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.

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.

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.

Early diagnosis and population prevention of coronavirus disease 2019.

PURPOSE OF REVIEW: To discuss the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection by nucleic acid and antibody testing as well as its contribution to mitigating the spread of coronavirus disease 2019. RECENT FINDINGS: Nucleic acid testing (NAT) is the firstly developed and most widely used diagnostic technique for SARS-CoV-2 infection. However, the sensitivity of SARS-CoV-2 RNA NAT assays is always unsatisfactory, mainly due to insufficient viral RNA in samples, especially when upper respiratory samples were used. Compared with NAT assays, serological tests are more convenient and less dependent on the quality of sample collection. But the sensitivity of antibody assays varies largely to test samples collected at different time after onset of symptoms. The diagnostic sensitivity can be significantly improved by combination of RNA and antibody testing. Due to the lack of effective drugs and vaccines, population prevention results mainly from timely triage and quarantine of SARS-CoV-2 infected individuals. Thus, extensive testing with NAT and antibody assays simultaneously is very important to constrain coronavirus disease 2019 epidemic. SUMMARY: Viral RNA testing combining with serological testing could improve the early diagnosis of SARS-CoV-2 infection, which has great value for clinical practice and public health.

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.

Heat inactivation decreases the qualitative real-time RT-PCR detection rates of clinical samples with high cycle threshold values in COVID-19.

SARS-CoV-2 has caused COVID-19 pandemic globally in the beginning of 2020, and qualitative real-time RT-PCR has become the gold standard in diagnosis. As SARSCoV-2 with strong transmissibility and pathogenicity, it has become a professional consensus that clinical samples from suspected patients should be heat inactivated at 56°C for 30 min before further processing. However, previous studies on the effect of inactivation on qualitative real-time RT-PCR were conducted with diluted samples rather than clinical samples. The aim of this study was to investigate whether heat inactivation on clinical samples before detection will affect the accuracy of qualitative real-time RT-PCR detection. All 46 throat swab samples from 46 confirmed inpatients were detected by qualitative real-time RT-PCR directly, as well as after heat inactivation. Heat-Inactivation has significantly influenced the qualitative detection results on clinical samples, especially weakly positive samples. The results indicate the urgency to establish a more suitable protocol for COVID-19 clinical sample's inactivation.

Serology characteristics of SARS-CoV-2 infection since exposure and post symptom onset

BACKGROUND: Timely diagnosis of SARS-CoV-2 infection is a prerequisite for treatment and prevention. The serology characteristics and complement diagnosis value of the antibody test to RNA test need to be demonstrated. METHOD: Serial sera of 80 patients with PCR-confirmed COVID-19 were collected at the First Affiliated Hospital of Zhejiang University, China. Total antibody (Ab), IgM and IgG antibodies against SARS-CoV-2 were detected, and the antibody dynamics during the infection were described. RESULTS: The seroconversion rates for Ab, IgM and IgG were 98.8%, 93.8% and 93.8%, respectively. The first detectible serology marker was Ab, followed by IgM and IgG, with a median seroconversion time of 15, 18 and 20 days post exposure (d.p.e) or 9, 10 and 12 days post onset (d.p.o), respectively. The antibody levels increased rapidly beginning at 6 d.p.o. and were accompanied by a decline in viral load. For patients in the early stage of illness (0-7 d.p.o), Ab showed the highest sensitivity (64.1%) compared to IgM and IgG (33.3% for both, p<0.001). The sensitivities of Ab, IgM and IgG increased to 100%, 96.7% and 93.3% 2 weeks later, respectively. When the same antibody type was detected, no significant difference was observed between enzyme-linked immunosorbent assays and other forms of immunoassays. CONCLUSIONS: A typical acute antibody response is induced during SARS-CoV-2 infection. Serology testing provides an important complement to RNA testing in the later stages of illness for pathogenic specific diagnosis and helpful information to evaluate the adapted immunity status of patients.