Sensitive Assay Research Articles

A Multiplex Recombinase-Aided qPCR Assay for Highly Sensitive and Rapid Detection of khe, blaKPC -2, and blaNDM -1 Genes in Klebsiella pneumoniae.

This study aimed to establish a highly sensitive and rapid single-tube, two-stage, multiplex recombinase-aided qPCR (mRAP) assay to specifically detect the khe, blaKPC-2, and blaNDM-1 genes in Klebsiella pneumoniae. mRAP was carried out in a qPCR instrument within 1 h. The analytical sensitivities of mRAP for khe, blaKPC-2, and blaNDM-1 genes were tested using recombinant plasmids and dilutions of reference strains. A total of 137 clinical isolates and 86 sputum samples were used to validate the clinical performance of mRAP. mRAP achieved the sensitivities of 10, 8, and 14 copies/reaction for khe, blaKPC-2, and blaNDM-1 genes, respectively, superior to qPCR. The Kappa value of qPCR and mRAP for detecting khe, blaKPC-2, and blaNDM-1 genes was 1, 0.855, and 1, respectively (p < 0.05). mRAP is a rapid and highly sensitive assay for potential clinical identification of khe, blaKPC-2, and blaNDM-1 genes in K. pneumoniae.

Sensitivity and Specificity of Selected Biomarkers and Their Combinations in the Diagnosis of Ovarian Cancer.

One of the greatest challenges in modern gynecological oncology is ovarian cancer. Despite the numerous studies currently being conducted, it is still sometimes detected at late clinical stages, where the prognosis is unfavorable. One significant contributing factor is the absence of sensitive and specific parameters that could aid in early diagnosis. An ideal screening test, in view of the low incidence of ovarian cancer, should have a sensitivity of greater than 75% and a specificity of at least 99.6%. To enhance sensitivity and specificity, diagnostic panels are being created by combining individual markers. The drive to develop better screening tests for ovarian cancer focuses on modern diagnostic methods based on molecular testing, which in turn aims to find increasingly effective biomarkers. Currently, researchers' efforts are focused on the search for a complementary parameter to those most commonly used that would satisfactorily enhance the sensitivity and specificity of assays. Several biomarkers, including microRNA molecules, autoantibodies, cDNA, adipocytokines, and galectins, are currently being investigated by researchers. This article reviews recent studies comparing the sensitivity and specificity of selected parameters used alone and in combination to increase detection of ovarian cancer at an early stage.

Fabrication of dextran-grafted magnetic polymer microspheres with low nonspecific protein adsorption as carriers for chemiluminescence immunoassays

Inhibiting the nonspecific protein adsorption on the surface of magnetic microspheres has become a pressing challenge for immunoassays. In this paper, a novel dextran-modified sandwich-type magnetic polymer microsphere was developed and demonstrated to be a promising carrier for chemiluminescence immunoassay (CLIA). Dextran-modified sandwich-type magnetic polymer microspheres (PFS-PGDN) were synthesized by surface-initiated atom transfer radical polymerization (SI-ATRP) and ring-opening reaction of epoxy and aminodextran on the surface of magnetic P(GMA-MMA)-NH2@Fe3O4@SiO2 (PFS) microspheres. Then, carboxylated dextran-modified sandwich-type magnetic polymer microspheres (PFS-PGDC) were prepared by the surface carboxylation of the microspheres, which allowed the prepared microspheres to better couple with nucleic acids or antibodies. It is found that after grafting dextran, magnetic PFS-PGDC microspheres could significantly reduce the nonspecific protein adsorption by over 90 %. The chemiluminescence immunoassay assay showed a reduction of the background value to a very low level, indicating that the magnetic spheres showed significant advantages in improving the sensitivity of the chemiluminescence assay. Moreover, the chemiluminescence sensitivity and signal values of the synthesized magnetic microspheres have reached high levels. Therefore, the magnetic microspheres synthesized here hold great promise for applications in the field of CLIA.

The Utilization of the SaLux19-Based Loop-Mediated Isothermal Amplification (LAMP) Assay for the Rapid and Sensitive Identification of Minute Amounts of a Biological Specimen.

Our research has developed a highly sensitive and simple assay to detect small amounts of animal and human biological material in less than 40 min. The handheld SaLux19 device developed at the Max Planck Institute of Experimental Medicine in Göttingen, Germany, was used to validate our concept. The proposed system uses isothermal amplification of DNA in a rapid assay format. Our results show that the assay can detect Sus scrofa nucleic acids with very high sensitivity and specificity. This detection system has potential for forensic scenarios.

Prevalence, characteristics, and virologic correlations of hepatitis delta (D) among patients with hepatitis B surface antigen in Mongolia.

Clinical and biochemical features of hepatitis delta virus (HDV) infections in Mongolia remain largely unknown. We aimed to investigate the clinical characteristics of HDV patients in Mongolia using several markers. The 143 hepatitis B surface antigen (HBsAg)-positive patients were divided into 122 HDV-positive and 21 HDV-negative patients by HDV RNA positivity. Subgroup analysis was performed between hepatitis B e antigen (HBeAg)-positive and -negative HDV-positive patients. Liver function, quantitative HBsAg (qHBsAg), anti-HDV Immunoglobulin (Ig) M, Mac-2 binding protein glycosylation isomer (M2BPGi), hepatitis B virus (HBV) DNA level, and HDV RNA level were tested. HDV RNA was positive in 85.3% (122/143) of patients showing anti-HDV IgG. Liver disease activity was higher in HDV-positive patients than in HDV-negative patients. The HDV-positive group included a higher proportion of patients with high qHBsAg and M2BPGi levels (p < 0.001). The positivity rate for anti-HDV IgM was significantly higher in the HDV-positive group (p < 0.001). HDV RNA levels showed an inverse correlation with qHBsAg levels in HBeAg-positive-HDV-positive patients (r = -0.49, p = 0.034), and a positive correlation with qHBsAg levels in HBeAg-negative patients (r = 0.35, p < 0.001). Hepatitis B virus (HBV) DNA and HDV RNA levels did not show any correlation. M2BPGi levels likewise did not correlate with HDV RNA levels. A high positivity rate for HDV RNA was observed for HBV patients in Mongolia using the highly sensitive HDV RNA assay. The positivity rate for anti-HDV IgM was high in HDV RNA-positive patients. Severity of liver disease and M2BPGi levels were both high in the HDV RNA-positive group.

A Sensitive Assay for Unbound Docetaxel Using Ultrafiltration plus HPLC-MS and Its Application to a Clinical Study.

Docetaxel, a taxane used in the treatment of solid tumours, exerts pharmacological activity when in its unbound form. We report a sensitive assay to quantify unbound docetaxel after oral administration of docetaxel plus encequidar (oDox+E). Unbound drug quantification is important due to its direct correlation with drug-related toxicity and therapeutic efficacy. We improve on the sensitivity of current assay methods and demonstrate the utility of the assay on a novel formulation of oral docetaxel. Ultrafiltration followed by high-performance liquid chromatography and tandem mass spectrometry (HPLC-MS/MS) was utilized. Long-term stability, precision, accuracy, and recovery experiments were conducted to validate the assay. Additionally, patient samples from a Phase I dose-escalation pharmacokinetic study were analyzed using the developed assay. The assay method exhibited long-term stability with an observed change between 0.8 and 6.9% after 131 days of storage at -60 °C. Precision and accuracy quality controls met the FDA acceptance criteria. An average recovery of 88% was obtained. Patient sample analysis demonstrated successful implementation of the assay. A validated sensitive assay was developed with an LLOQ of 0.084 ng/mL using 485 µL of human plasma. The sensitivity of the assay allowed quantification of unbound docetaxel concentrations in an early-phase oDox+E clinical study to compare it against IV docetaxel using pharmacokinetic modelling. Successful development of oDox+E represents an opportunity to replace the current IV docetaxel regimen with an oral regimen with lower cost, decreased side effects, and improve patient quality of life and experience.

Predicting the presence of infectious virus from PCR data: A meta-analysis of SARS-CoV-2 in non-human primates.

Researchers and clinicians often rely on molecular assays like PCR to identify and monitor viral infections, instead of the resource-prohibitive gold standard of viral culture. However, it remains unclear when (if ever) PCR measurements of viral load are reliable indicators of replicating or infectious virus. The recent popularity of PCR protocols targeting subgenomic RNA for SARS-CoV-2 has caused further confusion, as the relationships between subgenomic RNA and standard total RNA assays are incompletely characterized and opinions differ on which RNA type better predicts culture outcomes. Here, we explore these issues by comparing total RNA, subgenomic RNA, and viral culture results from 24 studies of SARS-CoV-2 in non-human primates (including 2167 samples from 174 individuals) using custom-developed Bayesian statistical models. On out-of-sample data, our best models predict subgenomic RNA positivity from total RNA data with 91% accuracy, and they predict culture positivity with 85% accuracy. Further analyses of individual time series indicate that many apparent prediction errors may arise from issues with assay sensitivity or sample processing, suggesting true accuracy may be higher than these estimates. Total RNA and subgenomic RNA showed equivalent performance as predictors of culture positivity. Multiple cofactors (including exposure conditions, host traits, and assay protocols) influence culture predictions, yielding insights into biological and methodological sources of variation in assay outcomes-and indicating that no single threshold value applies across study designs. We also show that our model can accurately predict when an individual is no longer infectious, illustrating the potential for future models trained on human data to guide clinical decisions on case isolation. Our work shows that meta-analysis of in vivo data can overcome longstanding challenges arising from limited sample sizes and can yield robust insights beyond those attainable from individual studies. Our analytical pipeline offers a framework to develop similar predictive tools in other virus-host systems, including models trained on human data, which could support laboratory analyses, medical decisions, and public health guidelines.

Analytical and clinical evaluation of the cobas Epstein–Barr virus test at a tertiary care cancer hospital

BackgroundEpstein–Barr Virus (EBV) viral loads in hematopoietic stem cell transplant (HSCT) recipients are typically monitored using quantitative molecular assays. The Cobas EBV test (Roche Molecular, Pleasanton, CA) has recently been FDA-cleared for the monitoring of EBV viral loads in plasma samples of transplant patients. In this study, we compared the viral loads obtained by a laboratory-developed test (EBV LDT) using Altona Analyte specific reagents (ASR) to those obtained on the Cobas EBV test. MethodsThe analytical performance of the assay was established using the EBV verification panel from Exact Diagnostics and the EBV ATCC strain B95-8. The clinical evaluation was performed using 343 plasma samples initially tested on the EBV LDT. ResultsThe analytical sensitivity (<18.8 IU/mL), precision (SD < 0.17 log) and linear range (35.0 IU/mL to 1E + 08 IU/mL) of the Cobas EBV assay established by the manufacturers were confirmed. The strength of the qualitative agreement was substantial between the cobas EBV and the EBV LDT (85.6 %; κ = 0.71) and almost perfect when discordant results were resolved (96.4 %; κ = 0.93). The quantitative agreement was moderate (82.9 %; κ = 0.53) with the viral load obtained on the Cobas EBV test being lower across the linear range of the two tests (mean log difference of 1.0). While the absolute values of the viral loads were markedly different, the overall trends observed in patients with multiple consecutive results were similar between the two tests. ConclusionsThe Cobas EBV test provides an accurate and valid, in vitro diagnostic (IVD) option for monitoring of EBV viral loads in transplant patients and should provide an opportunity for increased standardization and commutability of tests results across laboratories.

An update on the development of a bottlenose dolphin, Tursiops truncatus, immune reagent toolkit

There are extensive immunological reagents available for laboratory rodents and humans. However, for veterinary species there is a need for expansion of immunological toolkits, with this especially evident for marine mammals, such as cetaceans. In addition to their use in a research setting, immune assays could be employed to monitor the health status of cetaceans and serve as an adjunct to available diagnostic tests. Such development of specific and sensitive immune assays will enhance the proper care and stewardship of wild and managed cetacean populations. Our goal is to provide immune reagents and immune assays for the research community, clinicians, and others involved in care of bottlenose dolphins. This review will provide an update on our development of a bottlenose dolphin immunological toolkit. The future availability and continued development of these reagents is critical for improving wild and managed bottlenose dolphin population health through enhanced assessment of their responses to alterations in the marine environment, including pathogens, and improve our ability to monitor their status following vaccination.

Evaluating the effects of mefenoxam on taxonomic and functional dynamics of nontarget fungal communities during carrot cultivation

Ridomil Gold SL (45.3% a.i. mefenoxam) is a widely used chemical fungicide for the control of oomycetes. However, its impact on fungal communities remains unexplored. Therefore, the goal of this study was to examine the effects of mefenoxam on the temporal dynamics of fungal taxonomic and functional diversities during carrot cultivation under four treatment groups: mefenoxam application with and without Pythium inoculation, and untreated control groups with and without Pythium inoculation. Our in vitro sensitivity assay showed that the maximum recommended concentration of mefenoxam, 0.24 ppm, did not suppress the mycelial growth of P. irregulare. At 100 ppm, mycelial growth was only reduced by 11.4%, indicating that the isolate was resistant to mefenoxam. MiSeq sequencing data revealed transient taxonomic variations among treatments 2 weeks post-treatment. Mortierella dominated the fungal community in the mefenoxam-Pythium combination treatment, as confirmed through PCR using our newly designed Mortierella-specific primers. Conversely, mefenoxam-Pythium combination had adverse effects on Penicillium, Trichoderma, and Fusarium, and decrease the overall alpha diversity. However, these compositional changes gradually reverted to those observed in the control by the 12th week. The predicted ecological functions of fungal communities in all Pythium and mefenoxam treatments shifted, leading to a decrease in symbiotrophs and plant pathogen functional groups. Moreover, the community-level physiological profiling approach, utilizing 96-well Biolog FF microplates, showed discernible variations in the utilization of 95 diverse carbon sources among the treatments. Notably, arbutin, l-arabinose, Tween 80, and succinamic acid demonstrated a strong positive association with Mortierella. Our findings demonstrate that a single application of mefenoxam at its recommended rate triggers substantial taxonomic and functional shifts in the soil fungal community. Considering this impact, the conventional agricultural practice of repeated mefenoxam application is likely to exert considerable shifts on the soil ecosystem that may affect agricultural sustainability.

Development of recombinase polymerase amplification-based colorimetric detection assay for rapid identification of invasive cassava mealybug, Phenacoccus manihoti Matile-Ferrero

Phenacoccus manihoti Matile-Ferrero (Hemiptera: Pseudococcidae), is an economically important invasive cassava pest responsible for the massive devastation of cassava in Asia and African continent. Initially, identifying this invasive pest posed challenges because it closely resembled native mealybug species. Additionally, the traditional morphological identification process is labor-intensive and time-consuming. Detecting invasive pests at an early stage is crucial, hence development of a rapid detection assay is essential. In the current study, we have developed a simple, rapid, sensitive, and efficient molecular detection assay for P. manihoti based on Recombinase Polymerase Amplification (RPA). The primers for the RPA assay were designed using unique nucleic acid sequences of P. manihoti, and the protocol was standardized. Specificity test demonstrated that the RPA assay could amplify DNA of P. manihoti only, and no amplification was observed in six other mealybug species. The specificity of assay was confirmed using SYBR green-based colorimetric detection and gel electrophoresis where positive samples showed 195 bp amplicon size in P. manihoti samples. The assay successfully amplified P. manihoti DNA in thirty minutes at an annealing temperature of 41° C in a water bath and displayed a sensitivity of 72.5 picograms per microliter. The assay's simplicity, rapidity, and high sensitivity make it a valuable tool for detecting and monitoring P. manihoti in quarantine stations and facilitating in development of a portable diagnostic kit.

Inflammatory Protein Signatures as Predictive Disease-Specific Markers for Non-Alcoholic Steatohepatitis (NASH).

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic disease worldwide, consisting of a broad spectrum of diseases such as simple steatosis (NAFL), non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma. Hepatic inflammation plays a key role in the pathophysiology of NAFLD. Inflammatory mediators such as cytokines and chemokines are considered as contributing factors to NAFLD development and progression. In the present study, we aimed to investigate the inflammatory protein signatures as predictive disease-specific markers for non-alcoholic fatty liver disease (NAFLD). This cross-sectional study included healthy control (n = 64), NAFL (n = 109), and NASH (n = 60) human subjects. Serum concentrations of various cytokines and chemokines were evaluated using sensitive multiplex assays. We used principal component analysis (PCoA) to reveal distinct differences in the levels of cytokines and chemokines between each of the study groups. Further, a random forest classification model was developed to identify the panel of markers that could predict diseases. The protein-protein network analysis was performed to determine the various signaling pathways associated with the disease-specific panel of markers. Serum concentrations of TNF-α, IL-1β, IL-1ra, G-CSF, PDGF-BB, MCP-1, MIP-1a, MIP-1b, RANTES, eotaxin, IL-8 and IP-10 were significantly increased in NASH group as compared to control group. Furthermore, serum concentrations of IL-9 and IL-13 were significantly lower in the NASH group, whereas IL-2 levels were significantly decreased in the NAFL group when compared to the control group. PCoA results demonstrated statistically significant differences in cytokines and chemokines between each of the study groups (PERMANOVA p = 0.001; R2 = 0.102). RANTES, IL-1ra, MIP-1b, IL-2, and G-CSF could differentiate the NAFL group from the controls; G-CSF, IL-1ra, TNF-α, RANTES, and IL-9 could differentiate the NASH group from the controls; and G-CSF, IL-9, IL-13, eotaxin, and TNF- α could differentiate the NASH group from the NAFL group. Our protein-protein network revealed that these markers are involved in cytokine-cytokine receptor interaction, Th1 and Th2 cell differentiation, TNF, chemokine, JAK/STAT, P13K/Akt, TLR, NOD-like receptor, NF-kB, and adipocytokine signaling pathways which might be responsible for disease pathogenesis.Our study findings revealed a set of distinct cytokine and chemokine markers and they might be considered as biomarkers in distinguishing NASH from NAFL. Future multicentre studies with larger sample size are recommended to determine the potential utility of these panels of markers.

Phenotypic characterization and candidate gene analysis of a short kernel and brassinosteroid insensitive mutant from hexaploid oat (Avena sativa).

In an ethyl methanesulfonate oat (Avena sativa) mutant population we have found a mutant with striking differences to the wild-type (WT) cv. Belinda. We phenotyped the mutant and compared it to the WT. The mutant was crossed to the WT and mapping-by-sequencing was performed on a pool of F2 individuals sharing the mutant phenotype, and variants were called. The impacts of the variants on genes present in the reference genome annotation were estimated. The mutant allele frequency distribution was combined with expression data to identify which among the affected genes was likely to cause the observed phenotype. A brassinosteroid sensitivity assay was performed to validate one of the identified candidates. A literature search was performed to identify homologs of genes known to be involved in seed shape from other species. The mutant had short kernels, compact spikelets, altered plant architecture, and was found to be insensitive to brassinosteroids when compared to the WT. The segregation of WT and mutant phenotypes in the F2 population was indicative of a recessive mutation of a single locus. The causal mutation was found to be one of 123 single-nucleotide polymorphisms (SNPs) spanning the entire chromosome 3A, with further filtering narrowing this down to six candidate genes. In-depth analysis of these candidate genes and the brassinosteroid sensitivity assay suggest that a Pro303Leu substitution in AVESA.00010b.r2.3AG0419820.1 could be the causal mutation of the short kernel mutant phenotype. We identified 298 oat proteins belonging to orthogroups of previously published seed shape genes, with AVESA.00010b.r2.3AG0419820.1 being the only of these affected by a SNP in the mutant. The AVESA.00010b.r2.3AG0419820.1 candidate is functionally annotated as a GSK3/SHAGGY-like kinase with homologs in Arabidopsis, wheat, barley, rice, and maize, with several of these proteins having known mutants giving rise to brassinosteroid insensitivity and shorter seeds. The substitution in AVESA.00010b.r2.3AG0419820.1 affects a residue with a known gain-of function substitution in Arabidopsis BRASSINOSTEROID-INSENSITIVE2. We propose a gain-of-function mutation in AVESA.00010b.r2.3AG0419820.1 as the most likely cause of the observed phenotype, and name the gene AsGSK2.1. The findings presented here provide potential targets for oat breeders, and a step on the way towards understanding brassinosteroid signaling, seed shape and nutrition in oats.

Human and camel cystic echinococcosis - a polyclonal antibody-based sandwich ELISA for its serodiagnosis with molecular identification.

Cystic echinococcosis (CE) is an emergent neglected disease affecting human and animals in Egypt with a wide distribution and incidence. This study aimed to evaluate the use of a polyclonal antibody-based sandwich ELISA in the detection of Echinococcus granulosus antigen in human and camel sera. Hydatid cyst protoscoleces antigen (PsAg) was isolated from hydatid cysts collected from naturally infected camel livers and lungs. PsAg was used for immunization of rabbits to raise IgG polyclonal antibodies (IgG PsAb). IgG PsAb were then precipitated, purified using Protein-A Sepharose gel and labeled with horseradish peroxidase enzyme. We assayed the purity of the IgG PsAb, and the two prepared E. granulosus antigens CPsAg from camel cysts and HPsAg from human cysts by Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The resulted protein bands of the prepared CPsAg appeared at different molecular weights: 180, 90, 68, 54, 42 and 22kDa while, HPsAg shared with it in 4 common bands at 68, 54, 42, and 22kDa. The purified IgG PsAb had been resolved at two bands at 52kDa and at 32kDa. Sandwich ELISA were performed for the detection of circulating E. granulosus antigens in sera of human (n = 183) and camels (n = 190). The purified IgG PsAb showed strong reactivity against E. granulosus infected human and camel samples and no cross reactivity neither with free-healthy negative sera nor with others parasitic diseases (Schistosomiasis, Fascioliasis, Toxoplasmosis, Ancylostomiasis for human samples and Fascioliasis, ticks' infestation, Eimeriosis, Cryptosporidiosis, Nasal myiasis, Toxoplasmosis for camel samples). The sensitivity of the assay was 98.25% (56/57) and 96.9% (31/32) against human and camel samples, respectively. Specificity was 100% in both human and camel samples. Sandwich ELISA detected CE in 33.3% (24/72) and 55.6% (50/90) random human and camel samples, respectively. Indirect ELISA, using CPsAg, was used for detection of antibodies in positive human and camels' sera and detected 96.5% (55/57) and 93.8% (30/32) of human and camel samples, respectively. In our study, Genomic DNA was extracted from protoscoleces fluid of human liver hydatid cysts to identify the Echinococcus sp. isolate based on NADH dehydrogenase subunit 1 (NAD1) gene by Polymerase Chain Reaction (PCR) and the isolate (GenBank: OP785689.1) were identified as E. granulosus sensu lato genotype. In conclusion, Sandwich ELISA technique was found to be a potent and sensitive assay for detection of hydatid antigen in both human and camel samples.

Cascaded autocatalytic hairpin assembly molecular circuit for amplified fluorescent aptamer luteinising hormone assay

The quantitative detection of luteinising hormone (LH) is critical for the study of the physiological mechanism of reproductive function and the assessment of infertility and the clinical treatment of reproductive disorders. However, conventional approaches for LH detection are mostly based on an antibody recognition module with the limitations of sensitivity, simplicity and cost. The development of robust LH sensing methods is therefore highly demanded for facilitating the diagnosis of LH-related diseases. We establish a convenient, amplified and sensitive fluorescent aptamer LH assay based on new target-triggered and cascaded autocatalytic hairpin assembly (C-aCHA) circuit amplification means via initiator sequence replication. Target LH molecules bind the aptamers in the aptamer/initiator duplexes to release the initiator sequences, which trigger CHA formation of DNA three-way junctions (TWJs) and the unfolding of fluorescently quenched signal hairpins to show amplified fluorescence. The TWJs further activate another CHA cycle for the yield of more initiator sequences to form the C-aCHA circuit amplification cycles, which lead to the unfolding of many signal hairpins to exhibit substantially magnified fluorescence recovery for detecting LH down to 8.56 pM in the range from 10 pM to 50 nM. In addition, the monitoring of trace LH in diluted serums by this sensing approach has been also verified. Our LH assay clearly outperforms current existing antibody-based methods and the C-aCHA signal amplification strategy can be easily extended as a robust means for sensitively monitoring various biomolecular markers with simple replacement of the corresponding aptamers for diverse applications.

The dynamics of colicin E9 release from Escherichia coli in native conditions.

Colicin (Col) plasmid contains colicin encoding genes arranged in an operon controlled by an SOS inducible promoter. Therefore, any external stresses to the host cell can induce the expression of the downstream genes in the Col operon, including a lysis gene. The lysis protein is involved in the extracellular release of colicin through lysis of the producer cells, which causes a decline in culture turbidity. However, it is not yet known that E. coli cells with the native pColE9-J plasmid hold the same level of cell death at the population level following a set of induced conditions. In this study, using a mitomycin C sensitivity assay along with a live dead staining method of detection, we showed that the native pColE9-J plasmid, which unusually carries an extended Col operon (ColE9) containing two lysis genes, did not confer a rapid decline in the culture turbidity following induction with mitomycin C. Interestingly a subset of the cells suffered perturbation of their outer membrane, which was not observed from single lysis mutant (∆celE or ∆celI) cells. This observed heterogeneity in the colicin E9 release leading to differential outer membrane perforation may bring a competitive advantage to these cells in a mixed population.

Single-exon fetal RHD genotyping: a 31-month follow up in the obstetric population of Western Sweden.

The Rh blood group system is highly complex, polymorphic, and immunogenic. The presence of RHD gene variants in RhD negative pregnant women is a challenge in fetal RHD genotyping as it may influence the antenatal management of anti-D prophylaxis. The aim of this study was to determine the efficiency of a non-invasive single-exon approach in the obstetric population of Western Sweden in a 31-month follow up. The frequency and type of maternal RHD variants were explored and the relation to the ethnicity was elucidated. Discrepant results between fetal RHD genotyping and serological blood group typing of newborns were investigated and clarified. RHD exon 4 was analysed with quantitative real-time PCR technique in a total of 6,948 blood samples from RhD negative women in early pregnancy. All cases with suspected maternal RHD gene and discrepant results observed in newborn samples, were further investigated using both serological and molecular technologies. A total of 43 samples (0.6%) had inconclusive fetal genotyping result due the presence of a maternal RHD gene. These findings were in most cases (>66%) observed in pregnant women of non-European ancestry. Additionally, two novel RHD alleles were found. Seven discrepant results between fetal RHD genotype and serological RhD type of the newborns, were shown to be related to D antigen variants in newborns. Assay sensitivity was 99.95%, specificity 100%, and accuracy 99.97%. The single-exon approach for fetal RHD screening early in pregnancy is an appropriate choice in the population of Western Sweden, with a very low frequency of inconclusive results caused by the presence of maternal RHD gene variants. Due to the high sensitivity, specificity, and accuracy of the test, serological typing of neonates born to RhD negative women has no longer been performed at our laboratory since June 2023.

RT-QuIC detection of chronic wasting disease prion in platelet samples of white-tailed deer

BackgroundChronic wasting disease (CWD) is a prion disease of captive and free-ranging cervids. Currently, a definitive diagnosis of CWD relies on immunohistochemistry detection of PrPSc in the obex and retropharyngeal lymph node (RPLN) of the affected cervids. For high-throughput screening of CWD in wild cervids, RPLN samples are tested by ELISA followed by IHC confirmation of positive results. Recently, real-time quacking-induced conversion (RT-QuIC) has been used to detect CWD positivity in various types of samples. To develop a blood RT-QuIC assay suitable for CWD diagnosis, this study evaluated the assay sensitivity and specificity with and without ASR1-based preanalytical enrichment and NaI as the main ionic component in assay buffer.ResultsA total of 23 platelet samples derived from CWD-positive deer (ELISA + /IHC +) and 30 platelet samples from CWD-negative (ELISA-) deer were tested. The diagnostic sensitivity was 43.48% (NaCl), 65.22% (NaI), 60.87% (NaCl-ASR1) or 82.61% (NaI-ASR1). The diagnostic specificity was 96.67% (NaCl), 100% (NaI), 100% (NaCl-ASR1), or 96.67% (NaI-ASR1). The probability of detecting CWD prion in platelet samples derived from CWD-positive deer was 0.924 (95% CRI: 0.714, 0.989) under NaI-ASR1 experimental condition and 0.530 (95% CRI: 0.156, 0.890) under NaCl alone condition. The rate of amyloid formation (RFA) was greatest under the NaI-ASR1 condition at 10–2 (0.01491, 95% CRI: 0.00675, 0.03384) and 10–3 (0.00629, 95% CRI: 0.00283, 0.01410) sample dilution levels.ConclusionsIncorporation of ASR1-based preanalytical enrichment and NaI as the main ionic component significantly improved the sensitivity of CWD RT-QuIC on deer platelet samples. Blood test by the improved RT-QuIC assay may be used for antemortem and postmortem diagnosis of CWD.

Fluorescent Molybdenum Disulfide Quantum Dots for Sensitive Detecting Curcumin in Food Samples through FRET Mechanism.

A selective and sensitive fluorometric assay was developed for specific determination of curcumin (Cur) based on fluorescence resonance energy transfer (FRET) between molybdenum disulfide quantum dots (MoS2 QDs) and Cur. The MoS2 QDs were prepared via a one-step hydrothermal protocol using sodium molybdate dihydrate, L-cysteine (Cys) as precursors, and sodium cholate (SC) as a modification agent. The as-prepared MoS2 QDs possessed maximum fluorescence emission at 460nm with a 20% of fluorescence quantum yield (FQY). It was found that the fluorescence of MoS2 QDs could be quantitatively quenched by Cur through FRET mechanism. Therefore, Cur could be detected in the range of 0.1-20µg mL- 1 with a detection limit of 5 ng mL- 1. Additionally, the developed MoS2 QDs based fluorescent assay has been successfully applied for real food sample analysis with satisfactory results.

An ultrasensitive dual-mode approach for AFB1 detection: Colorimetric/label-free SERS aptasensor based on a self-assembled core-shell structured Ag@Au IP6 bifunctional nanozyme

A sensitive and accurate assay of aflatoxin B1 (AFB1) is critically important to ensure food safety. This work designed a dual-mode aptasensor for the convenient and sensitive detection of AFB1 in foodstuff. The aptasensor based on a bifunctional nanozymes was prepared by combining the hybridization chain reaction (HCR) amplification and alkaline phosphatase (ALP)-induced self-assembly reaction. The Ag@Au IP6 nanozyme demonstrated better peroxidase-like activity, effectively initiating the TMB−H2O2 color reaction and facilitating the generation of the blue oxidation product oxTMB. Furthermore, Ag@Au IP6 as a surface-enhanced Raman scattering (SERS) substrate featuring a core-shell structure can notably enhance the Raman signal of oxTMB. When AFB1 existed, the trigger probe exposed and initiated HCR, leading to the introduction of ALP, consequently mediating the generation of Ag@Au IP6 nanozymes, enhancing both SERS signal and colorimetric signal in the TMB-H2O2 system. Consequently, the dual-mode aptasensor is capable of achieving SERS/colorimetric detection with no need for the modification of signal molecules and the replacement of material or reagent. In on-site detection, the visual sensing assay could achieve with collecting and transforming the colorimetric signal to RGB value by a color-assist App in smartphones. Within optimal conditions, the detection limit of dual-mode aptasensor as low as 0.58 pg/L, and with a linear range from 2 pg/L to 200 pg/L, which can meet the needs for AFB1 precise quantification and on-site detection.