All-in-one microfluidic chip for 30-min quantitative point-of-care-testing of nucleic acids

Nucleic acid testing (NAT) is directly oriented to determining the genetic material of pathogens and is characterized by its high sensitivity and specificity, which are indispensable qualities in disease diagnosis. However, standard laboratory NAT methods require joint testing by highly trained inspectors using multiple instruments in professional laboratories. The entire process requires many manual steps, and the total testing time may range from 3 to 5h, indicating that these methods cannot be used to realize the demands of on-site rapid testing. In this study, we propose a microfluidic chip for the on-site and rapid detection of nucleic acids. We utilize dynamic sealing, ultrasound, and advanced control methods and integrate the entire process of reagent pre-storage, extraction, Real-time Quantitative polymerase chain reaction (qPCR), and fluorescence detection. The sensitivity of this system is in line with current clinical standards, and the nucleic acid quantification process is completed fully automated within 30min. Compared with conventional microfluidic chips, the proposed system has the advantages of high integration, low cost, and it may be produced at a high volume. Moreover, it can be used in a wide range of screening cases in the context of the COVID-19 pandemic and exhibits broad clinical application prospects.

The duration and breadth of antibody responses to 3-dose of inactivated COVID-19 vaccinations in healthy blood donors: An observational study.

Objectives: We aimed to evaluate the duration and breadth of antibodies elicited by inactivated COVID-19 vaccinations in healthy blood donors. Methods: We performed serological tests on 1,417 samples from 658 blood donors who received two (n=357), or three (n=301) doses of COVID-19 inactivated vaccine. We also accessed the change in antibody response before and after booster vaccination in 94 participants and their neutralization breadth to the current variants after the booster. Results: Following vaccination, for either the 2- or 3-dose, the neutralizing antibodies (nAbs) peaked with about 97% seropositivity approximately within one month but subsequently decreased over time. Of plasmas collected 6-8 months after the last immunization, the nAb seropositivities were 37% and 85% in populations with 2-dose and 3-dose vaccinations, respectively. The nAbs of plasma samples (collected between 2-6 weeks after the 3rd dose) from triple-vaccinated donors (n=94) showed a geometric mean titer of 145.3 (95% CI: 117.2 to 180.1) against the ancestral B.1, slightly reduced by 1.7-fold against Delta variant, but markedly decreased by 4-6 fold in neutralizing Omicron variants, including the sub-lineages of BA.1 (5.6-fold), BA.1.1 (6.0-fold), BA.2 (4.2-fold), B.2.12.1 (6.2-fold) and BA.4/5 (6.5-fold). Conclusion: These findings suggested that the 3rd dose of inactivated COVID-19 vaccine prolongs the antibody duration in healthy populations, but the elicited-nAbs are less efficient in neutralizing circulating Omicron variants.

Nasal irrigation efficiently attenuates SARS-CoV-2 Omicron infection, transmission and lung injury in the Syrian hamster model.

Recently, a new variant lineage of SARS-CoV-2, namely Omicron, became the dominant global circulating strain. The multiple antigenic mutations of Omicron largely decrease the efficiency of current vaccines and neutralizing antibodies, which highlights the need for more potent and reachable medical countermeasures. Here, we hypothesize that direct viral clearance by nasal irrigation might be a convenient and alternative option, and perform proof-of-concept experiments in the Syrian hamster model. Interestingly, Omicron shows a different dynamic in the changes of viral RNA, viral titers, and proinflammatory cytokines in nasal rinsing samples when compared with the prototype. Meanwhile, the levels of viral load and proinflammatory cytokines in nasal rinsing samples can indicate the severity of lung injury. Of note, daily nasal irrigation efficiently attenuates inflammation and lung injury in Omicron-infected hamsters by decreasing the viral loads in the respiratory tract organs. Moreover, daily nasal irrigation effectively suppresses viral transmission by close contact.

Infection, pathology and interferon treatment of the SARS-CoV-2 Omicron BA.1 variant in juvenile, adult and aged Syrian hamsters.

The new predominant circulating SARS-CoV-2 variant, Omicron, can robustly escape current vaccines and neutralizing antibodies. Although Omicron has been reported to have milder replication and disease manifestations than some earlier variants, its pathogenicity in different age groups has not been well elucidated. Here, we report that the SARS-CoV-2 Omicron BA.1 sublineage causes elevated infection and lung pathogenesis in juvenile and aged hamsters, with more body weight loss, respiratory tract viral burden, and lung injury in these hamsters than in adult hamsters. Juvenile hamsters show a reduced interferon response against Omicron BA.1 infection, whereas aged hamsters show excessive proinflammatory cytokine expression, delayed viral clearance, and aggravated lung injury. Early inhaled IFN-α2b treatment suppresses Omicron BA.1 infection and lung pathogenesis in juvenile and adult hamsters. Overall, the data suggest that the diverse patterns of the innate immune response affect the disease outcomes of Omicron BA.1 infection in different age groups.

Development of amplification system for point-of-care test of nucleic acid.

Nucleic acid testing (NAT) has been widely used in many fields such as medical diagnosis, food safety testing and forensic identification. However, it can only be carried out in professional laboratory because the test process is complicated and rigorous. In this paper, a nucleic acid amplification system based on polymerase chain reaction (PCR) was developed to meet the requirements of point-of-care testing (POCT) for nucleic acids. Firstly, the mechanical structure and electronic control system were designed and constructed. Secondly, an integral separation PID algorithm for temperature control and an intelligent temperature compensation method based on support vector regression (SVR) were proposed. Finally, temperature measurement and biological experiments were performed to prove the stability and availability of the nucleic acid amplification system. The results showed that the system achieved a rapid temperature change velocity of 4.5 °C/s, and the steady-state error was within ± 0.5 °C. The nucleic acids in samples of different concentrations were well amplified, the system can be used for quantitative detection of nucleic acid with the help of a fluorescence detection system, and has higher sensitivity than Tianlong PCR instrument.

Cell-based reporter assays for measurements of antibody-mediated cellular cytotoxicity and phagocytosis against SARS-CoV-2 spike protein.

The COVID-19 pandemic caused by SARS-CoV-2 infections has led to excess deaths worldwide. Neutralizing antibodies (nAbs) against viral spike protein acquired from natural infections or vaccinations contribute to protection against new- and re-infections. Besides neutralization, antibody-mediated cellular cytotoxicity (ADCC) and phagocytosis (ADCP) are also important for viral clearance. However, due to the lack of convenient methods, the ADCC and ADCP responses elicited by viral infections or vaccinations remain to be explored. Here, we developed cell-based assays using target cells stably expressing SARS-CoV-2 spikes and Jurkat-NFAT-CD16a/CD32a effector cells for ADCC/ADCP measurements of monoclonal antibodies and human convalescent COVID-19 plasmas (HCPs). In control samples (n = 190), the specificity was 99.5% (95%CI: 98.4-100%) and 97.4% (95%CI: 95.1-99.6%) for the ADCC and ADCP assays, respectively. Among 87 COVID-19 HCPs, 83 (sensitivity: 95.4%, 95%CI: 91.0-99.8%) and 81 (sensitivity: 93.1%, 95%CI: 87.8-98.4%) showed detectable ADCC (titer range: 7.4-1721.6) and ADCP activities (titer range: 4-523.2). Notably, both ADCC and ADCP antibody titers positively correlated with the nAb titers in HCPs. In summary, we developed new tools for quantitative ADCC and ADCP analysis against SARS-CoV-2, which may facilitate further evaluations of Fc-mediated effector functions in preventing and treating against SARS-CoV-2.

An encodable multiplex microsphere-phase amplification sensing platform detects SARS-CoV-2 mutations.

SARS-CoV-2 variants of concern (VOCs) contain several single-nucleotide variants (SNVs) at key sites in the receptor-binding region (RBD) that enhance infectivity and transmission, as well as cause immune escape, resulting in an aggravation of the coronavirus disease 2019 (COVID-19) pandemic. Emerging VOCs have sparked the need for a diagnostic method capable of simultaneously monitoring these SNVs. To date, no highly sensitive, efficient clinical tool exists to monitor SNVs simultaneously. Here, an encodable multiplex microsphere-phase amplification (MMPA) sensing platform that combines primer-coded microsphere technology with dual fluorescence decoding strategy to detect SARS-CoV-2 RNA and simultaneously identify 10 key SNVs in the RBD. MMPA limits the amplification refractory mutation system PCR (ARMS-PCR) reaction for specific target sequence to the surface of a microsphere with specific fluorescence coding. This effectively solves the problem of non-specific amplification among primers and probes in multiplex PCR. For signal detection, specific fluorescence codes inside microspheres are used to determine the corresponding relationship between the microspheres and the SNV sites, while the report probes hybridized with PCR products are used to detect the microsphere amplification intensity. The MMPA platform offers a lower SARS-CoV-2 RNA detection limit of 28 copies/reaction, the ability to detect a respiratory pathogen panel without cross-reactivity, and a SNV analysis accuracy level comparable to that of sequencing. Moreover, this super-multiple parallel SNVs detection method enables a timely updating of the panel of detected SNVs that accompanies changing VOCs, and presents a clinical availability that traditional sequencing methods do not.

Protocol for the clinical performance assessment of SARS - CoV - 2 antibody testing reagent

To date, the coronavirus disease 2019 (COVID-19) pandemic is impacting globally. COVID-19 is mainly diagnosed via viral nucleic acid testing, but with the disadvantages of unsatisfactory sensitivity and high requirements for expensive equipment and facility the operating settings. Compared with nucleic acid testing, antibody testing usually has advantages as wide popularization, convenient sample collection, easy to achieve high throughput. less workload, high reproducibility, and low cost, therefore it will be an efficient supplement for nucleic acid detection to confirm COVID-19. This protocol provided detailed design for the assessment of antibody testing reagent, including consideration for the study objectives, calculation of sample size, inclusion and exclusion criteria, blinding method, experimental specimen, ethical issues, study management and quality control, data management and statistical analysis. and results report and so on, aiming to assist the researchers to systematically assess the critical performance of antibody testing reagent prior to large-scale application of the antibody testing reagent, so that researchers could make reasonable choices among different antibody testing reagents according to their respective purposes.

Transferable, easy-to-use and room-temperature-storable PCR mixes for microfluidic molecular diagnostics.

As the outbreak of coronavirus disease 2019 (COVID-19), on-site molecular diagnosis is becoming increasingly important. In this study, a freeze-drying method was introduced for PCR reagents to meet the requirements of microfluidic molecular diagnosis. Using this method, PCR components were pre-mixed and freeze-dried as a bead, which could be transferred into microfluidic chips easily. As this bead only required reconstitution in water, operational steps of PCR were simplified, pipetting errors and errors associated with improper handling of wet reagents could also be reduced. In addition, 19 PCR mixes for different targets (including both RNA and DNA) detection were stable when stored at room temperature (18-25 °C) for 1-2 years and may be stored longer as activity monitoring remains ongoing. To shorten the stability testing time, accelerated stability testing at higher temperatures was proposed. The evaluation periods of the freeze-dried PCR mixes were shortened to less than one month when stored at 56 °C and 80 °C. When attempts were further tried to predict the shelf lives for freeze-dried PCR mixes, our findings challenged the classic view of the Q10 method as a prediction model for freeze-dried PCR mixes and confirmed for the first time that this prediction was influenced by different factors at varying degrees. These studies and findings are important for the development of molecular diagnosis at both central laboratories and resource-limited areas.

Rapid PCR powered by microfluidics: A quick review under the background of COVID-19 pandemic.

PCR has been widely used in different fields including molecular biology, pathogen detection, medical diagnosis, food detection and etc. However, the difficulty of promoting PCR in on-site point-of-care testing reflects on challenges relative to its speed, convenience, complexity, and even cost. With the emerging state-of-art of microfluidics, rapid PCR can be achieved with more flexible ways in micro-reactors. PCR plays a critical role in the detection of SARS-CoV-2. Under this special background of COVID-19 pandemic, this review focuses on the latest rapid microfluidic PCR. Rapid PCR is concluded in two main features, including the reactor (type, size, material) and the implementation of thermal cycling. Especially, the compromise between speed and sensitivity with microfluidic PCR is explored based on the system ratio of (thermal cycling time)/(reactor size). Representative applications about the detection of pathogens and SARS-CoV-2 viruses based on rapid PCR or other isothermal amplification are discussed as well.

Gender associates with both susceptibility to infection and pathogenesis of SARS-CoV-2 in Syrian hamster.

Epidemiological studies of the COVID-19 patients have suggested the male bias in outcomes of lung illness. To experimentally demonstrate the epidemiological results, we performed animal studies to infect male and female Syrian hamsters with SARS-CoV-2. Remarkably, high viral titer in nasal washings was detectable in male hamsters who presented symptoms of weight loss, weakness, piloerection, hunched back and abdominal respiration, as well as severe pneumonia, pulmonary edema, consolidation, and fibrosis. In contrast with the males, the female hamsters showed much lower shedding viral titers, moderate symptoms, and relatively mild lung pathogenesis. The obvious differences in the susceptibility to SARS-CoV-2 and severity of lung pathogenesis between male and female hamsters provided experimental evidence that SARS-CoV-2 infection and the severity of COVID-19 are associated with gender.

Antibody Responses to SARS-CoV-2 in Patients With Novel Coronavirus Disease 2019.

BACKGROUND: The novel coronavirus SARS-CoV-2 is a newly emerging virus. The antibody response in infected patients remains largely unknown, and the clinical value of antibody testing has not been fully demonstrated. METHODS: 173 patients with SARS-CoV-2 infection were enrolled. Their serial plasma samples (n = 535) collected during hospitalization were tested for total antibodies (Ab), IgM, and IgG against SARS-CoV-2. The dynamics of antibodies with disease progress were analyzed. RESULTS: Among 173 patients, the seroconversion rates for Ab, IgM, and IgG were 93.1%, 82.7%, and 64.7%, respectively. The reason for the negative antibody findings in 12 patients might be due to the lack of blood samples at the later stage of illness. The median seroconversion times for Ab, IgM, and then IgG were days 11, 12, and 4, respectively. The presence of antibodies was <40% among patients within 1 week of onset, and rapidly increased to 100.0% (Ab), 94.3% (IgM), and 79.8% (IgG) by day 15 after onset. In contrast, RNA detectability decreased from 66.7% (58/87) in samples collected before day 7 to 45.5% (25/55) during days 15-39. Combining RNA and antibody detection significantly improved the sensitivity of pathogenic diagnosis for COVID-19 (P < .001), even in the early phase of 1 week from onset (P = .007). Moreover, a higher titer of Ab was independently associated with a worse clinical classification (P = .006). CONCLUSIONS: Antibody detection offers vital clinical information during the course of SARS-CoV-2 infection. The findings provide strong empirical support for the routine application of serological testing in the diagnosis and management of COVID-19 patients.

Several FDA-Approved Drugs Effectively Inhibit SARS-CoV-2 Infection in vitro.

To identify drugs that are potentially used for the treatment of COVID-19, the potency of 1403 FDA-approved drugs were evaluated using a robust pseudovirus assay and the candidates were further confirmed by authentic SARS-CoV-2 assay. Four compounds, Clomiphene (citrate), Vortioxetine, Vortioxetine (hydrobromide) and Asenapine (hydrochloride), showed potent inhibitory effects in both pseudovirus and authentic virus assay. The combination of Clomiphene (citrate), Vortioxetine and Asenapine (hydrochloride) is much more potent than used alone, with IC50 of 0.34 µM.

Virus-Free and Live-Cell Visualizing SARS-CoV-2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors.

The ongoing corona virus disease 2019 (COVID-19) pandemic, caused by SARS-CoV-2 infection, has resulted in hundreds of thousands of deaths. Cellular entry of SARS-CoV-2, which is mediated by the viral spike protein and ACE2 receptor, is an essential target for the development of vaccines, therapeutic antibodies, and drugs. Using a mammalian cell expression system, a genetically engineered sensor of fluorescent protein (Gamillus)-fused SARS-CoV-2 spike trimer (STG) to probe the viral entry process is developed. In ACE2-expressing cells, it is found that the STG probe has excellent performance in the live-cell visualization of receptor binding, cellular uptake, and intracellular trafficking of SARS-CoV-2 under virus-free conditions. The new system allows quantitative analyses of the inhibition potentials and detailed influence of COVID-19-convalescent human plasmas, neutralizing antibodies and compounds, providing a versatile tool for high-throughput screening and phenotypic characterization of SARS-CoV-2 entry inhibitors. This approach may also be adapted to develop a viral entry visualization system for other viruses.

Robust neutralization assay based on SARS-CoV-2 S-protein-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressing BHK21 cells.

The global pandemic of coronavirus disease 2019 (COVID-19) is a disaster for human society. A convenient and reliable neutralization assay is very important for the development of vaccines and novel drugs. In this study, a G protein-deficient vesicular stomatitis virus (VSVdG) bearing a truncated spike protein (S with C-terminal 18 amino acid truncation) was compared to that bearing the full-length spike protein of SARS-CoV-2 and showed much higher efficiency. A neutralization assay was established based on VSV-SARS-CoV-2-Sdel18 pseudovirus and hACE2-overexpressing BHK21 cells (BHK21-hACE2 cells). The experimental results can be obtained by automatically counting the number of EGFP-positive cells at 12 h after infection, making the assay convenient and high-throughput. The serum neutralizing titer measured by the VSV-SARS-CoV-2-Sdel18 pseudovirus assay has a good correlation with that measured by the wild type SARS-CoV-2 assay. Seven neutralizing monoclonal antibodies targeting the receptor binding domain (RBD) of the SARS-CoV-2 S protein were obtained. This efficient and reliable pseudovirus assay model could facilitate the development of new drugs and vaccines.

Room-temperature-storable PCR mixes for SARS-CoV-2 detection.

OBJECTIVES: A novel coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) emerged in late 2019, causing an outbreak of pneumonia [coronavirus disease 2019 (COVID-19)] globally. Although the use of ready-made reaction mixes can enable more rapid PCR-based diagnosis of COVID-19, the need to transport and store these mixes at low temperatures presents challenges to already overburdened logistics networks. METHODS: Here, we present an optimized freeze-drying procedure that allows SARS-CoV-2 PCR mixes to be transported and stored at ambient temperatures, without loss of activity. Additive-supplemented PCR mixes were freeze-dried. The residual moisture of the freeze-dried PCR mixes was measured by Karl-Fischer titration. RESULTS: We found that the freeze-dried PCR mixes with ~1.2% residual moisture are optimal for storage, transport, and reconstitution. The sensitivity, specificity, and repeatability of the freeze-dried reagents were similar to those of freshly prepared, wet reagents. The freeze-dried mixes retained activity at room temperature (18 ~ 25 °C) for 28 days, and for 14 and 10 days when stored at 37 °C and 56 °C, respectively. CONCLUSION: The uptake of this approach will ease logistical challenges faced by transport networks and make more cold storage space available at diagnosis and hospital laboratories.