Molecular Diagnostics on Smartphone

During the COVID-19 pandemic, point-of-care genetic testing (POCT) devices were used for on-time and on-site detection of the virus, which helped to prevent and control the spread of the pandemic. Smartphones, which are widely used electronic devices with many functions, have the potential to be used as a molecular diagnostic platform for universal healthcare monitoring. Several integrated diagnostics platforms for the real-time and end-point detection of COVID-19 were developed using the functions of smartphones, such as the operating system, power, sound, camera, data storage, and display. These platforms use the 5V output power of smartphones, which can be amplified to power a micro-capillary electrophoresis system or a thin-film heater, and the CMOS camera of smartphones can capture the color change during a colorimetric loop-mediated isothermal amplification test and detect fluorescence signals. Smartphones can also be used with self-written web-based apps to enable automatic and remote pathogen analysis on POCT platforms. Our lab developed a handheld micro-capillary electrophoresis device for end-point detection of SARS-CoV-2, as well as an integrated smartphone-based genetic analyzer for the qualitative and quantitative colorimetric detection of foodborne pathogens with the help of a custom mobile app. © 2023 SPIE.

Advancements in portable instruments based on affinity-capture-migration and affinity-capture-separation for use in clinical testing and life science applications.

The shift from testing at centralized diagnostic laboratories to remote locations is being driven by the development of point-of-care (POC) instruments and represents a transformative moment in medicine. POC instruments address the need for rapid results that can inform faster therapeutic decisions and interventions. These instruments are especially valuable in the field, such as in an ambulance, or in remote and rural locations. The development of telehealth, enabled by advancements in digital technologies like smartphones and cloud computing, is also aiding in this evolution, allowing medical professionals to provide care remotely, potentially reducing healthcare costs and improving patient longevity. One notable POC device is the lateral flow immunoassay (LFIA), which played a major role in addressing the COVID-19 pandemic due to its ease of use, rapid analysis time, and low cost. However, LFIA tests exhibit relatively low analytical sensitivity and provide semi-quantitative information, indicating either a positive, negative, or inconclusive result, which can be attributed to its one-dimensional format. Immunoaffinity capillary electrophoresis (IACE), on the other hand, offers a two-dimensional format that includes an affinity-capture step of one or more matrix constituents followed by release and electrophoretic separation. The method provides greater analytical sensitivity, and quantitative information, thereby reducing the rate of false positives, false negatives, and inconclusive results. Combining LFIA and IACE technologies can thus provide an effective and economical solution for screening, confirming results, and monitoring patient progress, representing a key strategy in advancing diagnostics in healthcare.

Direct SARS-CoV-2 Detection System Utilizing Simple-to-Use Capillary Gel Electrophoresis Sample-to-Result.

We present a Direct SARS-CoV-2 Detection System that achieves sample-to-results in less than two hours in three simple steps. The Detection System includes Direct one-step Reverse Transcription PCR (RT-PCR) reagents (Qexp-MDx kit), a portable thermal cycler (Qamp-mini) with a pre-programmed chip and a simple-to-use Capillary Gel Electrophoresis system (Qsep Series Bio-Fragment Analyzer) with high fluorescence detection sensitivity to solve the problems associated with traditional real-time PCR (qPCR) systems which produces inaccurate test results with high false negative and false positive rates. The proposed simple-to-use detection platform can provide high detection sensitivity (identify less than 20 copies), fast results (less than 120 minutes) and cost-effective results which should be suitable for decentralized testing application of COVID-19.

ELECTROCHEMICAL CAPILLARY-FLOW DRIVEN IMMUNOASSAY FOR DETECTION OF SARS-COV-2 NUCLEOCAPSID PROTEIN

As the COVID-19 pandemic continues, there remains a need for fast, accurate and low-cost diagnostic tests to prevent outbreaks. We have developed an electrochemical capillary-flow driven immunoassay (eCaDI) capable of detecting SARS-CoV-2 nucleocapsid (N) protein in self-administered nasal samples at the point of care (POC). The low-cost device is made of polyester and adhesive films and provides sequential delivery of sample and reagents to a detection zone integrating a screen-printed carbon electrode (SPCE) modified with anti-N protein antibodies from a single addition of sample, automating the steps of an ELISA. The modified electrodes are highly sensitive and selective for COVID-19 N protein and were successfully applied to test clinical samples. The novelty of this work resides in the integration of sensitive electrochemical detection with pump-free capillary-flow assay, providing accuracy at the POC. Previously reported systems are slow and/or require multiple user steps reducing the utility for POC applications relative to the system reported here. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

Presence of monoclonal components in SARS-CoV-2 patients: Preliminary data

Introduction: electrophoresis of serum proteins (EF) is indicated for the identification and monitoring of monoclonal components (CM). It has been shown that interleukins play a role in the differentiation of B cells in plasma cells producing immunoglobulins;it has been also demonstrated that COVID-19 patients show a higher prevalence of CM in comparison to the general population. The aim of this work is to retrospectively evaluate the presence of CM in patients hospitalized with COVID-19, compared to a population of patients admitted in non-COVID-19 wards. Methods: EF was performed in the two groups of patients (COVID positive and negative) using capillary electrophoresis. Patients with previous plasma cell dyscrasias have been excluded. Results: the results show that in the COVID positive group, the incidence of CM is statistically higher compared to the COVID negative group (39.7% versus 13.3%). In one patient, the CM was no longer detectable when the swab became negative. Conclusions: the study confirmed that the viral infection produces detectable CM, probably transitory as shown by a case index. The pathogenesis of the phenomenon could be explained by the cytokine stimulus on B cells and by the interaction of the virus with the lymphocyte ACE 2 receptor. Larger studies are needed to confirm the presented data.

QUANTITATIVE AND QUALITATIVE EVALUATION OF MRNA VACCINES AFTER STERILE FILTRATION

Background and importance The importance of mRNA-based vaccines increased rapidly due to the COVID-19 pandemic. However, little is known on the challenges linked to handling shortages and extended stability of these new types of substance. Since vaccine remnants have to be discarded according to the Summary of Product Characteristics, we hypothesise that sterile filtration after pooling is suitable to save vaccine material for clinical application. Aim and objectives The aim of this pilot study was to compare quality parameters of remnants derived from ready-to-use mRNA vaccine solutions before and after sterile filtration. Therefore, we pooled mRNA vaccine solution remnants from Corminaty vials (BioNTech/Pfizer) and compared particle size, distribution and quantity of the lipoplexes. In addition, quantity and/or quality of the mRNA was determined. Material and methods Measurements of invisible particulates in the range 1-50 mm were performed by light obscuration according to the European Pharmacopoeia (10th edn). The size of lipoplexes was measured with nanoparticle tracking analysis (NTA) to determine hydrodynamic diameter and particle concentration. Dynamic light scattering was employed complementarily to the NTA technique to focus on particle size from 0.3 nm to 10 mm. The concentration, purity and integrity of the mRNA was analysed by ultraviolet (UV) spectrophotometry and capillary electrophoresis after mRNA purification. Results After pooling the remnants of the vials we found a substantial increase of particulates >1 mm when compared to fresh vaccine samples. This effect was likely due to contamination of the examined probes with particles from ambient air. As expected, all these particulates were eliminated by sterile filtration. Size distribution and concentration of the lipoplexes were comparable between unfiltered and filtered samples. With respect to the mRNA, we identified the fragment of interest in all examined samples. Sterile filtration did not change the concentration, purity and integrity of the mRNA. Conclusion and relevance Our results indicate that sterile filtration of mRNA-based vaccines eliminates particle contamination from the vaccine solution while the concentration of lipoplex nanoparticles was not altered. Moreover, neither the quantity nor quality of the mRNA was affected by the filtration process. The results of our pilot study provide the first data on the stability of mRNA vaccines and help to fill knowledge gap when dealing with these substances in hospital pharmacy.

Buccal swab testing with the AmplideX PCR/CE SMN1/2 Plus Kit that assesses copy number and critical mutations for SMA

Introduction: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease that results from mutation of the survival motor neuron 1 gene (SMN1) and the most common genetic cause of infant death. Approximately 95% of SMA cases are caused by a deletion in both alleles of exon 7 in the SMN1 gene. The copy number of the highly homologous SMN2 gene is an important predictor of the severity of SMA as it has been shown to decrease disease severity in a dose-dependent manner. SMN1 and SMN2 only differ by a few nucleotides, presenting a challenge in determining copy numbers. While carriers typically have one copy of SMN1, cis duplication of SMN1 can produce “silent carrier” (2 + 0) genotypes, which are often associated with two SMN1 variants, c.*3 + 80T>G and c.*211_*212del, that can improve the overall carrier detection rate. SMA treatments SPINRAZA®,, Evrysdi™, and ZOLGENSMA® achieve profound benefits on survival and motor milestones by modifying SMN2 splicing or using gene replacement with functional SMN genes. Early detection of SMA (including SMN2 copy number status) and identification of at-risk couples through carrier screening is critical to aid in early intervention and family planning decisions. We developed an accurate and robust single-tube PCR assay and companion software (AmplideX® PCR/CE SMN1/2 Plus Kit*) that uses capillary electrophoresis (CE) to quantify SMN1 and SMN2 copy numbers (0 to ≥4) and determines the presence/absence of the two SMN1 gene duplication “silent carrier” variants, c.*3 + 80T>G and c. *211_*212del, and the SMN2 disease modifier variant c.859G>C. The SMN1/2 Plus Kit has been previously validated for use with DNA isolated from blood. Here, we verify that DNA isolated from buccal swabs can also be used to determine SMN1 and SMN2 copy number and expanded content using this kit. Materials and Methods: A total of 60 DNA samples isolated from buccal swabs, with varying SMN1/2 copies and other positive and negative variants,were tested using the SMN1/2 Plus kit at a single site (Asuragen). Samples were tested in two cohorts: an initial cohort containing 17 samples isolated from buccal swabs with column or magnetic bead-based methods, and a second cohort of 43 samples isolated from matched blood and buccal samples using column-based methods. PCR products were generated using a Veriti thermal cycler and resolved on Applied Biosystems™ 3500xL, 3130xl, 3730xl, and SeqStudio™ Genetic Analyzers. Raw electrophoresis data (.fsa) files were directly imported into an assay-specific analysis module of the AmplideX® Reporter software that automates peak detection and sizebased classification, SMN1 and SMN2 exon 7 copy number quantification, detection of gene duplication and disease modifier variants, and sample- and batch-level quality control checks. Samples were analyzed using the default (kit calibrator) and user-defined calibration (UDC) (buccal DNA) workflows as described in the protocol. Results: For the initial cohort of 17 Buccal swab samples, SMN1 copy number calls were concordant with MLPA reference results (reported as 0, 1, 2, or ≥3) for 16/17 (94.1%) of samples with default calibration and 17/17 (100%) of samples with UDC. Further, concordance for carrier samples (1 SMN1 copy) were 7/7 (100%) using both methods. SMN2 copy numbercallswere concordant with MLPA reference results for 17/17 (100%) of samples with either default calibration or UDC. For the second cohort of 43 buccal swab samples with matched blood samples, SMN1 and SMN2 copy number calls were concordant with the results from the paired whole blood for at least 95% of samples assessed across the four different CE platforms. All variant status calls were concordant between the buccal swab and whole blood results. Conclusions: Here, we demonstrate that buccal swabs are a compatible DNA source for the quantification of 0, 1, 2, 3, and ≥4 gene copies of both SMN1 and SMN2 and the status determination of three clinically significant variants using the single-tube PCR/CE SMN1/2 Plus kit. Although d fault calibration yielded high rates of agreement between copy number results from buccal swabs and reference results, analyzing samples with user-defined calibration (i.e. calibrating to a buccal swab sample) modestly improved concordance. These results suggest that DNA samples isolated from buccal swabs are compatible with this assay and has implications for more facile sample collection and handling, particularly given the strain of COVID-19 on healthcare infrastructure.

Clinical chemistry and immunoassay test results: Harmonisation and standardization of discordant notes

However, it is this very set of tests that have come Hin question. First, what is the normal and acceptable range (upper & lower limits) of different Clinical Chemistry and Immunoassay biomarkers in a particular population has been debated in different scientific fora. This is because the level of markers can change in relation to other biochemicals, biomolecules and hormones, which themselves vary considerably with race, gender, age, different physiological conditions (like pregnancy, new-born) and other illnesses & interfering substances. The variation can be to an extent that each individual can seem to have their own set-point of these parameters. A second problem which the diagnosticians have to grapple with is the variability of test-results in itself;even a broadly similar set of instruments and methods can provide variable results. It is then a real challenge to physicians, to decide whether the patient is suffering from a disease or not, since other factors can also cause changes in test levels. Standardization and harmonization of clinical chemistry and immunoassay testingis therefore still a formidable challenge, due to the lack of proper reference intervals and sometimes due to standardized measurement procedures. Laboratory medicine community the world over has realized that, variability in test results in different platforms can create a lot of confusion to clinicians and the general population;harmonization of procedures is therefore the need of the hour. In this talk, I will provide few examples and technical solutions to address laboratory challenges and to take it forward from both Clinical Chemistry as well as Immunoassay platforms. To begin with, harmonization and standardization of TSH and other thyroid function tests are still a formidable challenge, due to the lack of proper reference intervals and standardized measurement procedures. It has been documented that even a broadly similar set of instruments and methods can give up to 40% more or less values in TSH levels. Based on a particular population's demographic variations, reference interval can be different for immunoassay like TSH. Therefore, we have verified the reference interval for the Indian population for TSH in our laboratory. We have screened 800 subjects, of which 630 healthy subjects were chosen in the study group for reference interval verification. The reference interval (90% Confidence interval) for TSH by non-parametric procedure (bootstrap) was 0.48- 4.52, and by parametric one (after transformation of the data) was 0.45-4.27 for the adult population, which is little different from the manufacturer's guidelines. Similarly, we have conducted a study of 2797 female patients and 2805 male patients in a six month period and have observed that, women have a greater risk of being 14% under-diagnosed of acute coronary syndrome, if we donot use gender specific cut off (Male: 32.3 pg/ml and Female: 14.6pg/ml for the Indian population), with high th sensitive troponin I assay near the 99 percentile of a reference control population. Therefore, implementation of sex-specific hs-cTnI assay was able to identify 14% of under-diagnosed women with ACS in 6 months period. This in turn also decreased the number of men being diagnosed by 3%. On a similar note, there has been a continuous challenge in the health care system in U.S, Europe and other countries to standardize and harmonize the HbA1c reporting: the decision on what to report in NGSP (%) and/or IFCC (mmol/mol) units along with eAG (in either mmol/L or mg/dL). This globalization places a responsibility on laboratory medicine specialists to work together to reduce the current variability in patient results, which arises from differences between units, methods and laboratory practices in different countries. Due to the standardization efforts of IFCC, NGSP, and also due to ongoing efforts of manufacturers and laboratories, the quality of HbA1c reporting has increased dramatically. Consequently, there has been a paradigm shift: HbA1c is now considered the gold standard, not only for monitoring, but also for diagnosis of diabetes. We have performed verification studies of HbA1c by different methods: HPLC, Capillary Electrophoresis, Enzymatic, Immunoassays in 200 samples and compared 60 samples with hemoglobinopathies. Finally, we have also explored harmonizing the clinical protocol based on the use of inflammatory and routine laboratory biomarkers in 2,654 COVID-19 patients. To explain the role of harmonizing routine laboratory parameters in disease monitoring, two adult males, two adult females and one adolescent girl were selected. These are representative examples of different manifestations of COVID 19, with Adult Respiratory Distress Syndrome (ARDS), Cardiac Injury, Neurological manifestations and Pediatric Multi system inflammatory syndrome (PIMS), admitted in the Intensive care unit (ICU) of the hospital, which will be discussed. Therefore, the road map for laboratory medicine will involve strategies for harmonizing, communicating and integrating with all stakeholders, like, clinicians, diagnosticians and IVD industry, in order to formulate guidelines for assisting in correct measurement, diagnosis and management of diseases.

Combining Direct PCR Technology and Capillary Electrophoresis for an Easy-to-Operate and Highly Sensitive Infectious Disease Detection System for Shrimp.

Infectious diseases are considered the greatest threat to the modern high-density shrimp aquaculture industry. Specificity, rapidity, and sensitivity of molecular diagnostic methods for the detection of asymptomatic infected shrimp allows preventive measures to be taken before disease outbreaks. Routine molecular detection of pathogens in infected shrimp can be made easier with the use of a direct polymerase chain reaction (PCR). In this study, four direct PCR reagent brands were tested, and results showed that the detection signal of direct PCR in hepatopancreatic tissue was more severely affected. In addition, portable capillary electrophoresis was applied to improve sensitivity and specificity, resulting in a pathogen detection limit of 25 copies/PCR-reaction. Juvenile shrimp from five different aquaculture ponds were tested for white spot syndrome virus infection, and the results were consistent with the Organization for Animal Health's certified standard method. Furthermore, this methodology could be used to examine single post larvae shrimp. The overall detection time was reduced by more than 58.2%. Therefore, the combination of direct PCR and capillary electrophoresis for on-site examination is valuable and has potential as a suitable tool for diagnostic, epidemiological, and pathological studies of shrimp aquaculture.

DNA aptamer selection for SARS-CoV-2 spike glycoprotein detection.

The rapid spread of SARS-CoV-2 infection throughout the world led to a global public health and economic crisis triggering an urgent need for the development of low-cost vaccines, therapies and high-throughput detection assays. In this work, we used a combination of Ideal-Filter Capillary Electrophoresis SELEX (IFCE-SELEX), Next Generation Sequencing (NGS) and binding assays to isolate and validate single-stranded DNA aptamers that can specifically recognize the SARS-CoV-2 Spike glycoprotein. Two selected non-competing DNA aptamers, C7 and C9 were successfully used as sensitive and specific biological recognition elements for the development of electrochemical and fluorescent aptasensors for the SARS-CoV-2 Spike glycoprotein with detection limits of 0.07 fM and 41.87 nM, respectively.

Pattern of serum protein capillary electrophoretogram in SARS- CoV-2 infection.

BACKGROUND AND AIMS: Monoclonal/biclonalgammopathy of unknown significance (MGUS/BGUS) is observed in COVID-19. This study was conducted to determine the changes in serum protein electrophoresis (SPEP) in COVID-19. MATERIALS AND METHODS: In this descriptive (cross-sectional) study, serum inflammatory markers (CRP, IL-6 and ferritin) were measured and SPEP was carried out by capillary electrophoresis method in 35 controls and 30 moderate & 58 severe COVID-19 cases. RESULTS: Serum inflammatory markers were increased in COVID-19 cases with severity. M-band(s), ß-γ bridging and pre-albumin band(s) on SPEP were observed in 15.5, 11 & 12% of severe cases and 3, 4 & 0% moderate COVID-19 cases respectively. Area under curve (AUC) of α 1 and α 2 bands of SPEP increased significantly in severe COVID-19. CONCLUSIONS: We conclude that SPEP changes like the appearance of M-band(s) indicating MGUS(BGUS), ß- γ bridging indicating the presence of fast-moving immunoglobulins, pre-albumin band indicating the rise in serum transthyretin level and the increase in AUC of α 1 and α 2 bands indicating the rise in positive acute phase reactants occur in COVID-19. The occurrence and magnitude of these changes are higher in severe COVID-19 than that in moderate COVID-19. The diagnostic and prognostic significance of these SPEP changes are worth exploring.

Clinical and Immunological Assessment of Patients with Sars-Cov-2 Infections

[Formula presented] Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induced coronavirus disease-2019 (COVID-19) has presented humanity with unprecedented challenges. Severe disease is associated with acute respiratory distress syndrome (ARDS), use of mechanical ventilation, ICU stay and prolonged hospitalization, The role of the immune system in the pathogenesis of COVID-19 disease is still unclear, which imposes limitations on identifying potential immunotherapy that can improve care for acute and chronic phases of COVID-19 in conjunction with current therapies. Research efforts are ongoing for more than 1 year to identify key immunological mechanisms involved in the disease process. While insightful, this knowledge is still incomplete and can be complemented with the assessment of immune response kinetics. Such assessment will help with the identification of early interventional modalities of immune cell regulation. With these considerations in mind, we aimed to assess several parameters of immune system regulation during the current medical care of patients with COVID-19. Methods: This is a pre-clinical prospective cohort study which involved laboratory-based assessments of blood samples obtained from COVID-19 patients and healthy volunteers. The study population was divided into three cohorts. Our first cohort included 18 years and older COVID-19 patients with respiratory complaints, oxygen (O2) saturations of less than or equal to 92 and pulmonary infiltrates on an imaging study or who were critically ill and required ventilatory support. Second cohort included 18 years and older COVID-19 patients who were hospitalized and did not require ventilatory support. Third cohort included participants with no prior diagnosis of COVID-19, or any recent viral respiratory symptoms including fever, cough or shortness of breath for the last 2 weeks. Patients with an established diagnosis of cancer or immunologic disorders were excluded. Blood specimens were collected over the period of hospitalization: specimen number 1 on day 1-3 of hospitalization, specimen number 2 on days 3-4 of hospitalization, specimen number 3 on days 5-7 of hospitalization, and specimen number 4 on 7-30 days after discharge. We performed capillary electrophoresis for serology and automated ELISA for cytokine measurement. We collected clinical data on patient demographic, clinical characteristics such as presence of any acute and chronic comorbidities and serum inflammatory markers C-Reactive Protein, D-Dimer and Ferritin. Results: We had 15 patients in cohort 1, 10 in cohort 2 and 15 in cohort 3. Patients in cohort 1 were older and had higher comorbidities. Males constituted a substantially high percentage of patients in cohort 1 and 2 (60% and 70% respectively). Patients had similar BMI in cohort 1 and 2. Total antibody levels were highest in cohort 1 but an upward trend over the course of hospitalization was noted in all cohorts. Most interesting pattern was noted in the context of antibodies against spike protein S1 receptor-binding domain (S1RBD) where patients in cohort 2 developed minimal S1RBD antibodies. Cohort 1 on average had higher levels of Interleukin 6(IL-6), Interleukin 8(IL-8), C-X-C motif chemokine ligand 10 (CXCL10) and other inflammatory cytokines except Interferon gamma (IFN-gamma) compared to Cohort 2. Remarkable difference in CXCL-10 levels was noted between the groups and healthy volunteers had the lowest levels. No significant difference in IFN-gamma was noted between cohorts and the levels quickly depleted over the course of the infection. Conclusion: Our analysis confirms that neutralizing antibodies do not correlate with lessened COVID-19 disease severity. Severe COVID-19 infection is secondary to ineffective innate immunity associated with immune overshoot. CXCL10 serves as a major component in triggering the cytokine storm that is a hallmark of SARS-CoV-2 infections. Our findings show an association between high levels of CXCL10 and more severe COVID-19 infection. There does not seem to be any s gnificant correlation with disease severity and IFN-gamma levels. [Formula presented] Disclosures: No relevant conflicts of interest to declare.

A handheld-type total integrated capillary electrophoresis system for SARS-CoV-2 diagnostics: Power, fluorescence detection, and data analysis by smartphone.

A micro-capillary electrophoresis (µCE) system is one of the widely adopted techniques in the molecular diagnostics and DNA sequencing due to the benefits of high resolution, rapid analysis, and low reagent consumption, but due to the requirements of bulky high-power suppliers and an expensive laser-induced fluorescence detector module, the conventional set-up of µCE system is not adequate for point-of-care (POC) molecular diagnostics. In this study, we constructed a miniaturized and integrated µCE system which can be manipulated by a smartphone. The smartphone not only powers two boost converters and an excited laser, but also controls the relay for the power switch. Moreover, the complementary metal-oxide-semiconductor (CMOS) camera of the smartphone was used for detecting the fluorescence signal of amplicons amplified with reverse transcription-polymerase chain reaction (RT-PCR). We also developed a web-based application so that the raw data of the recorded fluorescence intensity versus the running time can display typical capillary electropherograms on the smartphone. The total size of the hand-held µCE system was 9.6 cm [Width] × 22 cm [Length] × 15.5 cm [Height], and the weight was ∼1 kg, which is suitable for POC DNA testing. In the integrated smartphone-associated µCE system, we could accurately analyze two genes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), namely N gene and S gene along with two bracket ladders in 6 min to identify SARS-CoV-2. Such an advanced µCE platform can be applied for a variety of on-site molecular diagnostics fields with user-friendliness.

A new approach to identifying pathogens, with particular regard to viruses, based on capillary electrophoresis and other analytical techniques.

Fast determination, identification and characterization of pathogens is a significant challenge in many fields, from industry to medicine. Standard approaches (e.g., culture media and biochemical tests) are known to be very time-consuming and labor-intensive. Conversely, screening techniques demand a quick and low-cost grouping of microbial isolates, and current analysis call for broad reports of pathogens, involving the application of molecular, microscopy, and electromigration techniques, DNA fingerprinting and also MALDI-TOF methods. The present COVID-19 pandemic is a crisis that affects rich and poor countries alike. Detection of SARS-CoV-2 in patient samples is a critical tool for monitoring disease spread, guiding therapeutic decisions and devising social distancing protocols. The goal of this review is to present an innovative methodology based on preparative separation of pathogens by electromigration techniques in combination with simultaneous analysis of the proteome, lipidome, and genome using laser desorption/ionization analysis.

Capillary electrophoresis as a fragment screening tool to cross-validate hits from chromogenic assay: Application to FXIIa.

In this study, a partial-filling affinity capillary electrophoresis (pf-ACE) method was developed for the cross-validation of fragment hits revealed by chromogenic factor XIIa (FXIIa) assay. Chromogenic assay produces false positives, mainly due to spectrophotometric interferences and sample purity issues. pf-ACE was selected as counter-screening technology because of its separative character and the fact that the target does not have to be attached or tagged. The effects of protein plug length, applied voltage and composition of the running buffer were examined and optimized. Detection limit in terms of dissociation constant was estimated at 400 µM. The affinity evaluation was performed close to physiological conditions (pH 7.4, ionic strength 0.13 mol L-1) in a poly (ethylene oxide)-coated capillary of 75 µm internal diameter x 33 cm length with an applied voltage of 3 kV. This method uncovered chromogenic assay's false positives due to zinc contamination. Moreover, pf-ACE supported the evaluation of compounds absorbing at 405 nm.

Capillary electrophoresis of PCR fragments with 5´-labelled primers for testing the SARS-Cov-2.

Due to the huge demand for SARS-Cov-2 determination,alternatives to the standard qtPCRtestsare potentially useful for increasing the number of samples screened. Our aim was to develop a direct fluorescent PCR capillary-electrophoresis detection of the viral genome. We validated this approach on several SARS-Cov-2 positive and negative samples.We isolated the naso-pharingealRNA from 20 positive and 10 negative samples. The cDNA was synthesised and two fragments of the SARS-Cov-2 were amplified. One of the primers for each pair was 5´-end fluorochrome labelled. The amplifications were subjected to capillary electrophoresis in ABI3130 sequencers to visualize the fluorescent peaks.The two SARS-Cov-2 fragments were successfully amplified in the positive samples, while the negative samples did not render fluorescent peaks. In conclusion, we describe and alternative method to identify the SARS-Cov-2 genome that could be scaled to the analysis of approximately 100 samples in less than 5 h. By combining a standard PCR with capillary electrophoresis our approach would overcome the limits imposed to many labs by the qtPCR and increase the testing capacity.