Non-target Strains Research Articles

Phagemid-based capsid system for CRISPR-Cas13a antimicrobials targeting methicillin-resistant Staphylococcus aureus

In response to the escalating antibiotic resistance in multidrug-resistant pathogens, we propose an innovative phagemid-based capsid system to generate CRISPR-Cas13a-loaded antibacterial capsids (AB-capsids) for targeted therapy against multidrug-resistant Staphylococcus aureus. Our optimized phagemid system maximizes AB-capsid yield and purity, showing a positive correlation with phagemid copy number. Notably, an 8.65-fold increase in copy number results in a 2.54-fold rise in AB-capsid generation. Phagemids carrying terL-terS-rinA-rinB (prophage-encoded packaging site genes) consistently exhibit high packaging efficiency, and the generation of AB-capsids using lysogenized hosts with terL-terS deletion resulted in comparatively lower level of wild-type phage contamination, with minimal compromise on AB-capsid yield. These generated AB-capsids selectively eliminate S. aureus strains carrying the target gene while sparing non-target strains. In conclusion, our phagemid-based capsid system stands as a promising avenue for developing sequence-specific bactericidal agents, offering a streamlined approach to combat antibiotic-resistant pathogens within the constraints of efficient production and targeted efficacy.

Development and validation of a PMA-qPCR method for accurate quantification of viable Lacticaseibacillus paracasei in probiotics.

The effectiveness of probiotic products hinges on the viability and precise quantification of probiotic strains. This study addresses this crucial requirement by developing and validating a precise propidium monoazide combination with quantitative polymerase chain reaction (PMA-qPCR) method for quantifying viable Lacticaseibacillus paracasei in probiotic formulations. Initially, species-specific primers were meticulously designed based on core genes from the whole-genome sequence (WGS) of L. paracasei, and they underwent rigorous validation against 462 WGSs, 25 target strains, and 37 non-target strains across various taxonomic levels, ensuring extensive inclusivity and exclusivity. Subsequently, optimal PMA treatment conditions were established using 25 different L. paracasei strains to effectively inhibit dead cell DNA amplification while preserving viable cells. The developed method exhibited a robust linear relationship (R 2 = 0.994) between cycle threshold (Cq) values and viable cell numbers ranging from 103 to 108 CFU/mL, with an impressive amplification efficiency of 104.48% and a quantification limit of 7.30 × 103 CFU/mL. Accuracy assessments revealed biases within ±0.5 Log10 units, while Bland-Altman analysis demonstrated a mean bias of 0.058 Log10, with 95% confidence limits of -0.366 to 0.482 Log10. Furthermore, statistical analysis (p = 0.76) indicated no significant differences between theoretical and measured values. This validated PMA-qPCR method serves as a robust and accurate tool for quantifying viable L. paracasei in various sample matrices, including pure cultures, probiotics as food ingredients, and composite probiotic products, thereby enhancing probiotic product quality assurance and contributing to consumer safety and regulatory compliance.

Rapid identification of Bacteroides dorei using novel specific target revealed by pan-genome analysis and its application in food

Bacteroides dorei is a promising candidate for the next-generation probiotics (NGP), but its accurate identification remains challenging due to its close taxonomic relationship with other Bacteroides species. This study aimed to mine the novel specific molecular target for detecting Bacteroides dorei. Using pan-genome analysis and PCR validation, species-specific target genes with a size of 193 bp were identified, and the specificity of the designed primer pair (30 bp) for this molecular target was confirmed. The PCR assay demonstrated high applicability for detecting target and/or nontarget strains in three simulated food systems, yielding no false-positive or false-negative results. Additionally, a qPCR method was developed for quantifying Bacteroides dorei, with a linear detection range from 1 × 108 to 1 × 101 CFU/mL and an R2 value greater than 0.98. This method exhibited accurate and sensitive detection capabilities suitable for both food and feces samples. Overall, our study established PCR and qPCR assays for the rapid identification of Bacteroides dorei, advancing the exploration of Bacteroides species and the application of detection for NGPs in functional foods across various product forms.

Development of Tools to Detect and Identify Strains Belonging to the Pseudomonas syringae Species Complex Responsible for Vein Clearing of Zucchini

Vein clearing of zucchini (VCZ) is a disease caused by seedborne bacteria that affects young plants of Cucurbita pepo subsp. pepo. VCZ agents are distributed into four phylogenetic clusters within clades 2a and 2ba of phylogroup 2 of the Pseudomonas syringae species complex. All these strains are pathogenic to squash, but only certain strains can also attack melon and cucumber. Strains belonging to clades 2b and 2d are sometimes isolated from zucchini seeds but have not been associated with VCZ epidemics. Identification tools for VCZ agents are required to improve disease control. Primers were designed to implement a seven-gene multilocus sequence analysis (MLSA) scheme for a collection of 60 strains isolated from zucchini seeds. The MLSA showed a clear predominance of strains of cluster 2ba-A and the presence of VCZ strains in a fifth cluster (2ba-C). PCR tests were designed to characterize strains in the VCZ clusters, and a multiplex qPCR test was proposed to distinguish strains with a cucurbit host range extended to melon and cucumber, harboring hopZ5 and sylC, from other strains harboring avrRpt2 and sylC. Additional qPCR tests were also designed to gain insights into clade-2b and -2d strains that can be isolated from cucurbits. These tools evaluated in silico with the NCBI database and experimentally with a collection of 112 strains detected all target strains, except for the test dedicated to clade-2b strains, and excluded 96.7 to 100% of nontarget strains. These tools are intended to serve phylogenetic studies, epidemiological monitoring, and seed testing. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Specificity of the AMP-6000 Method for Enumerating Clostridium Endospores in Milk.

Enumeration of endospores of butyric acid-forming clostridia in cheese milk is an essential part of milk quality monitoring for cheese producers to avoid late blowing, severe spoilage caused by clostridia during ripening. However, due to the lack of an internationally standardized method, different methods are used and it is important to consider how the choice of method affects the results. This is particularly relevant when clostridial spore counts in milk are considered for quality payments. The aim of this study was to evaluate the specificity of the AMP-6000 method for the enumeration of endospores of cheese spoiling clostridia in milk. First, to assess the prevalence of Clostridium diversity and to determine potential non-target species, we identified isolates from positive reactions of the AMP-6000 method used to quantify clostridial endospores in raw milk and teat skin samples by MALDI-TOF MS. Based on these results, a strain library was designed to evaluate method inclusivity and exclusivity using pure cultures of target and non-target strains according to ISO 16140-2:2016. Most target Clostridium tyrobutyricum strains, as well as all tested C. butyricum and C. sporogenes strains were inclusive. However, C. beijerinckii may be underestimated as only some strains gave positive results. All non-target strains of bacilli and lysinibacilli, but not all paenibacilli, were confirmed to be exclusive. This study provides performance data to better understand the results of microbiological enumeration of butyric acid-forming clostridia in milk and serves as a basis for future methodological considerations and improvements.

Identification of antibiotic-resistance markers of Edwardsiella tarda using aptamers

Edwardsiella tarda poses significant challenges in the aquaculture sector due to its antibiotic resistance, leading to substantial economic loss in the aquaculture industry. Therefore, it is imperative to identify antibiotic-resistance markers to prevent and control E. tarda. In the present study, we used tetracycline-sensitive (S) and resistant (R) E. tarda strains and six non-target bacterial strains, to develop aptamers through the Systematic Evolution of Ligands by Exponential Enrichment technique. We then conducted comparative analysis of these aptamers based on their relative importance index values and selected six candidate sequences (M1, M13, M15, N57, N69, and N71) for affinity verification. The results showed that M1 had significantly higher affinity towards both the S and R strains compared to the non-target strains. M13 and M15 had significantly higher affinities for the S strain compared to the R strain and non-target strains, and N57, N69, and N71 had significantly higher affinities for R strains relative to the sensitive and non-target strains. Subsequent analysis indicated that N57 and N69 specifically recognized unique sites in the R strain. Therefore, our study presents a novel approach for detecting and controlling antibiotic-resistant strains of E. tarda and identification of drug-resistant markers in bacteria.

Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Ralstonia solanacearum Species Complex and Phylotype I in Solanaceous Crops

The Ralstonia solanacearum species complex (RSSC) causes bacterial wilt diseases, which affect a wide range of plant hosts, including Solanaceae, and is of major economic importance. Point-of-care (POC) techniques such as loop-mediated isothermal amplification (LAMP) enable the rapid and sensitive detection of plant pathogens and can be deployed directly in the field. For fast and reliable diagnosis on site, we optimized an RSSC-LAMP assay and developed a real-time LAMP assay targeting the phylotype I. Both LAMPs were highly specific, yielding negative results for a wide collection of nontarget strains and positive results for all target strains, except for two particular mulberry strains for the phylotype I assay. These results were supported by an extensive in silico analysis performed on 6,105 genomes of Burkholderiaceae. The two LAMP assays displayed high sensitivity on pure suspensions, with a detection limit at 103 and 104 CFU/ml for phylotype I and the RSSC, respectively. These thresholds correspond to theoretical quantities of as low as 5 and 50 CFU per reaction. Two simplified extraction methods were successfully used to detect the pathogen from different Solanaceae samples. We demonstrated that LAMP assays were operable on solanaceous crops during field surveys and varietal evaluation trials as rapid POC diagnostics tools, which can improve the disease management and control of the RSSC. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Rapid detection of Mucorales based on recombinase polymerase amplification and real-time PCR.

Mucormycosis, an invasive fungal disease with severe consequences, poses a significant threat to immunocompromised individuals. However, the timely and accurate identification of Mucorales infection continues to present difficulties. In this study, novel detection techniques utilizing recombinase polymerase amplification (RPA) and quantitative real-time polymerase chain reaction (qPCR) were developed, specifically targeting the mitochondrial rnl gene, in order to address this challenge. The specificity of the RPA and qPCR assay was assessed by adding genomic DNAs extracted from 14 non-targeted strains, as well as human and mouse blood. No false-positive results were observed. Additionally, genomic DNAs from 13 species in five genera of order Mucorales were tested and yielded positive results in both methods. To further evaluate the sensitivity of the assays, DNAs from Rhizopus oryzae, Mucor racemosus, Absidia glauca, Rhizomucor miehei, and Cunninghamella bertholletiae were utilized, with concentrations ranging from 1 ng/μL to 1 fg/μL. The limit of detection (LoD) for the RPA assay was determined to be 1 pg., with the exception of Rhizomucor miehei which had a LoD of 1 ng. The LoD for the qPCR assay varied between 10 fg and 1 pg., depending on the specific species being tested. Sensitivity analysis conducted on simulated clinical samples revealed that the LoD for RPA and qPCR assays were capable of detecting DNA extracted from 103 and 101 colony forming units (CFU) conidia in 200 μL of blood and serum, respectively. Consequently, the real-time RPA and qPCR assays developed in this study exhibited favorable sensitivity and specificity for the diagnosis of mucormycosis.

Validation of PVT VIABLE® for Detection of Legionella in Potable and Non-Potable Water: AOAC Performance Tested MethodSM 082303.

Phigenics Validation Test (PVT) VIABLE® (Viability Identification Assay by Legionella Enrichment) is a method to detect viable Legionella bacteria in building water systems. To evaluate PVT VIABLE against the ISO 11731:2017 Water Quality-Enumeration of Legionella reference method for the detection of viable Legionella species in potable and non-potable water. PVT VIABLE was tested for inclusivity (n = 50 strains of Legionella) and exclusivity (n = 30 non-target strains), robustness, and stability. A multi-laboratory instrument variation study was performed to evaluate the PCR data. The matrix study was performed on potable and non-potable water samples inoculated with Legionella pneumophila at a low (n = 20) and high fractional level (n = 5). Samples were analyzed using the PVT VIABLE and confirmed using ISO 11731:2017. Statistical analysis showed no difference between PVT VIABLE and ISO 11731:2017 for 100 mL test portions of potable and non-potable water. PVT VIABLE demonstrated high levels of specificity and sensitivity in the inclusivity and exclusivity study. Results of the robustness and stability studies demonstrated the method was sufficiently robust to handle small method changes and met the method claims of stability. PVT VIABLE allows the end user to obtain presumptive results for viable Legionella spp. contamination of potable and non-potable water in 2-3 days. PVT VIABLE provides viable Legionella results in 2-3 days versus 10-14 days for traditional spread plating. This novel diagnostic tool also differentiates between Legionella pneumophila sg1, Legionella pneumophila sg2-15, and Legionella spp. without the need for additional confirmation steps as outlined in ISO 11731:2017.

Molecular diagnosis of 5 silkworm strains endemic to South Korea using single-nucleotide polymorphisms selected from whole-genome sequences.

The domesticated silkworm, Bombyx mori Linnaeus (Lepidoptera: Bombycidae), often poses a challenge in strain identification due to similarities in morphology and genetic background. In South Korea, around 40 silkworm strains are classified as premium, including 5 endemic tri-molting strains: Goryeosammyeon, Sammyeonhonghoeback, Hansammyeon, Sun7ho, and Sandongsammyeon. These strains have potential for breeding programs in response to emerging industry demands, necessitating a reliable strain identification method. In this study, we established a molecular diagnosis approach for these 5 strains. We selected 2-4 single-nucleotide polymorphisms (SNPs) for each strain from whole-genome sequences of 39 strains, encompassing 37 previously studied and 2 newly added. These SNPs were utilized to construct decision trees for each endemic strain identification. The SNPs can be used to distinguish each target strain from the 38 nontarget strains by the tetra-primer amplification refractory mutation system-polymerase chain reaction, with the exception of HMS which needs the addition of PCR-restriction fragment length polymorphism method at the final step. This decision tree-based method using genomic SNPs, coupled with the 2 typing methods, produced consistent and accurate results, providing 100% accuracy. Additionally, the significant number of remaining SNPs identified in this study could be valuable for future diagnosis of the other strains.

Identification of the novel potential pathogen Trueperella pecoris with interspecies significance by LAMP diagnostics

Trueperella pecoris was described as a new species of the genus Trueperella in 2021 and might be pathogenic to various animal species. However, the lack of a suitable diagnostic test system stands in the way of epidemiological surveys to clarify possible causalities. In this study, a Loop-mediated Isothermal Amplification (LAMP) assay was developed and validated that was highly specific for T. pecoris. The assay provided an analytical sensitivity of 0.5 pg/25 µL and showed 100% inclusivity and exclusivity for 11 target and 33 non-target strains, respectively. Three different DNA extraction methods were evaluated to select the most LAMP-compatible method for cell disruption in pure and complex samples. Using an on-site applicable single-buffer DNA extraction with additional heating, the cell-based detection limit was 2.3 CFU/reaction. Finally, the LAMP assay was validated by means of artificially contaminated porcine lung tissue samples in which minimal microbial loads between 6.54 and 8.37 × 103 CFU per swab sample were detectable. The LAMP assay established in this study represents a suitable diagnostic procedure for identifying T. pecoris in clinical specimens and will help to collect epidemiological data on the pathogenicity of this species.

Rapid and sensitive detection of waterfowl mycoplasmas using TaqMan assays.

Waterfowl-specific mycoplasmas cause significant economic losses worldwide. However, only limited resources are available for the specific detection of three such bacteria, Mycoplasma anatis, M. anseris and M. cloacale. We developed species-specific TaqMan assays and tested their reliability across 20 strains of the respective target species as well as 84 non-target avian bacterial strains. Furthermore, we analysed 32 clinical DNA samples and compared the results with those of previously published conventional PCRs. The TaqMan assays showed 100% specificity and very high sensitivity, enabling the detection of target DNA as low as either 10 or 100 copies/μl concentration, depending on the assay. Importantly, we found that while the here developed TaqMan assays are reliable for species-specific detection of M. anatis, the previously published conventional PCR assay may give false positive results. In conclusion, the new assays are reliable, sensitive and suitable for clinical diagnostics of the target species.

Polyclonal antibodies inhibit growth of key cellulolytic rumen bacterial species.

Antibodies targeting specific bacterial species could allow for modification of the rumen microbial population to enhance rumen fermentation. However, there is limited knowledge of targeted antibody effects on rumen bacteria. Therefore, our objective was to develop efficacious polyclonal antibodies to inhibit the growth of targeted cellulolytic bacteria from the rumen. Egg-derived, polyclonal antibodies were developed against pure cultures of Ruminococcus albus 7 (anti-RA7), Ruminococcus albus 8 (anti-RA8), and Fibrobacter succinogenes S85 (anti-FS85). Antibodies were added to a cellobiose-containing growth medium for each of the three targeted species. Antibody efficacy was determined via inoculation time (0 h and 4 h) and dose response. Antibody doses included: 0 (CON), 1.3 × 10-4 (LO), 0.013 (MD), and 1.3 (HI) mg antibody per ml of medium. Each targeted species inoculated at 0 h with HI of their respective antibody had decreased (P < 0.01) final optical density and total acetate concentration after a 52 h growth period when compared with CON or LO. Live/dead stains of R. albus 7 and F. succinogenes S85 dosed at 0 h with HI of their respective antibody indicated a decrease (≥ 96%; P < 0.05) in live bacterial cells during the mid-log phase compared with CON or LO. Addition of HI of anti-FS85 at 0 h in F. succinogenes S85 cultures reduced (P < 0.01) total substrate disappearance over 52 h by at least 48% when compared with CON or LO. Cross-reactivity was assessed by adding HI at 0 h to non-targeted bacterial species. Addition of anti-RA8 or anti-RA7 to F. succinogenes S85 cultures did not affect (P ≥ 0.45) total acetate accumulation after 52 h incubation, indicating that antibodies have less of an inhibitory effect on non-target strains. Addition of anti-FS85 to non-cellulolytic strains did not affect (P ≥ 0.89) OD, substrate disappearance, or total VFA concentrations, providing further evidence of specificity against fiber-degrading bacteria. Western blotting with anti-FS85 indicated selective binding to F. succinogenes S85 proteins. Identification by LC-MS/MS of 8 selected protein spots indicated 7 were outer membrane proteins. Overall, polyclonal antibodies were more efficacious at inhibiting the growth of targeted cellulolytic bacteria than non-targeted bacteria. Validated polyclonal antibodies could serve as an effective approach to modify rumen bacterial populations.

A Label-Free Carbohydrate-Based Electrochemical Sensor to Detect Escherichia coli Pathogenic Bacteria Using D-mannose on a Glassy Carbon Electrode.

Controlling water and food contamination by pathogenic organisms requires quick, simple, and low-cost methods. Using the affinity between mannose and type I fimbriae in the cell wall of Escherichia coli (E. coli) bacteria as evaluation elements compared to the conventional plate counting technique enables a reliable sensing platform for the detection of bacteria. In this study, a simple new sensor was developed based on electrochemical impedance spectroscopy (EIS) for rapid and sensitive detection of E. coli. The biorecogniton layer of the sensor was formed by covalent attachment of p-carboxyphenylamino mannose (PCAM) to gold nanoparticles (AuNPs) electrodeposited on the surface of a glassy carbon electrode (GCE). The resultant structure of PCAM was characterized and confirmed using a Fourier Transform Infrared Spectrometer (FTIR). The developed biosensor demonstrated a linear response with a logarithm of bacterial concentration (R2 = 0.998) in the range of 1.3 × 10 1~1.3 × 106 CFU·mL-1 with the limit of detection of 2 CFU·mL-1 within 60 min. The sensor did not generate any significant signals with two non-target strains, demonstrating the high selectivity of the developed biorecognition chemistry. The selectivity of the sensor and its applicability to analysis of the real samples were investigated in tap water and low-fat milk samples. Overall, the developed sensor showed to be promising for the detection of E. coli pathogens in water and low-fat milk due to its high sensitivity, short detection time, low cost, high specificity, and user-friendliness.

Evaluation of the AmplifyRP XRT+ Kit for the Detection of Xylella fastidiosa by Recombinase Polymerase Amplification

Xylella fastidiosa is a quarantine priority pest in Europe and several other countries as it represents a major threat to many cultivated and wild plants. Early detection of the pathogen is needed to reduce potential losses and to stop the bacterial dissemination. Very few molecular methods are available for rapid field detection to identify the pathogen. Here, we evaluated a new rapid commercial detection test called AmplifyRP, based on molecular recombinase polymerase amplification (RPA). The AmplifyRP test was compared with the quantitative PCR (qPCR) Harper's assay, which represents the standard method used in most laboratories all over the world. Specificity was tested in vitro on a panel of target and nontarget strains. Additionally, the sensitivity of the RPA test was tested on X. fastidiosa genomic DNA, and the selectivity was compared between different host plant matrices spiked with X. fastidiosa cell suspensions. Although the qPCR assay showed a higher sensitivity than the RPA test, the latter appeared to be a very specific, rapid, and portable technique for which no skills are required to proceed. These findings should enable growers and inspectors to choose an appropriate diagnostic method. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Ultrafast, One-Step, Label-Based Biosensor Diagnosis Platform for the Detection of Mycobacterium tuberculosis in Clinical Applications.

Tuberculosis (TB) is a chronic infectious disease caused by the etiological agent Mycobacterium tuberculosis (MTB). Because the majority of TB patients come from poor economic backgrounds, the development of a simple, specific, low-cost, and highly sensitive detection method for the pathogen is extremely important for the prevention and treatment of this disease. In the current study, an efficient detection method for visual, rapid, and highly sensitive detection of MTB utilizing multiplex loop-mediated isothermal amplification combined with a label-based lateral flow immunoassay biosensor (mLAMP-LFIA) was developed. Three specific primer sets targeting the MTB genes IS6110 and mpb64 were successfully designed and synthesized for the LAMP assay. The optimal reaction conditions for the mLAMP-LFIA assay were confirmed to be 67 °C for 40 min. The mLAMP amplicons were intuitively verified using the LFIA biosensor within 5 min. The entire process, including clinical sample processing, amplification reaction, and product verification, was completed within 80 min. The limit of detection of the mLAMP-LFIA assay established in the current study was 100 fg per reaction for the genomic DNA of MTB H37Rv. The analytical specificity of the mLAMP-LFIA assay was one hundred percent, and no cross-reactions with non-target strains were detected. Compared with the GeneXpert test, the sensitivity of mLAMP-LFIA for 148 clinical specimens was 100% (97/97), and the specificity was 98.04% (50/51) in the preliminary evaluation of the clinical application. Hence, the mLAMP-LFIA method, targeting the IS6110 and mpb64 genes, is an ultrafast, one-step, low-cost, and highly sensitive detection method that could be used as a screening and/or diagnostic tool for MTB in the clinical setting, basic science laboratories, and especially in resource-poor regions.

Development of PCR rapid detection method for Listeria monocytogenes in oysters

To develop a polymerase chain reaction(PCR) method for rapid detection of Listeria monocytogenes in oysters without pre-enrichment. The combination of β-cyclodextrin and bentonite-coated activated carbon was used to remove PCR inhibitors from oyster samples, and the target gene inlB was used for the PCR subsequently. The specificity, sensitivity, and application of the developed method were verified, and the stability and application of the reagents stored under cryopreservation conditions were evaluated. The specificity of the developed PCR method was 100% for the detection of 130 target bacterial strains and 63 non-target bacterial strains. The method reduced the time required for Listeria monocytogenes detection to 4 h without pre-enrichment, and the detection limit was 10 CFU/25 g. The method was consistent with the conventional culture method on the detection rate and viable bacteria detection rate of Listeria monocytogenes in natural oyster samples(the coincidence rate was 100%). Additionally, the reagents could be used normally after storing at-20 ℃ for at least one year. The PCR method developed in this study has high specificity and sensitivity, and can be used for rapid, accurate detection of Listeria monocytogenes in oysters.

Development of a 3-plex droplet digital PCR for identification and absolute quantification of Salmonella and its two important serovars in various food samples

Salmonella is one of the most common foodborne pathogens that cause diarrhea in human, in which Enteritidis and Typhimurium are the top serovars frequently isolated from foodstuffs. A novel 3-plex droplet digital PCR (ddPCR) was successfully developed in this study for the simultaneous identification and absolute quantification of Salmonella and its two important serovars (Enteritidis and Typhimurium). The specificity of all the primers and probes was validated by testing 24 strains of Salmonella and 10 strains of other bacteria. Meanwhile, the anti-interference ability of this method was also evaluated using high proportion of non-target strains or various bacterial combinations. The detection limit of this 3-plex ddPCR was as low as 5 fg/μL for gDNA and 101 CFU/mL for pure cultures, respectively. Moreover, compared with quantitative PCR (qPCR), the detection limit of this ddPCR was much lower when it was applied to the detection of artificially contaminated food samples, which was 101 CFU/mL for lettuce, and 102 CFU/mL for milk and chicken juice samples, respectively. Additionally, a total of 100 retailed food samples were tested by this validated 3-plex ddPCR, which was also compared with traditional cultivation method. Notably, 19 of 20 samples showed consistent positive results by both methods, while the rest with the lowest concentration of Salmonella cells was detectable only by ddPCR. These results demonstrated that the 3-plex ddPCR established in this study presented highly specific, sensitive, and absolute quantitative capability, which had the potential to identify Salmonella, and to differentiate the two important serovars Enteritidis and Typhimurium in a wide variety of food samples.

Assessment of the ecotoxicological impact of captan@ZnO35–45nm and captan@SiO2 20–30nm nanopesticide on non-target soil microorganisms – A 100-day case study

Nanopesticide application should enable efficient pest management with smaller doses of an active ingredient. Nevertheless, the environmental risk assessment of nanopesticides is currently in its initial stages due to limited access to nanopesticides. Therefore, we synthesised nanofungicides with captan as an organic active ingredient and ZnO35–45nm or SiO2 20–30nm as nanocarriers (captan@ ZnO35–45nm and captan@ SiO2 20–30nm) and evaluated their environmental risk by testing different microbial parameters as its potential biomarkers. First, physicochemical analysis (SEM-EDS, XPS, and FTIR) confirmed the presence of captan in nanofungicides, and they maintained 43–61 % antifungal efficiency against pathogen fungi compared to captan. Second, a laboratory toxicity assay (spot test) showed that nanofungicides generally revealed 10–100-fold lower growth inhibition of non-target microbial strains compared to captan. Next, the effect of nanofungicides on the abundance, structure and function of non-target soil microorganisms was evaluated during the 100-day microcosm using orchard soil and compared to control, captan, and nanocarriers. The changes in the total number of bacteria, ammonia-oxidising bacteria (AOB) and fungi were enumerated using the copy number of the qPCR approach based on the copy number of 16S rRNA, amoA and ITS genes. The functional potential and microbial structure were estimated based on Community Level Physiological Profiles (CLPPs) and Phospholipid Fatty Acids (PLFAs) profiles. Generally, results indicated that nanofungicides affected soil microorganisms by changing, in different scale, various microbial parameters, but their negative effect was generally lower than pesticide. Although qPCR results revealed the harmful effect of all tested compounds on total bacteria number (16S rRNA) on day 42, and captan@ZnO35–45nm and nanocarrier SiO2 20–30nm still affected amoA gene copy number on day 100, but the total fungal abundance in orchard soil was not affected. Furthermore, the analyses of functional and structural microbial diversity indicated the recovery process that was the fastest for captan@SiO2 20–30nm nanofungicide. On the contrary, ZnO35–45nm increased and prolonged the negative effect of captan in synthesised nanofungicide and generally exerted a more profound and/or longer effect than SiO2 20–30nm nanocarrier. Therefore we conclude that SiO2 20–30nm has better potential to be used as a nanocarrier compared to ZnO35–45nm. More studies are needed but soil microorganisms as sensitive biomarkers should be used for environmental risk assessment of nanopesticides.

Rapid and Visual Determination of Cronobacter sakazakii in Powdered Infant Formula Using Competitive Annealing Mediated Isothermal Amplification (CAMP)

Cronobacter sakazakii (C. sakazakii) is a foodborne pathogen that causes serious infections, particularly in infants. Neonatal infections with this pathogen have an 80% mortality rate. One mechanism that C. sakazakii infects infants is through contaminated powdered infant formula (PIF). Here, based on the 16S rRNA gene, competitive annealing-mediated isothermal amplification (CAMP) is reported for the determination of C. sakazakii to reduce the risk of infection. The established CAMP method was able to accurately identify all target bacterial strains and did not yield false positive results with non-target bacterial strains. Furthermore, accelerating primers were designed and included in the CAMP reaction to enhance the amplification efficiency. The sensitivity of the assay for C. sakazakii in spiked PIF was 1.9 × 103 CFU/g. The CAMP assay with enrichment accurately detected the presence of C. sakazakii in PIF even at an initial inoculation level of 1 CFU/g. Additionally, to make the procedure simpler, hydroxynaphthol blue was employed as a visual indicator. For positive results, the reaction solution turns blue instead of violet, while the negative samples remain violet, which is suitable for rapid analysis in resource-limited settings.