OBJECTIVE: Thalassemia is a Mendelian-inherited blood disorder with severe consequences, including disability and mortality, making it a significant public health concern. Therefore, there is an urgent need for precise diagnostic technologies. We introduce two innovative diagnostic techniques for thalassemia, SNPscan and CNVplex, designed to enhance molecular diagnostics of thalassemia.
METHODS: The SNPscan and CNVplex assays utilize variations in PCR product length and fluorescence to identify multiple mutations. In the SNPscan method, we designed three probes per locus: two 5\' and one 3\', and incorporated allele identification link sequences into one of the 5\' probes to distinguish the alleles. The detection system was designed for 67 previously reported loci in the Chinese population for a specific genetic condition. CNVplex identifies deletion types by analyzing the specific positions of probes within the globin gene. This innovative approach enables the detection of six distinct deletional mutations, enhancing the precision of thalassemia diagnostics. We evaluated and refined the methodologies in a training cohort of 100 individuals with confirmed HBA and HBB genotypes. The validation cohort, consisting of 1647 thalassemia patients and 100 healthy controls, underwent a double-blind study. Traditional diagnostic techniques served as the control methods.
RESULTS: In the training set of 100 samples, 10 mutations (Hb QS, Hb CS, Hb Westmead, CD17, CD26, CD41-42, IVS-II-654, --SEA, -α3.7 and -α4.2) were identified, consistent with those identified by traditional methods. The validation study showed that SNPscan/CNVplex offered superior molecular diagnostic capabilities for thalassemia, with 100% accuracy compared to 99.43% for traditional methods. Notably, the assay identified three previously undetected mutations in 10 cases, including two deletion mutations (Chinese Gγ(Aγδβ)0 del and SEA-HPFH), and one non-deletion mutation (Hb Q-Thailand).
CONCLUSIONS: The SNPscan/CNVplex assay is a cost-effective and user-friendly tool for diagnosing thalassemia, demonstrating high accuracy and reliability, and showing great potential as a primary diagnostic method in clinical practice.

The ongoing co-circulation of multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains necessitates advanced methods such as high-throughput multiplex pseudovirus systems for evaluating immune responses to different variants, crucial for developing updated vaccines and neutralizing antibodies (nAbs). We have developed a quadri-fluorescence (qFluo) pseudovirus platform by four fluorescent reporters with different spectra, allowing simultaneous measurement of the nAbs against four variants in a single test. qFluo shows high concordance with the classical single-reporter assay when testing monoclonal antibodies and human plasma. Utilizing qFluo, we assessed the immunogenicities of the spike of BA.5, BQ.1.1, XBB.1.5, and CH.1.1 in hamsters. An analysis of cross-neutralization against 51 variants demonstrated superior protective immunity from XBB.1.5, especially against prevalent strains such as \"FLip\" and JN.1, compared to BA.5. Our finding partially fills the knowledge gap concerning the immunogenic efficacy of the XBB.1.5 vaccine against current dominant variants, being instrumental in vaccine-strain decisions and insight into the evolutionary path of SARS-CoV-2.

Real-time reverse transcription polymerase chain reaction (RT-PCR), a standard method recommended for the diagnosis of coronavirus disease 2019 (COVID-19) requires 2-4 h to get the result. Although antigen test kit (ATK) is used for COVID-19 screening within 15-30 min, the drawback is its limited sensitivity. Hence, a rapid one-step quadruplex real-time RT-PCR assay: termed ƩS COVID-19 targeting ORF1ab, ORF3a, and N genes of SARS-CoV-2; and Avocado sunblotch viroid (ASBVd) as an internal control was developed. Based on strategies including designing high melting temperature primers with short amplicons, applying a fast ramp rate, minimizing hold time, and reducing the range between denaturation and annealing/extension temperatures; the assay could be accomplished within 25 min. The limit of detection of ORF1ab, ORF3a, and N genes were 1.835, 1.310, and 1 copy/reaction, respectively. Validation was performed in 205 combined nasopharyngeal and oropharyngeal swabs. The sensitivity, specificity, positive predictive value, and negative predictive value were 92.8%, 100%, 100%, and 97.1%, respectively with 96.7% accuracy. Cohen\'s Kappa was 0.93. The newly developed rapid real-time RT-PCR assay was highly sensitive, specific, and fast, making it suitable for use as an alternative method to support laboratory diagnosis of COVID-19 in outpatient and emergency departments.

Proximity-dependent biotinylation is an important method to study protein-protein interactions in cells, for which an expanding number of applications has been proposed. The laborious and time-consuming sample processing has limited project sizes so far. Here, we introduce an automated workflow on a liquid handler to process up to 96 samples at a time. The automation not only allows higher sample numbers to be processed in parallel but also improves reproducibility and lowers the minimal sample input. Furthermore, we combined automated sample processing with shorter liquid chromatography gradients and data-independent acquisition to increase the analysis throughput and enable reproducible protein quantitation across a large number of samples. We successfully applied this workflow to optimize the detection of proteasome substrates by proximity-dependent labeling.

Extra-column band broadening can significantly reduce the performance of rapid ultra-high performance liquid chromatography-MS-based methods (UHPLC-MS). However, as we show here, UHPLC-MS/MS methods on short 2.1 mm i.d. columns can be optimized to reduce band broadening by simple procedures such as dispensing with the solvent divert valves placed between the column and the MS source. Vacuum jacketed columns have previously been shown to provide superior performance to conventional UHPLC-MS/MS by reducing on and post column band broadening. Here we have compared the optimized \"direct\" UHPLC approach for the high throughput (HT) bioanalysis of drugs and metabolites in biofluids such as urine and blood plasma with vacuum jacketed chromatography (VJC), using columns of the same geometry and packed with the same stationary phases. This study demonstrates that the performance of VJC was still superior to the direct UHPLC-MS/MS methods for rapid \"generic\" bioanalysis using gradient times of 0.25 to 5 min. Further investigations using microbore VJC-MS/MS, with 1 mm i.d. columns, for bioanalysis of the same biofluid samples showed that this format offers great promise for HT \"discovery\" drug and metabolite analysis/profiling. In addition the reduction of solvent use, by up to 90 % for methods when using microbore columns, can significantly contribute to improved sustainability and reducing costs per analysis.

The rise of mRNA as a novel vaccination strategy presents new opportunities to confront global disease. Double-stranded RNA (dsRNA) is an impurity byproduct of the in vitro transcription reaction used to manufacture mRNA that may affect the potency and safety of the mRNA vaccine in patients. Careful quantitation of dsRNA during manufacturing is critical to ensure that residual dsRNA is minimized in purified mRNA drug substances. In this work, we describe the development and implementation of a sandwich Enzyme-Linked Immunosorbent Assay (ELISA) to quantitate nanogram quantities of residual dsRNA contaminants in mRNA process intermediates using readily available commercial reagents. This sandwich ELISA developed in this study follows a standard protocol and can be easily adapted to most research laboratory environments. Additionally, a liquid handler coupled with an automated robotics system was utilized to increase assay throughput, improve precision, and reduce the analyst time requirement. The final automated sandwich ELISA was able to measure <10 ng/mL of dsRNA with a specificity for dsRNA over 2000-fold higher than mRNA, a variability of <15%, and a throughput of 72 samples per day.

We report the development and applications of a computer vision based reaction monitoring method for parallel and high throughput experimentation (HTE). Whereas previous efforts reported methods to extract bulk kinetics of one reaction from one video, this new approach enables one video to capture bulk kinetics of multiple reactions running in parallel. Case studies, in and beyond well-plate high throughput settings, are described. Analysis of parallel dye-quenching hydroxylations, DMAP-catalysed esterification, solid-liquid sedimentation dynamics, metal catalyst degradation, and biologically-relevant sugar-mediated nitro reduction reactions have each provided insight into the scope and limitations of camera-enabled high throughput kinetics as a means of widening known analytical bottlenecks in HTE for reaction discovery, mechanistic understanding, and optimisation. It is envisaged that the nature of the multi-reaction time-resolved datasets made available by this analytical approach will later serve a broad range of downstream efforts in machine learning approaches to exploring chemical space.

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Infectivity assays are the key analytical technology for the development and manufacturing of virus-based therapeutics. Here, we introduce a novel assay format that utilizes label-free bright-field images to determine the kinetics of infection-dependent changes in cell morphology. In particular, cell rounding is directly proportional to the amount of infectious virus applied, enabling rapid determination of viral titers in relation to a standard curve. Our kinetic infectious virus titer (KIT) assay is stability-indicating and, due to its sensitive readout method, provides results within 24 h post-infection. Compared to traditional infectivity assays, which depend on a single readout of an infection endpoint, cumulated analysis of kinetic data by a fit model results in precise results (CV < 20%) based on only three wells per sample. This approach allows for a high throughput with ~400 samples processed by a single operator per week. We demonstrate the applicability of the KIT assay for the genetically engineered oncolytic VSV-GP, Newcastle disease virus (NDV), and parapoxvirus ovis (ORFV), but it can potentially be extended to a wide range of viruses that induce morphological changes upon infection. The versatility of this assay, combined with its independence from specific instruments or software, makes it a promising solution to overcome the analytical bottleneck in infectivity assays within the pharmaceutical industry and as a routine method in academic research.

Developments in droplet microfluidics have facilitated an era of high-throughput, sensitive single-cell, or single-molecule measurements capable of tackling the heterogeneity present in biological systems. Relying on single emulsion (SE) compartments, droplet assays achieve absolute quantification of nucleic acids, massively parallel single-cell profiling, and more. Double emulsions (DEs) have seen recent interest for their potential to build upon SE techniques. DEs are compatible with flow cytometry enabling high-throughput multi-parameter drop screening and eliminate content mixing due to coalescence during lengthy workflows. Despite these strengths, DEs lack important technical functions that exist in SEs such as methods for adding reagents to droplets on demand. Consequently, DEs cannot be used for multistep workflows which has limited their adoption in assay development. Here, strategies to enable reagent addition and other active manipulations on DEs are reported by converting DE inputs to SEs on chip. After conversion, drops are manipulated using existing SE techniques, including reagent addition, before reforming a DE at the outlet. Device designs and operation conditions achieving drop-by-drop reagent addition to DEs are identified and used as part of a multi-step aptamer screening assay performed entirely in DE drops. This work enables the further development of multistep DE droplet assays.