Assay For Detection Research Articles

Graphene-Based Virus Enrichment Protocol Increases the Detection Sensitivity of Human Norovirus in Strawberry and Oyster Samples.

Human noroviruses (HuNoVs), the most prevalent viral contaminant in food, account for a substantial proportion of nonbacterial gastroenteritis cases. Extensive work has been focused on the diagnosis of HuNoVs in clinical samples, whereas the availability of sensitive detection methods for their detection in food is lacking. Here, we developed a virus enrichment approach utilizing graphene-based nanocomposites (CTAB-rGO-Fe3O4) that does not rely on large instruments and is suitable for on-site food pretreatment. The recovery efficiency of the developed virus enrichment procedure for serially diluted GII.4 norovirus ranged from 10.06 to 72.67% in strawberries and from 2.66 to 79.65% in oysters. Furthermore, we developed a real-time recombinase polymerase amplification (real-time RPA) assay, which can detect as low as 1.22 genome copies µL-1 of recombinant plasmid standard and has no cross-reactivity with genomes of astrovirus, rotavirus, adenovirus, and MS2 bacteriophage. Notably, the combined virus enrichment and real-time RPA detection assay enhanced the detection limits to 2.84 and 37.5 genome copies g-1 in strawberries and oysters, respectively, compared to those of qPCR. Our strategy, the graphene-based virus enrichment method combined with real-time RPA, presents a promising tool for sensitively detecting HuNoVs in food samples.

Development and Validation of Duplex PCR Assay for Simultaneous Detection and Differentiation of Mycoplasma gallisepticum and Mycoplasma synoviae in Poultry

Background: Mycoplasmosis caused by Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS), is an economically significant disease in both commercial and backyard poultry. The present work aimed at developing a duplex PCR assay for quick, accurate detection and differentiation of MG and MS and validating it for screening purpose. Methods: Duplex PCR assay was standardized with primers designed specific to the mgc2 gene for MG and hemagglutinin vlhA gene for MS with amplicon size of 150 bp and 787 bp, respectively. Validation of molecular detection of MG and MS with duplex PCR assay was performed with a total of 179 pooled oropharyngeal swab samples from backyard poultry flocks from different parts of India. Result: The duplex PCR assay showed, 133/179 (74.3%), 39/179 (21.78%) and 35/179 (19.55%) positivity for MG, MS and both MG and MS, respectively. Duplex PCR assay showed sensitivity of detection limit of 10-6 of 1 mg/ml of template DNA. Comparison of results of duplex PCR assay with single PCR of field samples showed perfect agreement between two assays (k= 0.90). The duplex PCR validated in the study would be useful in rapid screening/ surveillance, diagnosis and differentiation of MG and MS in field samples in cost-effective manner and to devise effective control strategies.

Achieving precise dual detection: One-tube reverse transcription-recombinase aided amplification (RT-RAA) combined with lateral flow strip (LFS) assay for RNA and DNA target genes from pepper mild mottle virus and Colletotrichum species in crude plant samples

Ensuring the detection sensitivity of both RNA-derived and DNA-derived target genes in a single reaction has posed a significant challenge for on-site detection of plant pathogens. This challenge was addressed by developing a one-tube dual RT-RAA assay combined with LFS for the rapid on-site detection of pepper mild mottle virus (PMMoV) and four Colletotrichum species causing anthracnose in Solanaceous crops. By testing four different combinations of primer groups, two combinations were precisely adjusted within the dual RT-RAA system to balance amplification efficiency and maintain consistent levels of amplification in crude plant samples. Utilizing commercially accessible small-scale equipment and following a streamlined optimization strategy, the assay achieved a limit of detection of 0.32 copies/μL of target genes in the reaction. Importantly, it demonstrated no cross-reactivity with other plant pathogens, thereby affirming the high sensitivity and specificity of the developed dual RT-RAA-LFS detection assay. Moreover, the entire process took only 25 min from sample collection to the visible presentation of results. The assay was validated with 60 field samples and 10 seed samples, producing results consistent with reverse transcription quantitative polymerase chain reaction (RT-qPCR). Notably, it successfully detected PMMoV in systemic leaves without visible symptoms three days post-inoculation, underscoring its effectiveness in early disease detection. This streamlined strategy offers a valuable approach for rapid, low-cost, and highly sensitive on-site simultaneous detection of RNA genome-contained PMMoV and DNA genome-contained Colletotrichum species.

A quantitative real-time PCR assay for rapid detection and quantification of Amyloodinium ocellatum parasites in seawater samples

Amyloodinium ocellatum is one of the most important ectoparasites of marine fish, causing amyloodiniosis and mass mortality in aquaculture. To date, no diagnostic method has been developed to detect and quantify parasite abundance in seawater. In this study, a quantitative real-time PCR (qPCR) assay using the ITS-F3 and ITS-R3 primer pair based on the internal transcribed spacers of nuclear ribosomal DNA (ITS rDNA) of A. ocellatum was successfully established for the detection and quantification of dinospores in seawater. The dinospores were collected by suction filtration of seawater using a vacuum pump, and examination by light microscopy and scanning electron microscopy (SEM) showed that the dinospores were adsorbed on the surface of the cellulose acetate membrane. The linear relationship between seawater parasite abundance and Ct values followed the model y = −3.0607 x + 29.919 with a coefficient of determination (R2) of 0.985. The results of the assay showed high reproducibility and no cross-reactivity with other aquatic pathogens. The assay successfully detected dinospores (100 % success rate) at concentrations of 8 and 10 cell per 300 mL of seawater. The protective fluid buffer effectively protected the dinospores DNA from degradation for over 96 h. The method was used to investigate the life span of dinospores in seawater and the incubation patterns of tomonts in the tank, the results showed that a small fraction of dinospores can survive for more than 7 days in seawater and the process of dinospores incubation from the tomonts persisted up to 84 h. This study provides a valuable quantitative diagnostic tool to assist in the early monitoring and understanding of disease progression in amyloodinosis.

An ultra-sensitive suboptimal protospacer adjacent motif enhanced rolling circle amplification assay based on CRISPR/Cas12a for detection of miR-183.

MicroRNAs (miRNAs) have been recognized as promising diagnostic biomarkers for Diabetic Retinopathy (DR) due to their notable upregulation in individuals with the condition. However, the development of highly sensitive miRNAs assays for the rapid diagnosis of DR in clinical settings remains a challenging task. In this study, we introduce an enhanced CRISPR/Cas12a assay, leveraging suboptimal PAM (sPAM)-mediated Cas12a trans-cleavage in conjunction with rolling circle amplification (RCA). sPAM was found to perform better than canonical PAM (cPAM) in the detection of Cas12a-mediated ssDNA detection at low concentrations and was used instead of canonical PAM (cPAM) to mediate the detection. The parameters of reactions have also been optimized. In comparison with cPAM, sPAM has higher sensitivity in the detection of ssDNA at concentrations lower than 10 pM by Cas12a. By replacing cPAM with sPAM in the padlock template of RCA, ultra-high sensitivity for miR-183 detection is achieved, with a detection limit of 0.40 aM. within 25min and a linear range spanning from 1 aM. to 1 pM. Our assay also exhibits exceptional specificity in detecting miR-183 from other miRNAs. Furthermore, the applicability of our assay for the sensitive detection of miR-183 in clinical serum samples is also validated. This study introduces a groundbreaking assay with excellent performance through a simple modification, which not only addresses existing diagnostic challenges, but also opens exciting new avenues for clinical diagnosis in the realm of DR.

Magnetic nanoparticles and quantum dots coupled immuno nano fluorescence assay for visual detection of HPV16-induced cervical cancer cells from cytology/biopsy samples

Biopsy-based histopathology and immunohistochemistry for cervical cancer detection are costly, time-consuming, and require expert personnel for data interpretation. We developed a simple magnetic nanoparticle (MNPs) and quantum dots (QD) coupled immuno nano fluorescence assay (MNPQDCINFA) for visual detection of HPV16-induced cervical cancer cells under UV light from cytology/biopsy samples exploiting host cancer cells expressing viral E7 protein as a biomarker. The E7 domain-specific polyclonal antibodies were generated against the 1–44 amino acid N-terminal (anti-domainN antibody) and 48–98 amino acid C-terminal domain (anti-domainC antibody). These antibodies were bioconjugated with nonfluorescent MNPs (60 % efficiency) and fluorescent QDs (66 % efficiency) to generate capturing (MNPs-anti-domainN antibody) and detecting (QDs-anti-domainC antibody) nano-complex, respectively. Assay conditions, such as concentration of capturing (20 μM) and detecting (50 nM) antibody nano-complexes and incubation duration (30 min), were standardized. The analytical sensitivity using pure HPV16 E7 protein was recorded up to 200 ng with very high specificity to differentiate from other HPV strains E7 proteins. The diagnostic performance characteristics with cytology samples showed 100 % sensitivity and specificity compared to immunofluorescence and biopsy-based histopathology analysis. The present invention can be effectively used for a quick, disposable, rapid cervical cancer cell detection system as an alternate test for immunofluorescence and histopathology.

Analytical Validation of Cxbladder® Detect, Triage, and Monitor: Assays for Detection and Management of Urothelial Carcinoma.

Background: Cxbladder® assays are reverse transcription-quantitative polymerase chain reaction (RT-qPCR) tests incorporating five genetic biomarkers (CDK1, MDK, IGFBP5, HOXA13, and CXCR2) to provide risk stratification for urothelial carcinoma (UC) in patients with hematuria or undergoing surveillance for recurrent disease. This study evaluated the analytical validity of the Cxbladder Detect, Triage, and Monitor assays. Methods: Pre-specified acceptance criteria, including the assays' fundamental aspects (sample and reagent stability, RNA extraction quality, RT-qPCR linearity, and analytical sensitivity and specificity), accuracy and precision, and reproducibility between laboratories. Results: Cxbladder had an analytical sensitivity of 12.5-31.1 RNA copies/mL urine for the CDK1, MDK, IGFBP5, and HOXA13 UC biomarkers and 68.9 RNA copies/mL for the inflammatory biomarker CXCR2. All the pre-specified analytical criteria were met. Cxbladder had diagnostic sensitivity, specificity, positive predictive value, and negative predictive values of 77%, 94%, 68%, and 96%, respectively, for Detect; 95%, 46%, 20%, and 98% for Triage; and 91%, 39%, 21%, and 96% for Monitor. Cxbladder had high analytical accuracy (≤10.63% inaccuracy across all biomarkers) and good reproducibility (>85% concordance between laboratories). Conclusions: Cxbladder accurately and reproducibly detects UC biomarker expression and can aid clinicians in risk stratification of hematuria patients or those undergoing surveillance for recurrent UC.

Integration of surface swab with optical microscopy for detection and quantification of bacterial cells from stainless-steel surfaces.

Swab sampling is a common method for recovering microbes on various environmental surfaces. Its successful application for a specific target depends on the proper swab method and the following detection assay. Herein, we evaluated critical factors influencing surface swab sampling, aiming to achieve the optimal detection and quantification performance of optical detection for bacterial cells on stainless-steel surfaces. Our results showed the recovery rate of Salmonella enterica (SE1045) cells from the 10×10cm2 stainless-steel surface reached up to 92.71±2.19% when using ammonia bicarbonate-moistened polyurethane foam swabs for gentle collection, followed by ultrasound-assisted release in NH4HCO3 solution. Among the six different foam swabs, the Puritan™ Sterile Large Foam Swab contributed the lowest background noise and highest recovery efficiency when integrated with the optical detection assay. Notably, our method exhibited a strong linear relationship (r2=0.9983) between the detected cell numbers and the theoretical number of SE1045 cells seeded on surfaces in the range of 104-107 Colony Forming Units (CFU), with a limit of detection of 7.2×104 CFU 100cm-2. This integration was completed within 2 h, exhibiting the applicable potential in various settings.

Development of new RT-PCR assays for the specific detection of BA.2.86 SARS-CoV-2 and its descendent sublineages

The SARS-CoV-2 BA.2.86 variant, also known as Pirola, has acquired over 30 amino acid changes in the Spike protein, evolving into >150 sublineages within ten months of its emergence. Among these, the JN.1, has been rapidly increasing globally becoming the most prevalent variant. To facilitate the identification of BA.2.86 sublineages, we designed the PiroMet-1 and PiroMet-2 assays in silico and validated them using BA.2.86 viral RNA and clinical samples to ascertain analytical specificity and sensitivity. Both assays resulted very specific with limit of detection of about 1–2 RNA copies/μL. The assays were then applied in a digital RT-PCR format to wastewater samples, combined with the OmMet assay (which identifies Omicron sublineages except BA.2.86 and its descendants) and the JRC-UCE.2 assay (which can universally recognize all SARS-CoV-2 variants). When used together with the OmMet and JRC-CoV-UCE.2 assays, the PiroMet assays accurately quantified BA.2.86 sublineages in wastewater samples. Our findings support the integration of these assays into routine SARS-CoV-2 wastewater surveillance as a timely and cost-effective complement to sequencing for monitoring the prevalence and spread of BA.2.86 sublineages within communities.

Hydrolysis probe assays for the detection of pathogenic Enterohemorrhagic Escherichia coli: Multi-Country validation study

Enterohemorrhagic Escherichia coli (EHEC) strains are foodborne pathogens frequently associated with outbreaks linked to red meat, posing significant global food safety concerns. While several commercial assays exist for detecting EHEC strains belonging to serogroups O26, O103, O111, and O121, they often fail to distinguish pathogenic strains from non-pathogenic strains lacking virulence genes. These non-pathogenic E. coli interfere with the accuracy of existing assays, resulting in false-positives rates between 81 – 100 % for beef products. This study aimed to standardize and validate four hydrolysis probe assays for the specific detection of pathogenic strains of E. coli O26, O103, O111, and O121. Primers and probes were designed to target single nucleotide polymorphisms conserved among pathogenic strains of each target serogroup. The standardized assays were validated using a comprehensive collection of pure culture and DNA samples (n = 248) from Canada, France, Switzerland, and the United States; laboratory-inoculated beef and spinach (n = 132), fractionally inoculated ground beef (n = 390), and samples from the federal red meat surveillance program (n = 166). The results demonstrated an overall accuracy of 94.8 % in predicting the virulence profile of pure culture strains. These four assays are a single-step PCR screening tool that can determine the presence of virulent strains. These assays provide food testing laboratories worldwide with a set of reliable methods for identifying pathogenic O26, O103, O111, and O121 strains. Thus, this assay will help to reduce product waste and financial losses caused by misleading positive results caused by presence of non-pathogenic strains.

Nitrogen-doped carbon quantum dots (NCQDs) detected to mercury ions in food monitoring

Using 2,3-diaminopyridine and citric acid as precursors, blue fluorescent nitrogen-doped carbon quantum dots (NCQDs) with a narrow size distribution (∼7.2 nm) were prepared and applied in the following assay for mercury ion detection at a weight ratio of 2,3-diaminopyridine:citric acid = 1:1 (0.2 g: 0.2 g, 20 mL for H2O), 220 °C, and 10 h. NCQDs was characterized by TEM, FT-IR, XPS, UV–Vis and EDS, and the prepared NCQDs display excitation-independent behavior due to less surface defects and uniform size. The optimal excitation and emission wavelengths of the NCQDs were 380 nm and 430 nm, respectively. Interestingly, the fluorescence of the NCQDs could be rapidly and selectively quenched by Hg2+ within 9 min at room temperature without further modification. Under optimal conditions, the limit of detection (LOD) was measured to be at the nanomolar level (42.4 nmol/L) with a linear range of 0–5.0 μmol/L, and fluorescence analysis of NCQDs was successfully used for the qualitative and quantitative analysis of mercury ions in food samples. Furthermore, our results revealed that fluorescence quenching occurred under the common fluences of the inner filter effect, and the static quenching effect was authenticated in the process in which Hg2+ coordinates with the NCQDs to form nonfluorescent complexes.

Development and validation of highly sensitive multiplex real-time RT-PCR assay for detection of classical swine fever virus genome

Classical swine fever (CSF) remains a challenge for pig farming industrial lover the world despite the measures taken. The last CS case in the Russian Federation was reported in 2020, however, the threat of the disease emerging still persists. A set of anti-epidemic measures including mainly preventive vaccination and annual diagnostic monitoring using molecular-genetic and serological methods is required for CSF virus introduction prevention and rapid eradication of potential disease out breaks. Therefore, areal-time reverse transcription-polymerase chain reaction using an internal control sample has been developed. Therefore, areal time reverse transcription-polymerase chain reaction using an internal control sample has been developed. Modified primers (locked nucleic acids containing conformationally blocked nucleosides) providing a higher affinity to the DNA matrix and physico-chemical stability and a FAM-labeled TaqMan probe were selected for 5’-untranslatedregion of the genome. The following validation parameters were defined: accuracy, repeatability, reproducibility, specificity and sensitivity. For comparative analysis of the developed as say sensitivity, swabs, samples of organs and tissues collected from pigs experimentally infected with an epizootic strain of the classical swine fever virus (spleen, kidney, liver, blood, lymph nodes, rectal and oral smears), animal-contaminated feed and virus-containing material with known virus titres were also tested in parallel with coded test systems No. x1 andx2. The developed assay was shown to have 100% diagnostic sensitivity and detection limit of 0,23 lgCCID50/cm3. Therewith, there sults of analysis of test systems No. x1, x2 based on above parameters were lower that could give rise to false positive real-time RT-PCR results and incorrect diagnosis. Thus, described assay can be used for extensive monitoring of classical swine fever in the Russian Federation

Abstract B054: Protease activity as a biomarker and potential target among pancreatic cancer patients

Abstract Background: The pancreas produces digestive proteases, which are often elevated in diseased states and capable of indiscriminate cell surface receptor cleavage. High levels of digestive protease activity have been implicated in the pathophysiology of pancreatic ductal adenocarcinoma (PDAC). We hypothesize that elevated digestive protease activity might downregulate or cleave immune cell receptors from the surface of cancer cells and contribute to the resistance of PDAC to immunotherapy. We have focused initially on major histocompatibility complex (MHC)-1, a ubiquitous receptor essential for antigen presentation, and CD155, an adhesion molecule over-expressed on cancer cells that regulates immune surveillance through binding to the TIGIT and CD226 receptors on leukocytes. Methods: The Rapid Assay for Protease Detection (RAPD) assay is a novel assay developed at our institution that utilizes fluorescent charge-changing peptide substrates to produce a positively charged fluorescent product fragment upon cleavage by the target protease. The RAPD assay was used to analyze protease activity levels in PDAC patients (N=50) and healthy (N=25) plasma samples. We also analyzed plasma samples obtained from an orthotopic KPC-derived mouse model for PDAC. We investigated the effects of specific proteases as well as plasma samples from PDAC and healthy subjects on MHC-1 and CD-155 receptor cleavage in cultured cells in vitro. Utilizing flow cytometry and immunofluorescent imaging, receptor loss was quantified for different proteases and plasma samples. Results: We observed elevated activity levels of digestive protease chymotrypsin as well as cathepsin-S and MMP-2 in the plasma of PDAC patients compared to healthy subjects. Similar patterns of protease activity were seen in plasma from tumor-bearing mice compared to non-tumor-bearing mice. Additionally, our results suggest loss of CD155 and MHC-1 from pancreatic cancer cells when incubated with trypsin and cathepsin-S. Incubation of cells with PDAC plasma also decreases the number of cells expressing MHC-1 and CD155 compared to control plasma, with greater differences observed in plasma with higher protease levels. Higher levels of cleaved CD155 are also detectable by ELISA in human and mouse PDAC plasma than in control plasma. Also, the protease activity (MMP2 and trypsin) correlates with plasma levels of CD155 and PD-L1. Conclusions: These studies begin to elucidate a possible role of digestive proteases in the ability of PDAC to evade the immune system. Ongoing experiments are exploring other immune receptors (including checkpoints) and whether specific protease inhibitors can block immune receptor cleavage. These experiments will inform subsequent experiments combining protease inhibitors with immunotherapy in mouse models. Citation Format: Utsav Joshi, Heather Farris, Jorge De La Torre De La Torre, kim Nguyen-Ta1, Michael Heller, Geert Schmid-Schonbein, Rebekah White. Protease activity as a biomarker and potential target among pancreatic cancer patients [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B054.

Detection of Sulfonamide Antibiotics Using an Elastic Hydrogel Microparticles-Based Optical Biosensor.

Sulfonamide antibiotics were the first synthetic antibiotics on the market and still have a broad field of application. Their extensive usage, wrong disposal, and limited degradation technologies in wastewater treatment plants lead to high concentrations in the environment, resulting in a negative impact on ecosystems and an acceleration of antibiotic resistance. Although lab-based analytical methods allow for sulfonamide detection, comprehensive monitoring is hampered by the nonavailability of on-site, inexpensive sensing technologies. In this work, we exploit functionalized elastic hydrogel microparticles and their ability to easily deform upon specific binding with enzyme-coated surfaces to establish the groundwork of a biosensing assay for the fast and straightforward detection of sulfonamide antibiotics. The detection assay is based on sulfamethoxazole-functionalized hydrogel microparticles as sensor probes and the biomimetic interaction of sulfonamide analytes with their natural target enzyme, dihydropteroate synthase (DHPS). DHPS from S. pneumoniae was recombinantly produced by E. coli and covalently coupled on a glass biochip using a reactive maleic anhydride copolymer coating. Monodisperse poly(ethylene glycol) hydrogel microparticles of 50 μm in diameter were synthesized within a microfluidic setup, followed by the oriented coupling of a sulfamethoxazole derivative to the microparticle surface. In proof-of-concept experiments, sulfamethoxazole, as the most used sulfonamide antibiotic in medical applications, was demonstrated to be specifically detectable above a concentration of 10 μM. With its straightforward detection principle, this assay has the potential to be used for point-of-use monitoring of sulfonamide antibiotic contaminants in the environment.

Detection Of CCR5 Delta-32 Mutation Using High-Resolution Melting Curve Analysis: Challenges and Facts.

The C-C chemokine receptor type 5 (CCR5) is a major co-receptor for human immunodeficiency virus (HIV). Some individuals carry the CCR5 delta-32 genetic polymorphism. People with homozygous CCR5 delta-32 gene are nearly completely resistant to HIV-1 infection. High-resolution melting curve (HRM) analysis is a post-PCR technique utilized for identifying genetic variations in a quick, affordable, and closed-tube assay. The objective of this study was to develop an HRM assay for easy detection of delta-32 mutations. DNA was extracted from peripheral blood mononuclear cells. HRM was performed to detect delta-32 mutation. The study investigated the impact of various factors, including annealing temperature, template concentration, touchdown PCR, additives, amplicon size, and program settings, on HRM Tm differentiation. It was expected that there would be a 4°C Tm difference between amplicons with and without delta-32 mutation, but the test showed a difference of only 2.3°C. In attempts to identify heterozygote delta-32 variants, a Tm difference of only 0.4°C could be achieved. Various modifications were applied, such as adjusting the template concentration, using touchdown PCR, and adding DMSO and glycerol. However, none of these changes helped to differentiate the Tm effectively, especially in delta-32 heterozygote samples. The HRM test identified four samples with heterozygote mutations in each HIV-infected (8.89%) and control (5.72%) groups. More importantly, this study showed that identifying the delta-32 mutation of the CCR5 gene using HRM assay is not as straightforward as previously suggested in some literature, and it requires special setup conditions.

Development and validation of a multi-marker liquid bead array assay for the simultaneous detection of PIK3CA and ESR1 hotspot mutations in single circulating tumor cells (CTCs)

BackgroundPIK3CA and ESR1 mutations are associated with progression and therapy resistance in metastatic breast cancer (MBC). CTCs are highly heterogeneous and their analysis at single cell level can provide unique information for mutational profiling and the existence of different sub-clones related to tumor progression. We have developed a novel multi-marker liquid bead array assay based on combination of an enzymatic mutation enrichment method, multiplex PCR-based assay, and liquid bead array technology for the simultaneous detection of PIK3CA and ESR1 hotspot mutations in liquid biopsy samples. We focus on single CTCs, however the assay can be used for bulk CTC and ctDNA analysis. Materials and methodsSingle CTCs were isolated from an ER+/HER2+ MBC patient from CellSearch® cartridges using the VyCAP Puncher System and subjected to whole genome amplification followed by nuclease-assisted minor-allele enrichment with probe-overlap (NaME-PrO) enrichment. The assay was validated for analytical sensitivity and specificity for the simultaneous detection of PIK3CA (E545K, E542K, H1047R, H1047L) and ESR1 (Y537S, Y537C, Y537N, D538G, L536H) mutations in single CTCs, while its clinical performance was evaluated on 22 single CTCs and three single white blood cells (WBCs). ResultsThe developed multi-marker liquid bead array assay is novel, highly specific and sensitive for both mutation panels. The assay can reliably detect mutation-allelic-frequencies (MAFs) as low as 0.1 %. The presence of PIK3CA and ESR1 mutations was detected in 13.6 % and 72.7 % of single CTCs, respectively. The developed assay is sample-saving since it requires only 2 μL of amplified DNA to check for nine hotspot PIK3CA and ESR1 mutations in a single cell. The developed liquid bead array assay (Luminex, US), based on a 96 microwell plate format, enables the simultaneous analysis of 96 single cells. ConclusionsThe developed novel multi-marker liquid bead array assay for the simultaneous detection of PIK3CA and ESR1 hotspot mutations in single CTCs is highly specific, highly sensitive, high-throughput, and sample-, cost-, and time-saving. This multi-marker liquid bead array assay can be extended to detect up to 100 mutations in many genes at once and can be applied for bulk CTC and ctDNA analysis.

Fluorescence assay for aflatoxin B1 based on aptamer-binding triggered DNAzyme activity.

As a kind of mycotoxin, aflatoxin B1 (AFB1), which is often found in agricultural products, poses a threat to human health. Developing asimple sensitive method for AFB1 detection is in great demand. Here, we reported an aptamer-based fluorescence assay for AFB1 detection by using DNAzyme to generate and amplify asignal. We redesigned a pair of DNA sequences, which originated from the anti-AFB1 aptamer and RNA-cleaving DNAzyme 10-23. In the absence of AFB1, the aptamer hybridized with the region of thesubstrate-binding arm of the DNAzyme, inhibiting the activity of theDNAzyme. In the presence of AFB1, the binding of AFB1 to theaptamer led to the displacement of theDNAzyme from theaptamer. The substrate-binding arm was unblocked, and the activity of theDNAzyme was restored for the hydrolysis of thefluorophore and quencher-labeled substrate, causing asignificant fluorescence increase. This assay could detect AFB1 in the dynamic range from 0.98 to 2000nmol/L with high selectivity, and the detection limit was 0.98nmol/L. Moreover, the assay was able to detect AFB1 in acomplex sample matrix. This work provides a useful tool for the analysis of AFB1.

Evaluation of cobas® Liat® Cdiff, STANDARD™ M10 C. difficile and Xpert® C. difficile BT assays for rapid detection of toxigenic Clostridioides difficile

We evaluated the analytical performance of three commercial molecular assays for rapid detection of Clostridioides difficile toxin B in stool samples. The results were compared with results from the BD MAX™ Cdiff assay. We analyzed forty negative and thirty-two positive stool samples with three rapid assays: Roche cobas® Liat® Cdiff, SD Biosensor STANDARD™ M10 C. difficile and Cepheid Xpert® C. difficile BT. The assays demonstrated a sensitivity of 96.9 %, 96.9 % and 100.0 % and a specificity of 100 %, 97.5 % and 97.5 %, respectively. There is limited data available on the analytical performance of the newly introduced STANDARD™ M10 C. difficile assay. In this study, all three rapid assays demonstrated similarly high analytical performance and can be used for detection of toxigenic C. difficile.

Recombinase-aid amplification combined with lateral flow detection assay for sex identification of the great white pelican (Pelecanus onocrotalus)

Sex identification in avian species is essential for biodiversity conservation and ecological studies. However, the sex of nearly half of the birds could not be identified based on their external appearance. It is difficult to visually identify sex to monitor the ecology and conservation of wild populations. In this study, we designed primer pairs for large white pelican using recombinase-based isothermal amplification combined with a lateral flow dipstick (RAA-LFD) assay for chromo-helicase-DNA binding protein (CHD) genes mapped to W chromosomes and an ultra-conserved element (UCE) located on chromosome 6, respectively. Our result showed that the raaW4-RAA-LFD can detect up to 0.1 ng of genomic DNA (gDNA) templates of female pelicans in 30 min at 39 ℃ and accurately distinguish female from male without any cross reactivity. RaaUCE2-RAA-LFD can amplify both male and female pelicans with a detection limit of 25 pg. To further evaluate the assay, 15 white pelicans of unknown sex were tested using the RAA-LFD assay and conventional polymerase chain reaction (PCR). The results of the raaW4-RAA-LFD assay were consistent with those of the conventional PCR. The developed RAA-LFD assay is equipped with field-deployable instruments and offers a field platform for rapid and reliable sex identification in pelicans.

A rapid, specific and ultrasensitive detection of the Chikungunya virus based on RT-RPA:CRISPR/Cas12a one-pot dual mode end-point detection system

BackgroundChikungunya (CHIK) is an underdiagnosed acute febrile illness (AFI) and an important cause of acute encephalitis syndrome (AES). Unavailaibility of rapid and sensitive molecular point-of-care tests (PoCTs) for CHIK at grass-root level, results in increased hospital burden, due to delayed diagnosis or misdiagnosis with other clinically relevant diseases. Since, no therapeutic intervention is readily available, accurate and differential diagnosis of CHIK is the only available option to initiate early supportive treatment. Thus, we aimed to develop a one-pot reverse transcription recombinase polymerase amplification (RT-RPA) mediated CRISPR/Cas12a based detection platform for rapid, specific, and ultrasensitive detection of chikungunya virus (CHIKV) in clinical samples. ResultsWe have successfully integrated CRISPR/Cas12a technology with reverse transcription recombinase polymerase amplification (RT-RPA) for the detection of Chikungunya virus (CHIKV). The developed assay enabled rapid detection of CHIKV within 35 min, requiring minimal handling process and instrumentation. Next, this assay demonstrated dual mode end-point detection capabilities, employing both fluorescence and lateral flow detection within a reaction. Our one-pot system allows the entire process to be completed without the need to open the reaction tube, thereby eliminating the risk of cross-contamination. Remarkably, the assay exhibits an analytical sensitivity of 412 zg μL−1 (≈1 copy), and 100 % clinical sensitivity and specificity for CHIKV. Furthermore, the developed assay demonstrated limit of detection of 8 gene copies of CHIKV. The assay demonstrates precise detection of CHIKV without any cross-reactivity with other pathogens commonly associated AFI or AES. SignificanceThe overall findings of this study indicate that the RT-RPA:CRISPR/Cas12a detection assay, with one-pot dual-mode detection approach enables rapid, specific and ultrasensitive molecular detection of CHIKV. This advancement holds significant potential for CHIKV detection in resource-limited settings, providing a robust tool for diagnosis and management of the disease. This developed assay may empower clinicians to initiate prompt supportive therapy for Chikungunya fever, thereby improving patient outcomes and public health responses.