Genetic response of Salmonella Typhimurium to trans-cinnamaldehyde assisted heat treatment and its correlation with bacterial resistance in different low moisture food components.

Our previous study found that water activity (aw)- and matrix-dependent bacterial resistance wasdeveloped in Salmonella Typhimurium during antimicrobial-assisted heat treatment in low moisture foods (LMFs) matrices. To better understand the molecular mechanism behind the observed bacterial resistance, gene expression analysis was conducted on S. Typhimurium adapted to different conditions with or without the trans-cinnamaldehyde (CA)-assisted heat treatment via quantitative polymerase chain reaction (qPCR). Expression profiles of nine stress-related genes were analyzed. The upregulation of rpoH and dnaK and downregulation of ompC were observed during bacterial adaptation in LMF matrices and the combined heat treatment, which likely contributed to the bacterial resistance during the combined treatment. Their expression profiles were partially consistent with the previously-observed effect of aw or matrix on bacterial resistance. The upregulation of rpoE, otsB, proV, and fadA was also observed during adaptation in LMF matrices and might contribute to desiccation resistance, but likely did not contribute to bacterial resistance during the combined heat treatment. The observed upregulation of fabA and downregulation of ibpA could not be directly linked to bacterial resistance to either desiccation or the combined heat treatment. The results may assist the development of more efficient processing methods against S. Typhimurium in LMFs.

Assessment of trans-cinnamaldehyde and eugenol assisted heat treatment against Salmonella Typhimurium in low moisture food components.

Increased thermal resistance of Salmonella at low water activity (aw) is a significant food safety concern in low-moisture foods (LMFs). We evaluated whether trans-cinnamaldehyde (CA, 1000 ppm) and eugenol (EG, 1000 ppm), which can accelerate thermal inactivation of Salmonella Typhimurium in water, can show similar effect in bacteria adapted to low aw in different LMF components. Although CA and EG significantly accelerated thermal inactivation (55 °C) of S. Typhimurium in whey protein (WP), corn starch (CS) and peanut oil (PO) at 0.9 aw, such effect was not observed in bacteria adapted to lower aw (0.4). The matrix effect on bacterial thermal resistance was observed at 0.9 aw, which was ranked as WP > PO > CS. The effect of heat treatment with CA or EG on bacterial metabolic activity was also partially dependent on the food matrix. Bacteria adapted to lower aw had lower membrane fluidity and unsaturated to saturated fatty acids ratio, suggesting that bacteria at low aw can change its membrane composition to increase its rigidity, thus increasing resistance against the combined treatments. This study demonstrates the effect of aw and food components on the antimicrobials-assisted heat treatment in LMF and provides an insight into the resistance mechanism.

Enhancement of Thermal Inactivation of Cronobacter sakazakii in Apple Juice at 58°C by Inclusion of Butyl Para-Hydroxybenzoate and Malic Acid.

ABSTRACT: After studies with powdered infant formula indicated that the enhancement of thermal inactivation of Cronobacter sakazakii by butyl para-hydroxybenzoate (BPB) was blocked by high protein concentrations, we hypothesized that BPB would retain its synergistic activity in foods with limited protein and lipid concentrations. This hypothesis was tested by examining the ability of BPB to enhance the thermal inactivation of C. sakazakii 607 at 58°C in commercial apple juice, including examining the effects of pH and possible synergistic effects with malic acid. Apple juice was adjusted to designated pH values of 3.2 to 9.0, supplemented with selected concentrations of BPB (≤125 ppm), inoculated with early-stationary-phase C. sakazakii 607, and thermally treated (58°C) for 15 min with a submerged coil apparatus. The same methods were used to study the enhancement of thermal inactivation by malic acid. Samples were plated on tryptic soy agar for recovery and enumeration. Survival curves were plotted, and D-values were calculated by linear regression and compared using the Tukey honestly significant difference test. BPB significantly enhanced thermal inactivation in a concentration dependent manner, with D-values of a few seconds at the original pH (3.8). The enhancement of thermal inactivation was pH dependent over the pH range of 3.4 to 9.0. Malic acid enhanced thermal inactivation; the pH was decreased from 3.8 to 3.2. These results support the hypothesis that BPB can enhance the thermal inactivation of C. sakazakii in low-protein and low-lipid foods.

Subtyping Evaluation of Salmonella Enteritidis Using Single Nucleotide Polymorphism and Core Genome Multilocus Sequence Typing with Nanopore Reads.

Whole-genome sequencing (WGS) for public health surveillance and epidemiological investigation of foodborne pathogens predominantly relies on sequencing platforms that generate short reads. Continuous improvement of long-read nanopore sequencing, such as Oxford nanopore technologies (ONT), presents a potential for leveraging multiple advantages of the technology in public health and food industry settings, including rapid turnaround and onsite applicability in addition to superior read length. Using an established cohort of Salmonella Enteritidis isolates for subtyping evaluation, we assessed the technical readiness of nanopore long read sequencing for single nucleotide polymorphism (SNP) analysis and core-genome multilocus sequence typing (cgMLST) of a major foodborne pathogen. By multiplexing three isolates per flow cell, we generated sufficient sequencing depths in <7 h of sequencing for robust subtyping. SNP calls by ONT and Illumina reads were highly concordant despite homopolymer errors in ONT reads (R9.4.1 chemistry). In silico correction of such errors allowed accurate allelic calling for cgMLST and allelic difference measurements to facilitate heuristic detection of outbreak isolates. IMPORTANCE Evaluation, standardization, and implementation of the ONT approach to WGS-based, strain-level subtyping is challenging, in part due to its relatively high base-calling error rates and frequent iterations of sequencing chemistry and bioinformatic analytics. Our study established a baseline for the continuously evolving nanopore technology as a viable solution to high-quality subtyping of Salmonella, delivering comparable subtyping performance when used standalone or together with short-read platforms. This study paves the way for evaluating and optimizing the logistics of implementing the ONT approach for foodborne pathogen surveillance in specific settings.

Evaluation of Potential for Butyl and Heptyl Para-Hydroxybenzoate Enhancement of Thermal Inactivation of Cronobacter sakazakii during Rehydration of Powdered Infant Formula and Nonfat Dry Milk.

ABSTRACT: In previous studies, parabens in model systems enhanced the thermal inactivation of foodborne pathogens, including Cronobacter sakazakii, Salmonella enterica serotype Typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes. However, few studies have been conducted to evaluate this phenomenon in actual food systems. In the present study, the potential enhancement of thermal inactivation of C. sakazakii by butyl para-hydroxybenzoate (BPB) was evaluated in powdered infant formula (PIF) and nonfat dry milk (NFDM) in dry and rehydrated forms. When PIF was rehydrated with water at designated temperatures (65 to 80°C) in baby bottles, BPB did not enhance thermal inactivation. When rehydrated NFDM and lactose solutions with BPB were inoculated and heated at 58°C, BPB enhancement of thermal inactivation of C. sakazakii was negatively associated with the concentration of NFDM solutions in a dose-dependent manner, whereas thermal inactivation was enhanced in the presence of lactose regardless of its concentration, suggesting an interaction between proteins and BPB. Fluorescence testing further indicated an interaction between BPB and the proteins in PIF and NFDM. In inoculated dry NFDM with and without BPB stored at 24 and 55°C for 14 days, BPB did not substantially enhance bacterial inactivation. This study suggests that BPB is not likely to enhance mild thermal bacterial inactivation treatments in foods that have appreciable amounts of protein.

Evaluation of multiplex nanopore sequencing for Salmonella serotype prediction and antimicrobial resistance gene and virulence gene detection.

In a previous study, Multiplex-nanopore-sequencing based whole genome sequencing (WGS) allowed for accurate in silico serotype prediction of Salmonella within one day for five multiplexed isolates, using both SISTR and SeqSero2. Since only ten serotypes were tested in our previous study, the conclusions above were yet to be evaluated in a larger scale test. In the current study we evaluated this workflow with 69 Salmonella serotypes and also explored the feasibility of using multiplex-nanopore-sequencing based WGS for antimicrobial resistance gene (AMR) and virulence gene detection. We found that accurate in silico serotype prediction with nanopore-WGS data was achieved within about five hours of sequencing at a minimum of 30× Salmonella genome coverage, with SeqSero2 as the serotype prediction tool. For each tested isolate, small variations were observed between the AMR/virulence gene profiles from the Illumina and Nanopore sequencing platforms. Taking results generated using Illumina data as the benchmark, the average precision value per isolate was 0.99 for both AMR and virulence gene detection. We found that the resistance gene identifier - RGI identified AMR genes with nanopore data at a much lower accuracy compared to Abricate, possibly due to RGI's less stringent minimum similarity and coverage by default for database matching. This study is an evaluation of multiplex-nanopore-sequencing based WGS as a cost-efficient and rapid Salmonella classification method, and a starting point for future validation and verification of using it as a AMR/virulence gene profiling tool for the food industry. This study paves the way for the application of nanopore sequencing in surveillance, tracking, and risk assessment of Salmonella across the food supply chain.

Assessment of butylparaben (4-hydroxybenzoic acid butyl ester)-assisted heat treatment against Escherichia coli O157:H7 and Salmonella enterica serotype Typhimurium in meat and bone meal.

Heat-resistant foodborne pathogens have been a concern in low-moisture foods and ingredients (LMFs). Due to low thermal conductivity of low moisture materials, thermal treatment is not efficient and may cause nutritional loss. This study investigated the enhancement of thermal treatment of meat and bone meal (MBM) at low water activity (aw ) by inclusion of butylparaben (BP) as a model antimicrobial compound. Stationary phase Escherichia coli O157:H7 (Shiga toxin-negative) or Salmonella enterica serotype Typhimurium was inoculated into MBM containing 0-2000 ppm BP and incubated at 55 or 60°C for up to 5 hr. A biphasic inactivation pattern was observed for both pathogens, indicating existence of potentially thermal resistant subpopulations. Addition of 1000 ppm BP to MBM (aw  = 0.4) significantly lowered the D-value at 55°C for E. coli O157:H7 (2.6 ± 0.5 hr) compared to thermal treatment alone (5.1 ± 0.6 h) during the treatment after the first 1 hr (p < 0.05), indicating that addition of BP accelerated the inactivation of thermal-resistant subpopulation of E. coli O157:H7 in MBM. Interestingly, similar enhancement in thermal inactivation upon addition of BP was not observed in either the sensitive or resistant subpopulation of S. Typhimurium at aw of 0.4 or 0.7, which is likely caused by the higher thermal resistance developed by S. Typhimurium within a low aw environment (aw  < 0.85). These results suggest that addition of certain antimicrobial compounds can improve the thermal processing efficiency in LMFs, while their efficiency against different pathogens may vary. PRACTICAL APPLICATION: Addition of appropriate food-grade compounds may help to improve thermal treatment efficiency in low moisture foods with varied efficiency against different pathogens. This approach has the potential to reduce the required heat treatment intensity while minimizing food safety risk.

Evaluation of Salmonella Serotype Prediction With Multiplex Nanopore Sequencing.

The use of whole genome sequencing (WGS) data generated by the long-read sequencing platform Oxford Nanopore Technologies (ONT) has been shown to provide reliable results for Salmonella serotype prediction in a previous study. To further meet the needs of industry for accurate, rapid, and cost-efficient Salmonella confirmation and serotype classification, we evaluated the serotype prediction accuracy of using WGS data from multiplex ONT sequencing with three, four, five, seven, or ten Salmonella isolates (each isolate represented one Salmonella serotype) pooled in one R9.4.1 flow cell. Each multiplexing strategy was repeated with five flow cells, and the loaded samples were sequenced simultaneously in a GridION sequencer for 48 h. In silico serotype prediction was performed using both SeqSero2 (for raw reads and genome assemblies) and SISTR (for genome assemblies) software suites. An average of 10.63 Gbp of clean sequencing data was obtained per flow cell. We found that the unevenness of data yield among each multiplexed isolate was a major barrier for shortening sequencing time. Using genome assemblies, both SeqSero2 and SISTR accurately predicted all the multiplexed isolates under each multiplexing strategy when depth of genome coverage ≥50× for each isolate. We identified that cross-sample barcode assignment was a major cause of prediction errors when raw sequencing data were used for prediction. This study also demonstrated that, (i) sequence data generated by ONT multiplex sequencing can be used to simultaneously predict serotype for three to ten Salmonella isolates, (ii) with three to ten Salmonella isolates multiplexed, genome coverage at ≥50× per isolate was obtained within an average of 6 h of ONT multiplex sequencing, and (iii) with five isolates multiplexed, the cost per isolate might be reduced to 23% of that incurred with single ONT sequencing. This study is a starting point for future validation of multiplex ONT WGS as a cost-efficient and rapid Salmonella confirmation and serotype classification tool for the food industry.

Evaluation of nanopore sequencing technology to identify Salmonella enterica Choleraesuis var. Kunzendorf and Orion var. 15+, 34.

Our previous study demonstrated that whole genome sequencing (WGS) data generated by Oxford Nanopore Technologies (ONT) can be used for rapid and accurate prediction of selected Salmonella serotypes. However, one limitation is that established methods for WGS-based serotype prediction, utilizing data from either ONT or Illumina, cannot differentiate certain serotypes and serotype variants with the same or closely related antigenic formulae. This study aimed to evaluate nanopore sequencing and additional data analysis for identification of Salmonella enterica Choleraesuis var. Kunzendorf and S. enterica Orion var. 15+, 34+, thus overcoming this limitation. Five workflows that combined different flow cells, library construction methods and basecaller models were evaluated and compared. The workflow that consisted of the R9 flow cell, rapid sequencing library construction kit and guppy basecaller with base modified model performed best for Single Nucleotide Polymorphism (SNP) analysis. With this workflow, 99.98% of matching identity between assembled genomes from ONT and that from Illumina was achieved. Less than five high-quality SNPs differed when comparing sequencing data between ONT and Illumina. SNP typing successfully identified Choleraesuis var. Kunzendorf. While prophage prediction further differentiated Orion var. 15+, 34+ from the other two Orion variants. Our study improves the readiness of ONT as a Salmonella subtyping and source tracking tool for food industry applications.

Synergistic Effects of Butyl Para-Hydroxybenzoate and Mild Heating on Foodborne Pathogenic Bacteria.

ABSTRACT: Although high-temperature heat treatments can efficiently reduce pathogen levels, they also affect the quality and nutritional profile of foods and increase the cost of processing. The food additive butyl para-hydroxybenzoate (BPB) was investigated for its potential to synergistically enhance thermal microbial inactivation at mild heating temperatures (54 to 58°C). Four foodborne pathogenic bacteria, Cronobacter sakazakii, Salmonella enterica Typhimurium, attenuated Escherichia coli O157:H7, and Listeria monocytogenes, were cultured to early stationary phase and then subjected to mild heating at 58, 55, 57, and 54°C, respectively, in a model food matrix (brain heart infusion [BHI]) containing low concentrations of BPB (≤125 ppm). The temperature used with each bacterium was selected based on the temperature that would yield an approximately 1- to 3-log reduction over 15 min of heating in BHI without BPB in a submerged coil system. The inclusion of BPB at ≤125 ppm resulted in significant enhancement of thermal inactivation, achieving 5- to >6-log reductions of the gram-negative strains with D-values of <100 s. A 3- to 4-log reduction of L. monocytogenes was achieved with a similar treatment. No significant microbial inactivation was noted in the absence of mild heating for the same time period. This study provides additional proof of concept that low-temperature inactivation of foodborne pathogens can be realized by synergistic enhancement of thermal inactivation by additives that affect microbial cell membranes.

Evaluation of real-time nanopore sequencing for Salmonella serotype prediction.

The use of whole genome sequencing (WGS) data generated by short-read sequencing technologies such as the Illumina sequencing platforms has been shown to provide reliable results for Salmonella serotype prediction. Emerging long-read sequencing platforms developed by Oxford Nanopore Technologies (ONT) provide an alternative WGS method to meet the needs of industry for rapid and accurate Salmonella confirmation and serotype classification. Advantages of the ONT sequencing platforms include portability, real-time base-calling and long-read sequencing. To explore whether WGS data generated by an ONT sequencing platform could accurately predict Salmonella serotypes, 38 Salmonella strains representing 34 serotypes were sequenced using R9.4 flow cells on an ONT sequencer for up to 2 h. The downstream bioinformatics analysis was performed using pipelines with different assemblers including Canu, Wdbtg2 combined with Racon, or Miniasm combined with Racon. In silico serotype prediction programs were carried out using both SeqSero2 (raw reads and genome assemblies) and SISTR (genome assemblies). The WGS data of the same strains were also obtained from Illumina Hiseq (200 x depth of coverage per genome) as a benchmark of accurate serotype prediction. Predictions using WGS data generated after 30 min, 45 min, 1 h, and 2 h of ONT sequencing time all matched the prediction results from Illumina WGS data. This study demonstrated the comparable accuracy of WGS-based serotype prediction between ONT and Illumina sequencing platforms. This study also sets a start point for future validation of ONT WGS as a rapid Salmonella confirmation and serotype classification tool for the food industry.

Assessment and Comparison of Molecular Subtyping and Characterization Methods for Salmonella.

The food industry is facing a major transition regarding methods for confirmation, characterization, and subtyping of Salmonella. Whole-genome sequencing (WGS) is rapidly becoming both the method of choice and the gold standard for Salmonella subtyping; however, routine use of WGS by the food industry is often not feasible due to cost constraints or the need for rapid results. To facilitate selection of subtyping methods by the food industry, we present: (i) a comparison between classical serotyping and selected widely used molecular-based subtyping methods including pulsed-field gel electrophoresis, multilocus sequence typing, and WGS (including WGS-based serovar prediction) and (ii) a scoring system to evaluate and compare Salmonella subtyping assays. This literature-based assessment supports the superior discriminatory power of WGS for source tracking and root cause elimination in food safety incident; however, circumstances in which use of other subtyping methods may be warranted were also identified. This review provides practical guidance for the food industry and presents a starting point for further comparative evaluation of Salmonella characterization and subtyping methods.

Enhancing plant productivity while suppressing biofilm growth in a windowfarm system using beneficial bacteria and ultraviolet irradiation.

Common problems in a windowfarm system (a vertical and indoor hydroponic system) are phytopathogen infections in plants and excessive buildup of biofilms. The objectives of this study were (i) to promote plant health by making plants more resistant to infection by using beneficial biosurfactant-producing Pseudomonas chlororaphis around the roots and (ii) to minimize biofilm buildup by ultraviolet (UV) irradiation of the water reservoir, thereby extending the lifespan of the whole system with minimal maintenance. Pseudomonas chlororaphis-treated lettuce grew significantly better than nontreated lettuce, as indicated by enhancement of color, mass, length, and number of leaves per head (p < 0.05). The death rate of the lettuce was reduced by ∼ 50% when the lettuce was treated with P. chlororaphis. UV irradiation reduced the bacteria (4 log reduction) and algae (4 log reduction) in the water reservoirs and water tubing systems. Introduction of P. chlororaphis into the system promoted plant growth and reduced damage caused by the plant pathogen Pythium ultimum. UV irradiation of the water reservoir reduced algal and biofilm growth and extended the lifespan of the system.

Salmonella internalization in mung bean sprouts and pre- and postharvest intervention methods in a hydroponic system.

Mung bean sprouts, typically consumed raw or minimally cooked, are often contaminated with pathogens. Internalized pathogens pose a high risk because conventional sanitization methods are ineffective for their inactivation. The studies were performed (i) to understand the potential of internalization of Salmonella in mung bean sprouts under conditions where the irrigation water was contaminated and (ii) to determine if pre- and postharvest intervention methods are effective in inactivating the internalized pathogen. Mung bean sprouts were grown hydroponically and exposed to green fluorescence protein-tagged Salmonella Typhimurium through maturity. One experimental set received contaminated water daily, while other sets received contaminated water on a single day at different times. For preharvest intervention, irrigation water was exposed to UV, and for postharvest intervention-contaminated sprouts were subjected to a chlorine wash and UV light. Harvested samples were disinfected with ethanol and AgNO3 to differentiate surface-associate pathogens from the internalized ones. The internalized Salmonella Typhimurium in each set was quantified using the plate count method. Internalized Salmonella Typhimurium was detected at levels of 2.0 to 5.1 log CFU/g under all conditions. Continuous exposure to contaminated water during the entire period generated significantly higher levels of Salmonella Typhimurium internalization than sets receiving contaminated water for only a single day (P < 0.05). Preintervention methods lowered the level of internalized Salmonella by 1.84 log CFU/g (P < 0.05), whereas postintervention methods were ineffective in eliminating internalized pathogens. Preintervention did not completely inactivate bacteria in sprouts and demonstrated that the remaining Salmonella Typhimurium in water became more resistant to UV. Because postharvest intervention methods are ineffective, proper procedures for maintaining clean irrigation water must be followed throughout production in a hydroponic system.

Impact of phytopathogen infection and extreme weather stress on internalization of Salmonella Typhimurium in lettuce.

Internalization of human pathogens, common in many types of fresh produce, is a threat to human health since the internalized pathogens cannot be fully inactivated/removed by washing with water or sanitizers. Given that pathogen internalization can be affected by many environmental factors, this study was conducted to investigate the influence of two types of plant stress on the internalization of Salmonella Typhimurium in iceberg lettuce during pre-harvest. The stresses were: abiotic (water stress induced by extreme weather events) and biotic (phytopathogen infection by lettuce mosaic virus [LMV]). Lettuce with and without LMV infection were purposefully contaminated with green fluorescence protein-labeled S. Typhimurium on the leaf surfaces. Lettuce was also subjected to water stress conditions (drought and storm) which were simulated by irrigating with different amounts of water. The internalized S. Typhimurium in the different parts of the lettuce were quantified by plate count and real-time quantitative PCR and confirmed with a laser scanning confocal microscope. Salmonella internalization occurred under the conditions outlined above; however internalization levels were not significantly affected by water stress alone. In contrast, the extent of culturable S. Typhimurium internalized in the leafy part of the lettuce decreased when infected with LMV under water stress conditions and contaminated with high levels of S. Typhimurium. On the other hand, LMV-infected lettuce showed a significant increase in the levels of culturable bacteria in the roots. In conclusion, internalization was observed under all experimental conditions when the lettuce surface was contaminated with S. Typhimurium. However, the extent of internalization was only affected by water stress when lettuce was infected with LMV.

Localization of viable Salmonella typhimurium internalized through the surface of green onion during preharvest.

Internalization of pathogens poses a tremendous health risk in the consumption of raw fresh produce, because conventional washing cannot remove pathogens effectively after internalization occurs. We investigated (i) the pattern of Salmonella internalization in different parts of green onions when it was contaminated on their surfaces, and (ii) whether environmental factors (extreme weather) affect the extent of Salmonell a internalization. Green onions were surface contaminated with three different levels of Salmonella Typhimurium (1, 3, and 5 log CFU per green onion). Each contamination group was irrigated with three different water volumes to mimic water stress and to determine if Salmonella Typhimurium internalization was localized in different parts of the plant. The plants were collected 2 days after contamination, and surface bacteria were inactivated with ethanol and silver nitrate. The plants were then cut into two parts, upper and lower. The internalized Salmonella Typhimurium in each part was visualized and confirmed with a laser scanning confocal microscope and was quantified with the plate count method and real-time quantitative PCR (qPCR). The results indicate that Salmonella Typhimurium can be taken up through the plant surface and transported from the upper to the lower part of the plant. The level of viable internalized Salmonella Typhimurium (plate count) was higher in the lower part than the level in the upper leafy part, especially when the leaves were contaminated with a high concentration of Salmonella (5 log CFU, P < 0.05), whereas the total internalized Salmonella Typhimurium (by qPCR) was higher in the upper part (P < 0.05) at the same contamination level. The discrepancy between these results suggests that most internalized Salmonella lost viability in the upper part but survived in the lower part. Water stress did not significantly change the extent of internalization in either location of green onion, whether detected via plate count or qPCR when the contamination occurred on the surface.

Co-expression of recombinant nucleoside phosphorylase from Escherichia coli and its application.

The genes encoding purine nucleoside phosphorylase (PNPase), uridine phosphorylase (UPase), and thymidine phosphorylase (TPase) from Escherichia coli K12 were cloned respectively into expression vector pET-11a or pET-28a. The recombinant plasmids were transformed into the host strain E. coli BL21(DE3) to construct four co-expression recombinant strains. Two of them had double recombinant plasmids (DUD and DAD) and the other two had tandem recombinant plasmid (TDU and TDA) in them. Under the repression of antibiotic, recombinant plasmids stably existed in host strains. Enzymes were abundantly expressed after induction with IPTG and large amount of target proteins were expressed in soluble form analyzed with SDS-PAGE. Compared with the host strain, enzyme activity of the recombinant strains had been notably improved. In the transglycosylation reaction, yield of 2,6-diaminopurine-2'-deoxyriboside (DAPdR) from 2,6-diaminopurine (DAP) and thymidine reached 40.2% and 51.8% catalyzed by DAD and TDA respectively; yield of 2,6-diaminopurine riboside (DAPR) from DAP and uridine reached 88.2% and 58.0% catalyzed by TDU and DUD respectively.

PCR-RFLP to detect codon 248 mutation in exon 7 of p53 tumor suppressor gene.

Individual genome DNA was extracted fast from oral swab and followed up with PCR specific for codon 248 of p53 tumor suppressor gene. MspI restriction mapping showed the G-C mutation in codon 248, which closely relates to cancer susceptibility. Students learn the concepts, detection techniques, and research significance of point mutations or single nucleotide polymorphisms in individual genomes.