45%) was higher than that in stallions (14.05%). In terms of horse breeds, the positive rates of self-propagating thoroughbred horses, half-bred horses, Ili horses and Yanqi horses were 43.22%, 28.81%, 14.72% and 11.24% respectively. In addition, S. abortus equi was more susceptible to juvenile and elderly horses, with positive rates of 70.00%and 41.86%, respectively, both of which were significantly higher than young (10.97%) and adult (19.79%) horses. Further, 9 strains of S. abortus equi were obtained through bacterial isolation, culture and identification, which were resistant to five antibiotics (Clarithromycin, Clindamycin, penicillin, Sulfamethoxazole and Rifampicin), and sensitive to 13 antimicrobial agents (Amoxicillin, Ciprofloxacin and Gentamicin, et al.). CONCLUSION: There was a high infection rate of S. abortus equi in Ili Prefecture and self-propagating thoroughbred horses, and juvenile or old mares were more susceptible, which will provide scientific basis for the prevention of S. abortus equi infection in different regions and breeds of horses in Xinjiang.

Development of a lateral flow dipstick test for the detection of 4 strains of Salmonella spp. in animal products and animal production environmental samples based on loop-mediated isothermal amplification.

OBJECTIVE: This study aimed to develop loop-mediated isothermal amplification (LAMP) combined with lateral flow dipstick (LFD) and compare it with LAMP-AGE, polymerase chain reaction (PCR), and standard Salmonella culture as reference methods for detecting Salmonella contamination in animal products and animal production environmental samples. METHODS: The SalInvA01 primer, derived from the InvA gene and designed as a new probe for LFD detection, was used in developing this study. Adjusting for optimal conditions by temperature, time, and reagent concentration includes evaluating the specificity and limit of detection. The sampling of 120 animal product samples and 350 animal production environmental samples was determined by LAMP-LFD, comparing LAMP-AGE, PCR, and the culture method. RESULTS: Salmonella was amplified using optimal conditions for the LAMP reaction and a DNA probe for LFD at 63°C for 60 minutes. The specificity test revealed no cross-reactivity with other microorganisms. The limit of detection of LAMP-LFD in pure culture was 3×102 CFU/mL (6 CFU/reaction) and 9.01 pg/µL in genomic DNA. The limit of detection of the LAMP-LFD using artificially inoculated in minced chicken samples with 5 hours of pre-enrichment was 3.4×104 CFU/mL (680 CFU/reaction). For 120 animal product samples, Salmonella was detected by the culture method, LAMP-LFD, LAMP-AGE, and PCR in 10/120 (8.3%). In three hundred fifty animal production environmental samples, Salmonella was detected in 91/350 (26%) by the culture method, equivalent to the detection rates of LAMP-LFD and LAMP-AGE, while PCR achieved 86/350 (24.6%). When comparing sensitivity, specificity, positive predictive value, and accuracy, LAMP-LFD showed the best results at 100%, 95.7%, 86.3%, and 96.6%, respectively. For Kappa index of LAMP-LFD, indicated nearly perfect agreement with culture method. CONCLUSION: The LAMP-LFD Salmonella detection, which used InvA gene, was highly specific, sensitive, and convenient for identifying Salmonella. Furthermore, this method could be used for Salmonella monitoring and primary screening in animal products and animal production environmental samples.

Prevalence of biofilm forming Salmonella in different seafood contact surfaces of fishing boats, fish landing centres, fish markets and seafood processing plants.

The prevalence of biofilm forming Salmonella on different seafood contact surfaces was investigated. Out of 384 swab samples, 16.14 % and 1 % were confirmed biochemically and molecularly as Salmonella respectively. One out of four isolates was from the boat deck, and three were from the seafood processing plant. Salmonella was more prevalent in January, June, and September months. Different assays investigated the biofilm forming ability of isolates. Two out of four isolates have shown strong biofilms, and the others were moderate biofilm formers by microtitre plate assay. In the CRA assay, three isolates showed 'rdar' morphotype, and one showed 'bdar' morphotype. All isolates were positive for gcpA gene (~1700 bp), a critical gene found in Salmonella biofilms. The microbial load of Salmonella biofilms on different contact surfaces were determined, stainless steel and HDPE were found prone to biofilms. With this, a suitable mechanism shall be formulated to control the biofilms of Salmonella.

Detection of invA, sivH, and agfA Virulence Genes in Salmonella spp. Isolated from Broiler Breeder Farms in Alborz Province, Iran.

Salmonellosis, among poultry infectious diseases, not only imposes economic losses in the field of poultry breeding but also is considered a zoonotic disease. This study aimed to investigate the presence of invA, sivH, and agfA virulence genes in Salmonella species. The present study was conducted on 30 Salmonella strains. Samples were cultured on selective and differential media, and afterward, the isolates were serotyped using specific antisera based on the Kauffman-White table. Subsequently, the samples were analyzed to detect invA, sivH, and agfA genes by polymerase chain reaction technique. The results indicated that 30 (100%) isolates had invA and agfA virulence genes and 28 (93.33%) isolates had a sivH virulence gene. The highest frequency of serotypes was related to Salmonella infantis. Among the studied serotypes, Salmonella uno and Salmonella O35 lacked the sivH virulence gene, unlike other serotypes. The findings of this study could pave the way for Salmonella monitoring and be used as a pattern to detect Salmonella bacteria-bearing genes encoding invasion and fimbria.

Isolation, molecular detection and antimicrobial susceptibility profile of Salmonella from raw cow milk collected from dairy farms and households in southern Ethiopia.

BACKGROUND: Salmonella is one of the foodborne pathogens affecting public health around the globe. A cross-sectional bacteriological study was conducted from December 2019 to November 2020. This study aimed to isolate, molecularly detect and determine antibiotic susceptibility patterns of Salmonella from raw cows' milk collected from dairy farms and households in Hawassa, Arsi Negele, and Dale districts. MATERIALS AND METHODS: A total of 384 raw milk samples were collected using a simple random sampling technique. Standard bacteriological and biochemical tests were used to isolate Salmonella. The positive samples were further confirmed by the molecular test. Kirby-Bauer disk diffusion method was used for antimicrobial susceptibility testing of Salmonella. RESULTS: Using bacteriological and biochemical detection tests, Salmonella was isolated from 10.42% (N = 40) of the total sample. However, in molecular detection, only 32 of the 40 isolates were confirmed to be Salmonella using PCR test. The prevalence was 8.54, 12.69, and 10.46% in Hawassa, Dale, and Arsi Negele districts, respectively. Bacteriological prevalence did not vary significantly between the districts (P > 0.05). Likewise, no significant (P > 0.05) variation was observed in the Salmonella isolation rate between households (12.5%) and farms (8.33%) as well as between dry (8.85%) and wet (11.98%) seasons. Based on herd size, the isolation rate of Salmonella was significantly higher (P < 0.05) in large-scale farms (19.51%) than in small (5.1%) or medium (5.6%) scale dairy farms. The result of the antibiotic susceptibility test showed that Salmonella isolates were 100% resistant to ampicillin, while they were 100% sensitive to ciprofloxacin. Multi-drug resistance (MDR) was demonstrated in all isolates. CONCLUSION: This study showed that Salmonella is widespread in the raw milk samples and developing MDR, which may be of public health concern in the study area. It is therefore important that dairy farmers and raw milk sellers in the study area take serious measures to avoid contamination of the milk with Salmonella spp. In addition, the active commitment of the animal health departments in the respective districts to sensitizing dairy farmers and the sensible use of antibiotics at the farm level can help to reduce the antibiotic resistance of Salmonella spp.

Rapid, Sensitive, Specific, and Visual Detection of Salmonella in Retail Meat with Loop-Mediated Isothermal Amplification, Targeting the invA Gene.

ABSTRACT: Salmonella is one of the major pathogenic bacteria causing foodborne diseases. The rapid detection of Salmonella in food is of great significance to food safety. In this study, the loop-mediated isothermal amplification (LAMP) method was developed, and primers were designed targeting the invA gene of Salmonella. Standard samples of recombinant invA-plasmid and 100 retail meat samples were tested by LAMP and compared with the results tested by conventional PCR and the routine Chinese National Food Safety Standard-Microbiological Examination of Food-Examination of Salmonella, respectively. The results showed that Salmonella strains of eight different serotypes were amplified successfully by the developed LAMP assay, and it was 1,000-fold more sensitive than conventional PCR, with the analytical sensitivity of 8 × 102 copies per µL of the standard sample of invA-plasmid. The results were visualized directly by adding calcein and MnCl2 in the LAMP reaction tube, and the positively amplified products turned green after an incubation of 2 min. In parallel detection, the positive rate of Salmonella by the LAMP assay was highly correlated with the routine Chinese national standard method. The results of the study demonstrated that the developed LAMP assay is a simple, rapid, strongly specific, highly sensitive, and visual detection method for Salmonella.

Detection of Campylobacter jejuni and Salmonella typhimurium in chicken using PCR for virulence factor hipO and invA genes (Saudi Arabia).

Campylobacter jejuni and Salmonella typhimurium are the leading causes of bacterial food contamination in chicken carcasses. Contamination is particularly associated with the slaughtering process. The present study isolated C. jejuni and S. typhimurim from fifty chicken carcass samples, all of which were acquired from different companies in Riyadh, Saudi Arabia. The identification of C. jejuni was performed phenotypically by using a hippurate test and genetically using a polymerase chain reaction with primers for 16S rRNA and hippurate hydrolase (hipO gene). For the dentification of S. typhimurim, a serological Widal test was carried out using serum anti-S. typhimurium antibodies. Strains were genetically detected using invA gene primers. The positive isolates for C. jejuni showed a specific molecular size of 1448 bp for 16S rRNA and 1148 bp for hipO genes. However, the positive isolates of the invA gene exhibited a specific molecular size at 244 bp using polymerase chain reaction (PCR). Comparing sequencing was performed with respect to the invA gene and the BLAST nucleotide isolates that were identified as Salmonella enterica subsp. enterica serovar typhimurium strain ST45, thereby producing a similarity of 100%. The testing identified C.jejuni for hippuricase, GenBank: Z36940.1. While many isolates of Salmonella spp. that contained the invA gene were not necessarily identified as S. typhimurim, the limiting factor for the Widal test used antiS. typhimurum antibodies. The multidrug resistance (MDR) of C. jejuni isolates in chickens was compared with the standard C. jejuni strain ATCC 22931. Similarly, S. typhimurium isolates were compared with the standard S. typhimurium strain ATCC 14028.

Salmonella Gallinarum in Small-Scale Commercial Layer Flocks: Occurrence, Molecular Diversity and Antibiogram.

Salmonella Gallinarum is one of the most important bacterial pathogens associated with diminished egg production in poultry. The aim of this study was to understand the occurrence, molecular traits and antimicrobial resistance patterns of Salmonella Gallinarum strains isolated from small-scale commercial layer flocks with low level biosecurity standards in Bangladesh. A total of 765 samples, including cloacal swabs (535), visceral organs (50), and droppings (180), were collected from chickens of 12 layer flocks in 11 districts. Salmonella Gallinarum was isolated and characterized through culture-based method, followed by biochemical tests, sero-grouping, PCR assays, sequencing, and antibiogram. The identity of biochemically detected isolates of Salmonella Gallinarum was confirmed via genus-specific 16S rRNA gene based PCR, followed by invA and spvC genes based PCR assays. Occurrence of Salmonella Gallinarum was detected in overall 25.75% (197/765) samples, with a significantly (p < 0.05) higher incidence in visceral organs (42%) in comparison to cloacal swab (24%) and droppings (26%). Sequencing and subsequent phylogenetic analysis of invA and spvC genes in representative strains of Salmonella Gallinarum revealed a close genetic lineage, with a sequence similarity of 98.05-99.21% and 97.51-99.45%, respectively, to previously published sequences of the corresponding genes from the same serogroup strains. Remarkably, 66.5% (131/197) of the isolated strains of Salmonella Gallinarum were found to be resistant to 3 to 6 antimicrobial agents, and interpreted as multidrug resistant (MDR). The findings of this study underscore an inherent need of appropriate control measures to curb the widespread incidence of MDR Salmonella Gallinarum in small-scale commercial layer flocks, thereby, facilitating enhanced egg production and further support to the food security and safety in low resource settings.

Development of novel visual detection methodology for Salmonella in meat using saltatory rolling circle amplification.

AIM: The aim of this study was to develop a saltatory rolling circle amplification (SRCA) assay for rapid, simple and visual detection of Salmonella in meat. METHODS AND RESULTS: Saltatory rolling circle amplification assay was established using simple PCR primers targeting the invA gene of Salmonella enterica. The specificity of the SRCA assay was determined using 28 Salmonella and 15 non-Salmonella strains. The analytical sensitivity of the developed SRCA, conventional and real-time PCR assays were 70 fg, 7 pg and 700 fg S. enterica DNA per tube, respectively. The limit of detection (LoD) of the SRCA assay was 40 CFU per gram of meat without enrichment and 4 CFU per gram after including 6 h brief enrichment step. The detection limits of 40 CFU per gram and 4 CFU per gram of meat were achieved within 165 min and 9 h, respectively (including DNA extraction). To assess the real-world relevance of the SRCA assay, it was used to screen Salmonella from the field pork samples (n = 82). The same samples were also tested with culture (ISO 6579: 2002) method, conventional and real-time PCR assays. Using the developed assay with 6-h enrichment step, it could give accurate results as that of the culture method. CONCLUSIONS: The results of this study showed that the SRCA assay is a rapid, simple, sophisticated equipment-free and user-friendly method for accurate detection of Salmonella in meat foods. To our information, this is the first study to deploy SRCA assay for screening foods for Salmonella. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed SRCA assay is cost-effective, easy-to-perform and equipment-free; therefore, it has the potential to replace other molecular detection methods for regular screening of Salmonella in foods in field laboratories.

Development and Evaluation of the Rapid and Sensitive RPA Assays for Specific Detection of Salmonella spp. in Food Samples.

Salmonella spp. is among the main foodborne pathogens which cause serious foodborne diseases. An isothermal real-time recombinase polymerase amplification (RPA) and lateral flow strip detection (LFS RPA) were used to detect Salmonella spp. targeting the conserved sequence of invasion protein A (invA). The Real-time RPA was performed in a portable florescence scanner at 39°C for 20 min. The LFS RPA was performed in an incubator block at 39°C for 15 min, under the same condition that the amplifications could be inspected by the naked eyes on the LFS within 5 min. The detection limit of Salmonella spp. DNA using real-time RPA was 1.1 × 101 fg, which was the same with real-time PCR but 10 times higher than that of LFS RPA assay. Moreover, the practicality of discovering Salmonella spp. was validated with artificially contaminated lamb, chicken, and broccoli samples. The analyzing time dropped from 60 min to proximately 5-12 min on the basis of the real-time and LFS RPA assays compared with the real-time PCR assay. Real-time and LFS RPA assays' results were equally reliable. There was no cross-reactivity with other pathogens in both assays. In addition, the assays had good stability. All of these helped to show that the developed RPA assays were simple, rapid, sensitive, credible, and could be a potential point-of-need (PON) test required mere resources.

Detection of invA virulence gene of multidrug-resistant Salmonella species isolated from the cloacal swab of broiler chickens in Blitar district, East Java, Indonesia.

BACKGROUND AND AIM: The increasing number of multidrug-resistant (MDR) Salmonella species on poultry farms in Indonesia has caused concern regarding human health. This study was conducted to determine the presence of the virulence gene invA in MDR Salmonella species isolated from the cloacal swab of broiler chickens in Blitar district, East Java Province, Indonesia. MATERIALS AND METHODS: Cloacal swab samples were collected by purposive sampling from 15 farms in four districts. Isolation and identification of bacteria were performed using standard microbiological techniques. Confirmation of MDR isolates was done using five different classes of antibiotics, including the beta-lactam, aminoglycoside, fluoroquinolone, phenicol, and monobactam groups. An antibiotic susceptibility test was conducted using the Kirby-Bauer disk diffusion method, and a polymerase chain reaction method was used to screen for the presence of invA. RESULTS: It was observed that 32.26% (50/155) of the samples were positive for Salmonella species. Of these 50 Salmonella isolates, 7 (14%) were identified as MDR strains. An important finding was the detection of invA in all the seven MDR Salmonella strains (100%) isolated from the cloacal swab of broiler chickens in Blitar district, East Java Province. CONCLUSION: Veterinarians have an extremely important role in monitoring the use of antibiotics in farm animals to mitigate the rapid spread of MDR organisms in our environment, which can otherwise cause serious economic losses and also public health issues.

Evaluation of different target genes for the detection of Salmonella sp. by loop-mediated isothermal amplification.

The loop-mediated isothermal amplification (LAMP) technique was used to investigate six salmonella-specific sequences for their suitability to serve as targets for the pathogen identification. Sequences selected for designing LAMP primers were genes invA, bcfD, phoP, siiA, gene62181533 and a region within the ttrRSBCA locus. Primers including single nucleotide polymorphisms were configured as degenerate primers. Specificity of the designed primer sets was determined by means of 46 salmonella and 32 other food- and waterborne bacterial reference species and strains. Primers targeting the ttrRSBCA locus showed 100 % inclusivity of target and exclusivity of other test species and strains. Other primer sets revealed deficiencies, especially regarding Salmonella enterica subsp. II-IV and Salmonella bongori. Additionally, primers targeting the siiA gene failed to detect S. enterica subsp. enterica serotypes Newport and Stanley, whereas bcfD primers did not amplify DNA of S. enterica subsp. enterica serotype Schleissheim. TtrRSBCA primers, providing short detection times and constant melting temperatures of amplification products, achieved best overall performance.

Antibiogram, Virulence Genes, and Biofilm-Forming Ability of Clinical Salmonella enterica Serovars: An In Vitro Study.

Salmonella enterica serovar Typhi and Salmonella Paratyphi are causative agents of enteric fever. Salmonella Typhi persists as a biofilm on gallstones. Hence, we studied the biofilm formation, antibiogram, and virulence genes of S. enterica serovars. Antibiogram of S. enterica serovars from human blood and stool samples were studied by Kirby-Bauer disk diffusion method and biofilm by microtiter plate method. We studied the minimum inhibitory concentration of the isolates by Vitek-2 semiautomated system. Polymerase chain reaction was done to detect invA and spvC genes. Of the 55 isolates studied, 36 (65.45%) were Salmonella Typhi, 13 (23.63%) were Salmonella Paratyphi A, 2 (3.64%) were Salmonella Typhimurium, and 4 (7.28%) were Salmonella spp. Resistance to ciprofloxacin and nalidixic acid were found to be 81.8% and 92.7%, respectively. Chloramphenicol and cotrimoxazole-susceptible strains were 98.18%. One each of Salmonella Typhi, Salmonella Paratyphi A, and S. enterica isolates formed weak biofilm at 28°C. However, at 37°C eight Salmonella Typhi produced weak biofilm in the presence of bile. One Salmonella Paratyphi A and two Salmonella spp. formed weak biofilm in the absence of bile. All the isolates had the invA gene. Salmonella Typhimurium had invA and spvC genes. Bile may contribute to biofilm formation and persistence of the Salmonella Typhi on gallstones, which may lead to carrier state. Changing antibiotic susceptibility pattern of Salmonella serovars is observed in our geographic area. The presence of invA and spvC genes indicate the ability of invasiveness and intracellular survival.

The mRNA expression of ompF, invA and invE was associated with the ciprofloxacin-resistance in Salmonella.

Salmonella developed drug-resistance under durative antibiotic pressures pressure. The widespread prevalence of Salmonella has been associated with not only drug-resistance but also pathogenicity. Outer membrane porin proteins (OMPs) are critical for the drug resistance of bacteria. Virulence genes in Salmonella pathogenicity islands (SPIs) play key roles in the virulence of bacteria. In this study, we analyzed the expression levels of three critical genes in ciprofloxacin-resistant strains and ciprofloxacin-susceptible strains of Salmonella, including outer membrane porin protein F (ompF), virulence genes invA and invE. In the clinical ciprofloxacin-resistant strains of Salmonella, the expression level of ompF was decreased. Meanwhile, the expression levels of invA and invE were decreased except for only one strain, indicating generally decreased virulence. These results were also verified with ciprofloxacin-induced resistant strains. Thus, it was informative for understanding the drug-resistance in Salmonella. Monitoring drug-resistance and virulence relevant genes would be significant in the prevention and control of salmonellosis.

Detection of Salmonella Enterica in Egg Yolk by PCR on a Microfluidic Disc Device Using Immunomagnetic Beads.

Salmonella enterica is a pathogenic bacterium that causes foodborne illness. One of the vehicle foods of S. enterica are chicken eggs. Efficient collection of the bacterium is necessary to detect it specifically. We developed a method to detect S. enterica by PCR on a microfluidic disc device using a fluorescent probe. Salmonella enterica cells were isolated in the microchambers on the device, followed by thermal lysis and PCR targeting with the invA gene, a gene specific to S. enterica, were observed by measurement of the fluorescent signal that resulted from gene amplification. However, the developed method was unable to discriminate viable cells from dead cells. Consequently, in this study, magnetic beads modified with anti-Salmonella antibody were utilized to detect viable Salmonella cells from egg yolk prior to PCR on the device. While using the antibody-modified beads, egg yolk components, which inhibit PCR, were removed. The collected cells were subsequently detected by PCR of the invA gene on a microfluidic disc device. This method enabled the detection of viable cells without the inhibition of PCR by any egg component. S. enterica was detected at 5.0×104 cells mL-1 or at a higher concentration of egg yolk within 6 h including the sampling time.

Molecular diversity of the invA gene obtained from human and egg samples.

BACKGROUND AND AIM: Salmonellosis is one of the most common foodborne bacterial diseases in the world. The great majority of Salmonella infections in humans are foodborne with Salmonella enterica and Salmonella Typhimurium accounting for a major part of the problem. The objective of this study was to investigate the presence of invA gene in strains of Salmonellae isolated from eggs and diarrheal swabs from human cases. In addition, the relationship between invA gene nucleotide sequences from different sources (human stool and egg samples) have been studied through phylogenetic tree. MATERIALS AND METHODS: One hundred and seventy eggs (eggshell and its contents) and 160 stool swabs samples were collected from four poultry farms and medical hospital in Giza Governorate. RESULTS: The study reported the presence of two Salmonella strains in eggshell surface with an overall isolation rate of 1.2 and 0% of the egg content. Salmonella Enteritidis and Salmonella Typhimurium were isolated from eggshell surface with an incidence of 50% for each strain. Six salmonella strains were isolated from human stool with an incidence of 3.75%; the isolated strains are S. Typhimurium, S. Enteritidis, Salmonella Virchow, Salmonella Haifa, and Salmonella Kentucky with an incidence of 33.3%, 16.6%, 16.6%, 16.6%, and 16.6%, respectively. Among eight Salmonella strains, invA gene was detected with percentage of 50%. The phylogenetic analysis of the sequences invA gene, from two isolates included in this study and five isolates retrieved from GenBank showed that sequence from human, layer hens, egg, and water in the same clusters. CONCLUSION: Close relation between drinking contaminated water and layer hens and contaminated water is one such source.

Electrochemical detection of Salmonella using an invA genosensor on polypyrrole-reduced graphene oxide modified glassy carbon electrode and AuNPs-horseradish peroxidase-streptavidin as nanotag.

A rapid and sensitive electrochemical biosensor was constructed to detect Salmonella using invA gene biosensor. The biosensing was based on polyrrole-reduced graphene oxide (PPy-rGO) nanocomposite modified glassy carbon electrode (GCE) and signal amplification with horseradish peroxidase-streptavidin biofunctionalized gold nanoparticles (AuNPs-HRP-SA). PPy-rGO was prepared at 60 °C by chemical reduction of PPy-functionalized graphene oxide (PPy-GO) that was synthesized by in situ polymerization at room temperature. The detection signal was amplified via enzymatic reduction of H2O2 in the presence of hydroquinone (HQ) using AuNPs-HRP-SA as nanotag. Under optimal conditions, the differential pulse voltametric (DPV) signal from the biosensor was linearly related to the logarithm of target invA gene concentrations from 1.0 × 10-16 to 1.0 × 10-10 M, and the limit of detection (LOD) was 4.7 × 10-17 M. The biosensor can also detect Salmonella in the range of 9.6 to 9.6 × 104 CFU mL-1, with LOD of 8.07 CFU mL-1. The biosensor showed good regeneration ability, acceptable selectivity, repeatability and stability, which bode well as an alternative method for Salmonella screening.

Rapid and Sensitive Detection of Salmonella in Chickens Using Loop-Mediated Isothermal Amplification Combined with a Lateral Flow Dipstick.

Salmonellosis is a highly contagious bacterial disease that threatens both human and poultry health. Tests that can detect Salmonella in the field are urgently required to facilitate disease control and for epidemiological investigations. Here, we combined loop-mediated isothermal amplification (LAMP) with a chromatographic lateral flow dipstick (LFD) to rapidly and accurately detect Salmonella. LAMP primers were designed to target the Salmonella invA gene. LAMP conditions were optimized by adjusting the ratio of inner to outer primers, MgSO4 concentration, dNTP mix concentration, amplification temperature, and amplification time. We evaluated the specificity of our novel LAMP-LFD method using six Salmonella species and six related non-Salmonella strains. All six of the Salmonella strains, but none of the non-Salmonella strains, were amplified. LAMP-LFD was sensitive enough to detect concentrations of Salmonella enterica subsp. enterica serovar Pullorum genomic DNA as low as 89 fg/µl, which is 1,000 times more sensitive than conventional PCR. When artificially contaminated feed samples were analyzed, LAMP-LFD was also more sensitive than PCR. Finally, LAMP-LFD gave no false positives across 350 chicken anal swabs. Therefore, our novel LAMP-LFD assay was highly sensitive, specific, convenient, and fast, making it a valuable tool for the early diagnosis and monitoring of Salmonella infection in chickens.