Assay For Detection Research Articles

Efficiency of MTT and Trypan Blue Assays for Detection of Viability and Recovery of Different Frozen Cell Lines

Efficiency of MTT and Trypan Blue Assays for Detection of Viability and Recovery of Different Frozen Cell Lines

Development and preliminary assessment of the iFIND TBR: all-in- one molecular diagnostic assay for rapid detection of Mycobacterium tuberculosis and rifampicin resistance

IntroductionEarly and accurate diagnosis of tuberculosis (TB) is crucial for initiating timely treatment and preventing new infections. In this study, we introduced the iFIND TBR assay, an automated all-in-one tuberculosis detection approach that simultaneously detect Mycobacterium tuberculosis (MTB) and rifampicin (RIF) resistance.MethodsThe limits of detection (LOD), sensitivity, specificity, and RIF-R rpoB mutation detection of the iFIND TBR were tested on Mycobacterium tuberculosis DNA or sputum samples spiked with known numbers of M.tuberculosis H37Rv. Frozen clinical samples from patients suspected of having TB were also tested.ResultsThe LOD of the iFIND TBR for MTB detection were 13.34 CFU/ml (95% CI, 11.71-16.47), and for RIF resistance was 109.79CFU/mL (95% CI, 95-138.19). The iFIND TBR assay accurately distinguish MTB strains from non-tuberculous mycobacteria (NTM) without any cross reactivity. Testing on 157 clinical sputum samples, compared with the bacteriologically TB standard, the overall sensitivity and specificity of the iFIND TBR was 100% (95%CI, 94.64, 100) and 85.29% (95% CI, 74.61, 92.72), respectively. When assessing RIF susceptibility, the iFIND TBR achieved a sensitivity of 98.15% (95% CI, 90.11–99.95) and a specificity of 85.71% (95% CI, 67.33–95.97), compared with phenotypic drug susceptibility testing. Discordant RIF susceptibility results were more frequently observed in samples exhibiting heteroresistance.DiscussionThese findings demonstrate that iFIND TBR assay performs well in detecting TB and RIF resistance, and shows promise as a point-of-care tool in resource-limited areas.

Multigeographic clinical assessment of a molecular diagnostic assay for detection of key codons to predict decreased susceptibility or resistance to cephalosporins in Neisseria gonorrhoeae.

Cephalosporin resistance in Neisseria gonorrhoeae has severely compromised the efficacy of World Health Organization (WHO)-recommended therapies. This study aimed to methodologically evaluate the optimized Six-CodonPlus assay, and additionally conducted a multicenter evaluation to assess its clinical application, especially for predicting antimicrobial resistance (AMR). For methodological evaluation, 397 sequence-known N. gonorrhoeae isolates were evaluated for specificity, 17 nongonococcal isolates were assessed for cross-reactivity, 159 uncultured urogenital swabs and urine samples were evaluated for sensitivity at the clinical level. For multicenter evaluation, 773 isolates with confirmed phenotypic data and 718 clinical urogenital swabs collected from four geographical cities were, respectively, utilized for the evaluation of AMR-prediction strategies and the clinical application of the assay. The assay accurately identified specific single-nucleotide polymorphisms in resistance-associated genes, the detection limits dropped to 10 copies/reaction for individual targets. The specificity reached 100% and no cross-reactivity occurred with double-target confirmation. The assay could be directly applied to clinical samples containing over 20 copies/reaction. Multicenter evaluation formulated two optimal strategies for decreased susceptibility prediction in specific scenarios, and one tactic for prediction of resistance and identification of FC428-like strains. High sensitivity of 86.84% (95% CI, 71.11-95.05) and specificity of 99.59% (95% CI, 98.71-99.89) for resistance prediction were demonstrated for ceftriaxone (CRO). Regarding N. gonorrhoeae identification among multicenter swabs, specificity reached 97.53% (95% CI, 95.49-98.69), and sensitivity reached 93.77% (95% CI, 90.04-96.22). The Six-CodonPlus assay exhibited excellent detection performance and formulated optimal AMR-related prediction strategy with regional adaptability, providing critical information for population screening and clinical treatment.

Expression of Immunodominant gI Protein of Duck Enteritis Virus and its Evaluation for ELISA

Background: Duck plague (DP), also known as duck viral enteritis (DVE) caused by Anatid alpha herpesvirus-1, is the major infectious viral disease reported in India very often with significant economic losses due to high morbidity and mortality. The genome is approximately 158,091 bp with a genomic arrangement pattern (UL-IRS-US-TRS) that corresponds to type-D herpesvirus. The incubation period of the disease in domestic ducks ranges from 3 to 7 days and the mortality varies from 5-100%. It has been reported that the strategies to monitor and control DVE were often frustrating because the disease tends to establish a latent infection in waterfowl and vaccination failure. The present study was taken as an attempt to develop indirect ELISA using developed recombinant gI protein to diagnose DEV in field condition. Methods: In this study, we selected a membrane glycoprotein protein I (US7) gene, that plays an important role in virion sorting, cell-cell spread and binding of IgG Fc receptor. The gI protein was cloned with pET-32a (+) and expressed in a prokaryotic (E. coli) expression system. Further, recombinant protein was purified by nickel column affinity chromatography and refolded by dialysis. The obtained concentrated protein was then evaluated for its antigenicity and reliability in an Indirect-Enzyme-linked immunosorbent assay (ELISA) for DEV antibody detection in duck sera. Result: A panel of positive, negative and vaccinal serum was evaluated which indicated the potential use of the protein in the development of a serological assay for sero- surveillance of duck plague infection.

NanoString nCounter-Based Assay for Detection of Fusion-Associated Salivary Gland Tumors.

Salivary gland tumors include numerous subtypes that vary from benign to highly aggressive, with many showing overlapping histopathological features that can make diagnosis challenging. Most subtypes express driver fusion genes that are tumor specific, and detection of such fusions is useful for differentiating amongst specific diagnoses, determining appropriate tumor grading, and guiding effective treatment. Currently, fusions can be detected by FISH, RT-PCR or through next-generation sequencing approaches, all of which are highly effective methodologies but can be costly or time consuming. We developed a rapid NanoString nCounter platform-based assay to detect salivary gland tumor fusions using a combination of fusion junction-specific probes and an approach through differential exon expression analysis. The assay includes 68 junction-specific probes and analysis of exon expression across 9 fusion-associated genes in a single multiplex assay. Out of 55 retrospective and 171 prospective cases assayed, we accurately detected the majority of cases of pleomorphic adenoma, adenoid cystic carcinoma, cribriform adenocarcinoma, clear cell carcinoma, secretory carcinoma and NUT-rearranged carcinoma, including cases of these tumor types arising in non-salivary gland sites, with the major drawback being an inability to detect MAML2-containing mucoepidermoid samples. With mucoepidermoid carcinoma excluded, the assay shows an overall sensitivity of 96.1% and specificity of 100%. We show that the majority of salivary gland tumor fusions can be effectively detected with a single rapid NanoString based assay, which can serve as a useful adjunctive tool for routine diagnostic head and neck pathology. The assay is low cost with a rapid turnaround time (30h total assay time per sample batch, with minimal technician input required) compared to alternate detection methods.

Donor-derived cell-free DNA versus left ventricular global longitudinal strain in noninvasive detection of heart allograft injury

Abstract Introduction Invasive endomyocardial biopsy (EMB) is still considered the gold standard method for monitoring heart transplant (HTx) rejection. However, it is associated with potentially serious complications, and its histopathologic interpretation has high interobserver variability. Thus, noninvasive methods for surveillance of the transplanted organ remains of great interest. The noninvasive donor-derived cell-free DNA is a marker of graft injury which can indicate acute rejection, even before the histopathological diagnosis of rejection detected by EMB. The assay has a high negative predictive value for acute rejection. Left ventricular global longitudinal strain (LV GLS) is an emerging marker of subclinical myocardial injury which plays a crucial role in the evaluation of several cardiac diseases; however, its role in HTx injury has so far been poorly investigated. Purpose We aimed to investigate the efficacy of a local laboratory-run dd-cfDNA assay-based heart transplant rejection surveillance programme, and the prognostic and discriminatory performance of LV GLS for subsequent heart allograft injury. Methods HTx recipients without a history of allograft rejection requiring intensification of immunosuppressive therapy were transitioned from our EMB-based surveillance protocol to dd-cfDNA-based rejection surveillance. We assessed the percentage of dd-cfDNA to total cell-free DNA. Injury cut-off was defined by a dd-cfDNA level ≥0.20%, while the severe injury threshold was at ≥0.35%. LV ejection fraction was calculated using 2D Simpson’s method. To derive LV GLS, we analyzed apical 4-chamber, 2-chamber, and long-axis images by 2D speckle tracking echocardiography. Results A total of 244 dd-cfDNA samples were analyzed from 46 patients in fifteen runs performed monthly between October 2022 and December 2023. The dd-cfDNA fraction remained below the injury cut-off in most patients (81%). There were no missed rejection episodes. 88% of routine EMBs that would have otherwise been performed were safely avoided. Eighteen for-cause EMBs were performed based on dd-cfDNA levels suggesting injury. One EMB verified moderate cellular rejection (ISHLT grade 2R) that required steroid pulse therapy. Two EMBs proved mild cellular rejection, one EMB confirmed mild antibody-mediated rejection, while one EMB proved mixed rejection. Thirteen EMBs found no rejection. Mean LVEF was preserved (57.6%±7.6%). Mean LV GLS was -15.1%±2.5% in the whole cohort, while -15.6%±4.7% in recipients with HTx rejection on indication EMB. There was no significant correlation between dd-cfDNA and LV GLS (n=159). Conclusion The noninvasive dd-cfDNA assay is effective in ruling out acute rejection and safely decreases the necessity of invasive surveillance EMBs after HTx. LV GLS is less sensitive in comparison with the dd-cfDNA assay for detection of myocardial injury in the early stages of graft rejection in HTx recipients at low risk for rejection.

Exploring the diagnostic potential of miRNA signatures in the Fabry disease serum: A comparative study of automated and manual sample isolations.

Fabry disease, an X-linked lysosomal storage disorder caused by galactosidase α (GLA) gene mutations, exhibits diverse clinical manifestations, and poses significant diagnostic challenges. Early diagnosis and treatment are crucial for improved patient outcomes, pressing the need for reliable biomarkers. In this study, we aimed to identify miRNA candidates as potential biomarkers for Fabry disease using the KingFisher™ automated isolation method and NanoString nCounter® miRNA detection assay. Clinical serum samples were collected from both healthy subjects and Fabry disease patients. RNA extraction from the samples was performed using the KingFisher™ automated isolation method with the MagMAX mirVanaTM kit or manually using the Qiagen miRNeasy kit. The subsequent NanoString nCounter® miRNA detection assay showed consistent performance and no correlation between RNA input concentration and raw count, ensuring reliable and reproducible results. Interestingly, the detection range and highly differential miRNA between the control and disease groups were found to be distinct depending on the isolation method employed. Nevertheless, enrichment analysis of miRNA-targeting genes consistently revealed significant associations with angiogenesis pathways in both isolation methods. Additionally, our investigation into the impact of enzyme replacement therapy on miRNA expression indicated that some differential miRNAs may be sensitive to treatment. Our study provides valuable insights to identify miRNA biomarkers for Fabry disease. While different isolation methods yielded various detection ranges and highly differential miRNAs, the consistent association with angiogenesis pathways suggests their significance in disease progression. These findings lay the groundwork for further investigations and validation studies, ultimately leading to the development of non-invasive and reliable biomarkers to aid in early diagnosis and treatment monitoring for Fabry disease.

Optimization of loop mediated isothermal amplification assay (LAMP) for detection of chloroquine resistance in P. vivax malaria

Chloroquine is still used as a first-line treatment for uncomplicated Plasmodium vivax malaria in India and resistance to this therapy can act as a major hurdle for malaria elimination. It is difficult to monitor drug-efficacy and drug resistance through in vivo and in vitro studies in case of Plasmodium vivax so analysis of molecular markers serves as an important tool to track resistance. Molecular methods that are currently in use for detecting single nucleotide polymorphisms in resistant genes including Polymerase chain reaction (PCR), Realtime-Polymerase chain reaction require highly sophisticated labs and are time consuming. So, with this background the study has been designed to optimize Loop Mediated Isothermal Amplification Assay to detect single nucleotide polymorphisms in chloroquine resistance gene of Plasmodium vivax in field settings. Eighty-eight Plasmodium vivax positive samples were collected. Pvmdr1 gene was amplified for all the samples and sequenced. Obtained sequences were analyzed for the presence of single nucleotide polymorphisms in the target gene. Further Loop Mediated Isothermal Amplification Assay primer sets were designed for the target mutants and the assay was optimized. Clinical as well as analytical sensitivity and specificity for the assay was calculated. Double mutants with variations at T958M and F1076L were detected in 100% of the Plasmodium vivax clinical isolates with haplotype M958 Y976 Y1028 L1076. Designed primers for Loop Mediated Isothermal Amplification Assay successfully detected both the mutants (T958M and F1076L) in 100% of the isolates and do not show cross-reactivity with other strains. So, the assay was 100% sensitive and specific for detecting single nucleotide polymorphisms in the target Pvmdr1 gene. Limit of detection was found to be 0.9 copies/µl and lowest DNA template concentration detected by designed assay was 1.5 ng/µL. Observed prevalence of single nucleotide polymorphisms in Pvmdr 1 gene is indicating a beginning of trend towards chloroquine resistance in Plasmodium vivax. The present study optimized LAMP for detecting single nucleotide polymorphisms in Plasmodium vivax cases in field settings, thus would help in finding significant hubs of emerging chloroquine drug resistance and ultimately helping in the management of suitable antimalarial drug policy.

A highly sensitive and specific real-time quantitative polymerase chain reaction assay for Perkinsus marinus detection in oysters

Oyster aquaculture is one of the fastest-growing food production industries worldwide; however, it faces a significant challenge from the protist Perkinsus marinus, particularly in the USA. Although several quantitative molecular diagnostic methodologies are available for identifying diseases caused by P. marinus, the primer pairs used therein led to non-specific identification of other Perkinsus spp. Hence, a quantitative real-time PCR (Pm-qPCR) assay specific for P. marinus was developed using a TaqMan-based probe with the internal quencher in this study. A primer pair and probe specific to P. marinus were designed from a hypothetical protein of P. marinus collected from the whole-genome shotgun sequence database of the National Center for Biotechnology Information (NCBI). In silico analysis using homologous sequences of P. olseni and P. chesapeaki confirmed the high specificity of primers designed in this study. The Pm-qPCR assay was performed using seven different strains of P. marinus, P. olseni, and P. chesapeaki, revealing high specificity and sensitivity for detecting only P. marinus strains. In conclusion, it was demonstrated that Pm-qPCR can effectively and accurately diagnose P. marinus with high specificity and sensitivity. This assay is promising for monitoring oyster health and disease management in ecosystems and aquaculture.

A novel methylation-detection panel for HPV associated high-grade squamous intraepithelial lesion and cervical cancer screening

Cervical cancer (CC) was considered to be the most common gynaecological cancer, with an estimated 342,000 deaths worldwide each year, as the majority of patients were diagnosed at an advanced stage of the disease. The purpose of this study was to evaluate the predictive value of multi-locus methylation assay for the early detection of CC. The cervical exfoliated cell samples from 492 HPV-positive females with cervical lesions were collected and subjected to methylation detection of gene FAM19A4, EPB41L3 and PAX1 after bisulfite conversion. The levels of gene methylation in patients with different severity of cervical lesions were evaluated and compared. The receiver-operating characteristic (ROC) curve was established and efficacy indexes such as sensitivity, specificity and area under the curve (AUC) were calculated to assess the diagnostic value of DNA methylation detection at multiple gene loci for CC. The methylation levels of FAM19A4, EPB41L3 and PAX1 were significantly increased with the grade of cervical squamous intraepithelial lesions. The sensitivities of FAM19A4, EPB41L3 and PAX1 alone for high-grade squamous intraepithelial lesion (HSIL) and CC diagnosis were 84.6%, 86.3% and 88.0%, respectively; when three markers were combined by a logistic regression model, the sensitivity was 88.0%, with a high specificity of 97.7% and AUC of 0.957 (95% CI 0.937–0.977). Methylation status of FAM19A4, EPB41L3 and PAX1 were highly specific and effective for monitoring the progression of cervical lesions and the tri-gene methylation assay could be used as a triage tool for CC early screening.

Adaptation of a Model Spike Aptamer for Isothermal Amplification-Based Sensing

Isothermal amplification (IA) techniques like rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP) have gained significant attention in recent years due to their ability to rapidly amplify DNA or RNA targets at a constant temperature without the need for complex thermal cycling equipment. Such technologies, combined with colorimetric systems rendering visual confirmation of the amplification event, are ideal for the development of point-of-need detection methods suitable for field settings where access to specialized laboratory equipment is limited. The utility of these technologies, thus far limited to DNA and RNA targets, could be broadened to a wide range of targets by using aptamers. Composed of DNA or RNA themselves, aptamers can bind to substances, including proteins, metabolites, and inorganic substances. Their nucleic acid nature can potentially allow them to serve as a bridge, extending the reach of DNA/RNA-centric technologies to the broader molecular world. Indeed, the change in aptamer conformation occurring during ligand interaction can be used to elaborate ligand-responding RCA or LAMP templates. By using an existing aptamer targeting SARS-CoV-2 Spike protein as a model, we explored the possibility of establishing ligand-responsive IA systems. Our study used aptamers with simple sequence modifications as templates in LAMP assays and hyperbranched RCA (HRCA) by exploiting the dynamic nature of the model aptamer to trigger these IA systems. Importantly, our work uniquely demonstrates that this aptamer’s dynamic response to ligand binding can regulate both RCA and LAMP processes. This novel approach of using aptamer conformational changes to trigger LAMP paves the way for new aptamer-based detection assays. Our system detects 50 nM of Spike protein, with LAMP occurring within 30 min in the presence of Spike. The colorimetric readout showed clear results, allowing for the detection of Spike protein presence.

Evaluation of five different methods for diagnosis of Helicobacter pylori from fecal samples.

Accurate detection of Helicobacter pylori and its antimicrobial resistance is essential for eradication of the infections. The aim of this study was to compare five different CE-IVD marked assays in detection of H. pylori from 268 clinical stool samples. Samples were considered positive for H. pylori when at least three of the five tests were positive. Amplified IDEIA Hp StAR (Oxoid) and Premier Platinum HpSA PLUS (Meridian Bioscience Inc.) assays showed sensitivity of 100% [95% CI (confidence interval): 87-100] and LIAISON® Meridian H. pylori SA (DiaSorin) of 83.3% (95% CI: 66-93). Specificities of the assays were 94.5% (95% CI: 91-97), 95.4%; (95% CI: 92-97), and 97.1% (95% CI: 94-99) respectively. Amplidiag® H. pylori + ClariR (Mobidiag) assay showed 93.3% (95% CI: 78-99) and Allplex™ H. pylori & ClariR Assay (Seegene Inc.) 36.7% (95% CI: 22-55) sensitivity, while specificity of both was 97.9% (95% CI: 95-99). The Amplidiag® and Allplex™ assays concordantly detected clarithromycin resistance in positive for H. pylori samples. The Amplidiag® assay showed the highest accuracy, namely 97.4% (95% CI: 95-99). These data provide helpful information for planning laboratory diagnostics of H. pylori and detection of clarithromycin resistance from stool samples.

Establishment of Animal Infection Model of Spirometra Mansoni and Identification of Spirometra Mansoni by Enzyme-Linked Immunosorbent Assay.

Objective: Spirometra mansoni is a crucial zoonotic parasite. Its larvae are more harmful than adult worms due to their ability to migrate through the host's tissues and organs. Therefore, it is necessary to establish an animal model of spargana for observing pathological changes and exploring diagnostic techniques. Methods: In this study, we infected Kunming mice and cats without any pathogens by feeding sparganum (with the scolex and neck) in order to understand the infection cycle of S. mansoni and explore the preservation host of sparganosis. The infection of S. mansoni was determined by fecal detection and enzyme-linked immunosorbent assay (ELISA). Results: In the model of cats, the eggs of S. mansoni were found in the feces ten days after the infection. The serum-specific IgG antibodies against S. mansoni were positive in experimental groups (mice and cats), and after sixty days, the S. mansoni worms isolated from experimental groups were collected. Conclusion: In conclusion, the experimental results show that mice and cats can be stably infected with S. mansoni through feeding sparganum (with the scolex and neck). The infection method of this study has the potential to establish a practical model for investigating the diagnostic process of S. mansoni, laying the groundwork for application and development. ELISA was used to diagnose mice and cats infected with sparganosis mansoni, providing a case for non-invasive identification of animal sparganosis.

Dual Recognition Strategy-Based Ratiometric Electrochemical Assay for Ultrasensitive and Accurate Detection of Specific Circulating Tumor Cells.

Sensitive, specific, and accurate detection of circulating tumor cells (CTCs) is of great importance in the diagnosis and prognosis of cancer. Herein, an ultrasensitive ratiometric electrochemical biosensor was designed with a dual recognition strategy for highly specific and accurate detection of circulating MCF-7 human breast cancer cells based on gold film-modified porous organic cages loaded with ferrocene (Au/Fc@POCs) as the substrate and methylene blue-encapsulated covalent organic frameworks (MB@COFs) as the label material, producing two independent electrochemical signals from the Fc and MB probes, respectively. As the concentration of MCF-7 cells increases, the electrochemical signal of MB enhances significantly while the oxidation signal of Fc decreases remarkably. Under optimal experimental conditions, the ratios (IMB/IFc) between the double signals showed a broad dynamic range of 10 to 1 × 107 cells/mL with an effectively lower detection limit of 1 cells/mL (S/N = 3). Furthermore, the biosensor was able to accurately enumerate MCF-7 cells in human serum samples with excellent results. In this work, the developed ratiometric electrochemical biosensor offers a reliable and sensitive strategy for the quantitative determination of circulating MCF-7 human breast cancer cells as well as an effective approach for the clinical detection of rare cancer cells, especially in early stage cancer diagnosis.

A rapid and visual detection assay for Senecavirus A based on recombinase-aided amplification and lateral flow dipstick

BackgroundSenecavirus A (SVA) is a newly pathogenic virus correlated with the acute death of piglets and vesicular lesions in pigs. The further prevalence of SVA will cause considerable economic damage to the global pig farming industry. Therefore, rapid and accurate diagnostic tools for SVA are crucial for preventing and controlling the disease.MethodsWe designed multiple primer pairs targeting the most conserved region of the SVA 3D gene and selected those with the highest specificity. Nfo-probes were subsequently developed based on the highest specificity primer pairs. Subsequently, the recombinase-assisted amplification (RAA) reaction was completed, and the reaction temperature and duration were optimized. The RAA amplicons were detected using a lateral flow device (LFD). Finally, a rapid and intuitive RAA-LFD assay was established against SVA.ResultsThe SVA RAA-LFD assay can be performed under reaction conditions of 35°C within 17 minutes, with results observable to the naked eye. We then evaluated the performance of this method. It exhibited high specificity and no cross-reaction with the other common swine pathogens. The lowest detectable limits of this method for the plasmid of pMD18-SVA-3D, DNA amplification product, and viral were 3.86×101 copies/µL, 8.76×10-7 ng/µL, and 1×100.25 TCID50/mL, respectively. A total of 44 clinical samples were then tested using the RAA-LFD, PCR, and RT-qPCR methods. The results demonstrated a consistent detection rate between the RAA-LFD and RT-qPCR assays.ConclusionThe SVA RAA-LFD assay developed in our study exhibits excellent specificity, sensitivity, and time-saving attributes, making it ideally suited for utilization in lack-instrumented laboratory and field settings.

Development and Evaluation of Real-Time Quantitative PCR Assays for Detection of Phellinus noxius Causing Brown Root Rot Disease.

Brown root rot disease (BRRD) is a highly destructive tree disease. Early diagnosis of BRRD has been challenging because the first symptoms and signs are often observed after extensive tissue colonization. Existing molecular detection methods, all based on the internal transcribed spacer (ITS) region, were developed without testing against global Phellinus noxius isolates, other wood-decay fungi, or host plant tissues. This study aimed to develop SYBR Green real-time quantitative PCR (qPCR) assays for P. noxius. The primer pair Pn_ITS_F/Pn_ITS_R targets the ITS, and the primer pair Pn_NLR_F/Pn_NLR_R targets a P. noxius-unique group of homologous genes identified through a comparative genomics analysis. The homologous genes belong to the nucleotide-binding-oligomerization-domain-like receptor (NLR) superfamily. The new primer pairs and a previous primer pair G1F/G1R were optimized for qPCR conditions and tested for specificity using 61 global P. noxius isolates, 5 other Phellinus species, and 22 non-Phellinus wood-decay fungal species. Although all three primer pairs could detect as little as 100 fg (approximately 2.99 copies) of P. noxius genomic DNA, G1F/G1R had the highest specificity and Pn_NLR_F/Pn_NLR_R had the highest efficiency. To avoid false positives, the cutoff quantification cycle values were determined as 34 for G1F/G1R, 29 for Pn_ITS_F/Pn_ITS_R, and 32 for Pn_NLR_F/Pn_NLR_R. We further validated these qPCR assays using Ficus benjamina seedlings artificially inoculated with P. noxius, six tree species naturally infected by P. noxius, rhizosphere soil, and bulk soil. The newly developed qPCR assays provide sensitive detection and quantification of P. noxius, which is useful for long-term monitoring of BRRD status.

Triplex real-time PCR ZKIR-T assay for simultaneous detection of the Klebsiella pneumoniae species complex and identification of K. pneumoniae sensu stricto.

The pathogens of the Klebsiella pneumoniae species complex are widespread in food and animals and are among the main pathogens responsible for multidrug resistant infections in humans. In this study, we developed a highly sensitive detection assay that enables detection of this group of bacteria, with the simultaneous identification of the most common and clinically important species. This triplex one-reaction assay was shown to be highly sensitive and precise, enabling fast screening of varied samples for the presence of KpSC and K. pneumoniae sensu stricto.

UBL3 Interacts with PolyQ-Expanded Huntingtin Fragments and Modifies Their Intracellular Sorting.

UBL3 (Ubiquitin-like 3) is a protein that plays a crucial role in post-translational modifications, particularly in regulating protein transport within small extracellular vesicles. While previous research has predominantly focused on its interactions with α-synuclein, this study investigates UBL3's role in Huntington's disease (HD). HD is characterized by movement disorders and cognitive impairments, with its pathogenesis linked to toxic, polyglutamine (polyQ)-expanded mutant huntingtin fragments (mHTT). However, the mechanisms underlying the interaction between UBL3 and mHTT remain poorly understood. To elucidate this relationship, we performed hematoxylin and eosin (HE) staining and immunohistochemistry (IHC) on postmortem brain tissue from HD patients. Gaussia princeps-based split-luciferase complementation assay and co-immunoprecipitation were employed to confirm the interaction between UBL3 and mHTT. Additionally, we conducted a HiBiT lytic detection assay to assess the influence of UBL3 on the intracellular sorting of mHTT. Finally, immunocytochemical staining was utilized to validate the colocalization and distribution of these proteins. Our findings revealed UBL3-positive inclusions in the cytoplasm and nuclei of neurons throughout the striatum of HD patients. We discovered that UBL3 colocalizes and interacts with mHTT and modulates its intracellular sorting. These results suggest that UBL3 may play a significant role in the interaction and sorting of mHTT, contributing to the understanding of its potential implications in the pathophysiology of Huntington's disease.

ImageJ and smartphone app chemistry analyzer using image-based colorimetric assay for quantitative detection of melamine

Abstract A smartphone-assisted microchemistry analyzer with an image-based colorimetric assay using ImageJ and a smartphone app detection method was successfully developed for the quantitative detection of melamine (MEL). The color changes of the MEL in the colloidal gold lateral flow immunoassay strip were captured and analyzed using a smartphone-controlled analyzer with an LED light source and a smartphone camera. The smartphone camera and light source were used to read the colorimetric signal from the strip, and a smartphone app was written and installed onto the smartphone. The quantitative analysis was validated with ImageJ and smartphone app colorimetric analysis. The limits of detection (LODs) for ImageJ and the smartphone app were calculated as 0.30 and 0.07 mg/L, respectively. The highly quantitative relationships between the MEL concentrations and the optical density and gray value of the ImageJ and smartphone app detection method. The designed image-based biosensor is successfully applied to detect MEL in solution of standard MEL and commercial milk samples.

Loop-mediated isothermal amplification detection of Phytophthora kernoviae, Phytophthora ramorum, and the P. ramorum NA1 lineage on a microfluidic chip and smartphone platform.

Rapid, field-deployable assays such as loop-mediated isothermal amplification (LAMP) are critical for detecting nursery and forest pathogens like Phytophthora ramorum and P. kernoviae to prevent pathogen spread. We developed and validated four LAMP assays for genus-level detection of Phytophthora spp., species-level detection of P. kernoviae and P. ramorum and lineage-level detection of the P. ramorum NA1 lineage. Cross reactivity of the two species-specific LAMP assays was evaluated using a set of 18 Phytophthora spp. known to infect nursery crop hosts. The correct target species were detected by the species-level LAMP assays. The Phytophthora spp. LAMP assay was evaluated against 27 Phytophthora spp. and other bacterial and fungal pathogens and reacted with all the Phytophthora spp. evaluated but no other bacterial or fungal species. The limit of detection (LOD) of the P. kernoviae LAMP was 100 fg/µl and the LOD of the P. ramorum LAMP assay was 1 pg/µl of DNA. The NA1 LAMP assay was tested against the NA1, NA2, EU1, and EU2 lineages of P. ramorum and was lineage-specific but had a higher LOD (100pg/µl) than the species-specific LAMP assays. Both P. ramorum and P. kernoviae LAMP assays were highly precise (>0.94) in detecting the respective pathogens in symptomatic rhododendron leaves and co-inoculation experiments. The set of four LAMP assays were run in tandem on a microfluidic chip and smartphone platform and can be used in the field to detect and monitor spread of these regulatory Phytophthora spp. in forest and/or nursery settings.