On-Chip Nucleic Acid Purification Followed by ddPCR for SARS-CoV-2 Detection.

We developed a microfluidic chip integrated with nucleic acid purification and droplet-based digital polymerase chain reaction (ddPCR) modules to realize a 'sample-in, result-out' infectious virus diagnosis. The whole process involved pulling magnetic beads through drops in an oil-enclosed environment. The purified nucleic acids were dispensed into microdroplets by a concentric-ring, oil-water-mixing, flow-focusing droplets generator driven under negative pressure conditions. Microdroplets were generated with good uniformity (CV = 5.8%), adjustable diameters (50-200 µm), and controllable flow rates (0-0.3 µL/s). Further verification was provided by quantitative detection of plasmids. We observed a linear correlation of R2 = 0.9998 in the concentration range from 10 to 105 copies/µL. Finally, this chip was applied to quantify the nucleic acid concentrations of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The measured nucleic acid recovery rate of 75 ± 8.8% and detection limit of 10 copies/µL proved its on-chip purification and accurate detection abilities. This chip can potentially be a valuable tool in point-of-care testing.

An integrated microfluidic chip for nucleic acid extraction and continued cdPCR detection of pathogens.

This paper introduces an enclosed microfluidic chip that integrates sample preparation and the chamber-based digital polymerase chain reaction (cdPCR). The sample preparation of the chip includes nucleic acid extraction and purification based on magnetic beads, which adsorb nucleic acids by moving around the reaction chambers to complete the reactions including lysis, washing, and elution. The cdPCR area of the chip consists of tens of thousands of regularly arranged microchambers. After the sample preparation processes are completed, the purified nucleic acid can be directly introduced into the microchambers for amplification and detection on the chip. The nucleic acid extraction performance and digital quantification performance of the system were examined using synthetic SARS-CoV-2 plasmid templates at concentrations ranging from 101-105 copies per µL. Further on, a simulated clinical sample was used to test the system, and the integrated chip was able to accurately detect SARS-CoV-2 virus particle samples doped with interference (saliva) with a detection limit of 10 copies per µL. This integrated system could provide a promising tool for point-of-care testing of pathogenic infections.

Recent advances in cascade isothermal amplification techniques for ultra-sensitive nucleic acid detection.

Nucleic acid amplification techniques have always been one of the hot spots of research, especially in the outbreak of COVID-19. From the initial polymerase chain reaction (PCR) to the current popular isothermal amplification, each new amplification techniques provides new ideas and methods for nucleic acid detection. However, limited by thermostable DNA polymerase and expensive thermal cycler, PCR is difficult to achieve point of care testing (POCT). Although isothermal amplification techniques overcome the defects of temperature control, single isothermal amplification is also limited by false positives, nucleic acid sequence compatibility, and signal amplification capability to some extent. Fortunately, efforts to integrating different enzymes or amplification techniques that enable to achieve intercatalyst communication and cascaded biotransformations may overcome the corner of single isothermal amplification. In this review, we systematically summarized the design fundamentals, signal generation, evolution, and application of cascade amplification. More importantly, the challenges and trends of cascade amplification were discussed in depth.

Ultrafast Nucleic Acid Detection Equipment with Silicon-Based Microfluidic Chip.

Recently, infectious diseases, such as COVID-19, monkeypox, and Ebola, are plaguing human beings. Rapid and accurate diagnosis methods are required to preclude the spread of diseases. In this paper, an ultrafast polymerase chain reaction (PCR) equipment is designed to detect virus. The equipment consists of a silicon-based PCR chip, a thermocycling module, an optical detection module, and a control module. Silicon-based chip, with its thermal and fluid design, is used to improve detection efficiency. A thermoelectric cooler (TEC), together with a computer-controlled proportional-integral-derivative (PID) controller, is applied to accelerate the thermal cycle. A maximum of four samples can be tested simultaneously on the chip. Two kinds of fluorescent molecules can be detected by optical detection module. The equipment can detect viruses with 40 PCR amplification cycles in 5 min. The equipment is portable, easily operated, and low equipment cost, which shows great potential in epidemic prevention.

A Miniaturized Silicon Lab-on-Chip for Integrated PCR and Hybridization Microarray for High Multiplexing Nucleic Acids Analysis.

A silicon lab-on-chip, for the detection of nucleic acids through the integrated PCR and hybridization microarray, was developed. The silicon lab-on-chip manufactured through bio-MEMS technology is composed of two PCR microreactors (each volume 11.2 µL) and a microarray-hybridization microchamber (volume 30 µL), fluidically connected by buried bypass. It contains heaters and temperature sensors for the management and control of the temperature cycles during the PCR amplification and hybridization processes. A post-silicon process based on (i) plasmo-O2 cleaning/activation, (ii) vapor phase epoxy silanization, (iii) microarray fabrication and (iv) a protein-based passivation step was developed and fully characterized. The ssDNA microarray (4 rows × 10 columns) composed of 400 spots (spot size-70 ± 12 µm; spot-to-spot distance-130 ± 13 µm) was manufactured by piezo-dispense technology. A DNA microarray probe density in the range of 1310 to 2070 probe µm-2 was observed, together with a limit of detection of about 19 target µm-2. The performances of the silicon lab-on-chip were validated by the detection of the beta-globin gene directly from human blood. Remarkable sensitivity, multiplexing analysis and specificity were demonstrated for the detection of beta-globin and mycobacterium tuberculosis sequences.

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.

The Dangers of Using Cq to Quantify Nucleic Acid in Biological Samples: A Lesson From COVID-19.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA quantities, measured by reverse transcription quantitative PCR (RT-qPCR), have been proposed to stratify clinical risk or determine analytical performance targets. We investigated reproducibility and how setting diagnostic cutoffs altered the clinical sensitivity of coronavirus disease 2019 (COVID-19) testing. METHODS: Quantitative SARS-CoV-2 RNA distributions [quantification cycle (Cq) and copies/mL] from more than 6000 patients from 3 clinical laboratories in United Kingdom, Belgium, and the Republic of Korea were analyzed. Impact of Cq cutoffs on clinical sensitivity was assessed. The June/July 2020 INSTAND external quality assessment scheme SARS-CoV-2 materials were used to estimate laboratory reported copies/mL and to estimate the variation in copies/mL for a given Cq. RESULTS: When the WHO-suggested Cq cutoff of 25 was applied, the clinical sensitivity dropped to about 16%. Clinical sensitivity also dropped to about 27% when a simulated limit of detection of 106 copies/mL was applied. The interlaboratory variation for a given Cq value was >1000 fold in copies/mL (99% CI). CONCLUSION: While RT-qPCR has been instrumental in the response to COVID-19, we recommend Cq (cycle threshold or crossing point) values not be used to set clinical cutoffs or diagnostic performance targets due to poor interlaboratory reproducibility; calibrated copy-based units (used elsewhere in virology) offer more reproducible alternatives. We also report a phenomenon where diagnostic performance may change relative to the effective reproduction number. Our findings indicate that the disparities between patient populations across time are an important consideration when evaluating or deploying diagnostic tests. This is especially relevant to the emergency situation of an evolving pandemic.

A glucose meter interface for point-of-care gene circuit-based diagnostics.

Recent advances in cell-free synthetic biology have given rise to gene circuit-based sensors with the potential to provide decentralized and low-cost molecular diagnostics. However, it remains a challenge to deliver this sensing capacity into the hands of users in a practical manner. Here, we leverage the glucose meter, one of the most widely available point-of-care sensing devices, to serve as a universal reader for these decentralized diagnostics. We describe a molecular translator that can convert the activation of conventional gene circuit-based sensors into a glucose output that can be read by off-the-shelf glucose meters. We show the development of new glucogenic reporter systems, multiplexed reporter outputs and detection of nucleic acid targets down to the low attomolar range. Using this glucose-meter interface, we demonstrate the detection of a small-molecule analyte; sample-to-result diagnostics for typhoid, paratyphoid A/B; and show the potential for pandemic response with nucleic acid sensors for SARS-CoV-2.

CRISPR-based diagnostics.

The accurate and timely diagnosis of disease is a prerequisite for efficient therapeutic intervention and epidemiological surveillance. Diagnostics based on the detection of nucleic acids are among the most sensitive and specific, yet most such assays require costly equipment and trained personnel. Recent developments in diagnostic technologies, in particular those leveraging clustered regularly interspaced short palindromic repeats (CRISPR), aim to enable accurate testing at home, at the point of care and in the field. In this Review, we provide a rundown of the rapidly expanding toolbox for CRISPR-based diagnostics, in particular the various assays, preamplification strategies and readouts, and highlight their main applications in the sensing of a wide range of molecular targets relevant to human health.

Stretch-driven microfluidic chip for nucleic acid detection.

Molecular diagnosis is an essential means to detect pathogens. The portable nucleic acid detection chip has excellent prospects in places where medical resources are scarce, and it is also of research interest in the field of microfluidic chips. Here, the article developed a new type of microfluidic chip for nucleic acid detection where stretching acts as the driving force. The sample entered the chip by applying capillary force. The strain valve was opened under the action of tensile force, and the spring pump generated the power to drive the fluid to flow to the detection chamber in a specific direction. The detection of coronavirus disease 2019 (COVID-19) was realized on the chip. The RT-LAMP amplification system was adopted to observe the liquid color in the detection chamber to decide whether the sample tested positive or negative qualitatively.

Plasmonic Biosensors for Single-Molecule Biomedical Analysis.

The rapid spread of epidemic diseases (i.e., coronavirus disease 2019 (COVID-19)) has contributed to focus global attention on the diagnosis of medical conditions by ultrasensitive detection methods. To overcome this challenge, increasing efforts have been driven towards the development of single-molecule analytical platforms. In this context, recent progress in plasmonic biosensing has enabled the design of novel detection strategies capable of targeting individual molecules while evaluating their binding affinity and biological interactions. This review compiles the latest advances in plasmonic technologies for monitoring clinically relevant biomarkers at the single-molecule level. Functional applications are discussed according to plasmonic sensing modes based on either nanoapertures or nanoparticle approaches. A special focus was devoted to new analytical developments involving a wide variety of analytes (e.g., proteins, living cells, nucleic acids and viruses). The utility of plasmonic-based single-molecule analysis for personalized medicine, considering technological limitations and future prospects, is also overviewed.

Perceived Effectiveness, Safety, and Attitudes Toward the Use of Nucleic Tests of SARS-CoV-2 Among Clinicians and General Public in China.

Objective: To assess whether there is a knowledge gap about the use of test kits for residents and to explore the knowledge, attitudes, and practices of using test kits in China during the coronavirus disease 2019 (COVID-19) epidemic. Method: An online-based, nationwide, and cross-sectional study was conducted. A total of 1,167 respondents were recruited from June 19 to July 2, 2020. All participants completed a validated questionnaire written in Chinese. Electronic consent was obtained from all participants upon their agreement to commence the questionnaire. Perceived efficacy, safety, and their attitudes toward the use of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing kits were measured. Result: The majority of the study respondents were female [749 (64.2%)], aged 31-40 years old [372 (31.9%)], and located in mainland China [1,137 (97.4%)]. The majority of the respondents held a positive view toward the introduction of the fast-track approval policy for novel coronavirus testing products (6.16 ± 1.30) as well as toward putting more investment in scientific research and biomedicine to improve the detection accuracy of detection kits (5.94 ± 1.55) in China. The respondents valued the detection accuracy more as opposed to the detection time of the testing kits (4.66 ± 2.00), whereas few participants agreed that in the research and development process, detection accuracy could be sacrificed to speed up production and coverage capacity (3.02 ± 2.04). Conclusion: The majority of the participants have a basic knowledge of the detection methods of the SARS-CoV-2 virus and the types of test kits, as well as great confidence in China's domestic production of test kits and decisions. However, how basic knowledge, high compliance, and positive attitudes play a role in easing the tension of the pandemic still remains unknown.

Post-lockdown SARS-CoV-2 nucleic acid screening in nearly ten million residents of Wuhan, China.

Stringent COVID-19 control measures were imposed in Wuhan between January 23 and April 8, 2020. Estimates of the prevalence of infection following the release of restrictions could inform post-lockdown pandemic management. Here, we describe a city-wide SARS-CoV-2 nucleic acid screening programme between May 14 and June 1, 2020 in Wuhan. All city residents aged six years or older were eligible and 9,899,828 (92.9%) participated. No new symptomatic cases and 300 asymptomatic cases (detection rate 0.303/10,000, 95% CI 0.270-0.339/10,000) were identified. There were no positive tests amongst 1,174 close contacts of asymptomatic cases. 107 of 34,424 previously recovered COVID-19 patients tested positive again (re-positive rate 0.31%, 95% CI 0.423-0.574%). The prevalence of SARS-CoV-2 infection in Wuhan was therefore very low five to eight weeks after the end of lockdown.

Detection of SARS-CoV-2 RNA residue on object surfaces in nucleic acid testing laboratory using droplet digital PCR.

The rapid development of global COVID-19 pandemic poses an unprecedented challenge to the safety and quality of laboratory diagnostic testing. Little is known about the laboratory surface areas and operation behaviors that may cause potential contamination in SARS-CoV-2 nucleic acid testing. This study aims to provide reference basis for the improvement of laboratory disinfection programs and personal operating protocols. In this study, we compared the qRT-PCR and ddPCR in detecting of residual virus that existed on the object surfaces from sample transportation and reception related facilities, testing related instruments, personal protective equipment and other facilities in nucleic acid testing laboratory. All samples were negative by qRT-PCR, in contrast, 13 of 61 samples were positive for SARS-CoV-2 by ddPCR. The areas with highest density of SARS-CoV-2 nucleic acid were the outer gloves of operator A (37.4 copies/cm2), followed by door handle of 4 °C refrigerator (26.25 copies/cm2), goggles of operator A (22.16 copies/cm2), outer cover of high speed centrifuge (19.95 copies/cm2), inner wall of high speed centrifuge (14.70 copies/cm2) and others. We found that all the positive objects were directly or indirectly contacted by the operator's gloved hands, suggesting that hands contact was the main transmission pathway that led to laboratory environmental contamination. In summary, ddPCR has an advantage over qRT-PCR in tracing laboratory contamination. We evaluated the risk areas and operation behaviors that may easily cause contamination, and provided recommendation for improving the laboratory disinfection programs and personal operating specifications.

Clinical features of COVID-19 convalescent patients with re-positive nucleic acid detection.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is a pandemic that has rapidly spread worldwide. Increasingly, confirmed patients being discharged according to the current diagnosis and treatment protocols, follow-up of convalescent patients is important to knowing about the outcome. METHODS: A retrospective study was performed among 98 convalescent patients with COVID-19 in a single medical center. The clinical features of patients during their hospitalization and 2-week postdischarge quarantine were collected. RESULTS: Among the 98 COVID-19 convalescent patients, 17 (17.3%) were detected positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid during 2-week postdischarge quarantine. The median time from discharge to SARS-CoV-2 nucleic acid re-positive was 4 days (IQR, 3-8.5).The median time from symptoms onset to final respiratory SARS-CoV-2 detection of negative result was significantly longer in re-positive group (34 days [IQR, 29.5-42.5]) than in non-re-positive group (19 days [IQR, 16-26]). On the other hand, the levels of CD3-CD56 + NK cells during hospitalization and 2-week postdischarge were higher in re-positive group than in non-re-positive group (repeated measures ANOVA, P = .018). However, only one case in re-positive group showed exudative lesion recurrence in pulmonary computed tomography (CT) with recurred symptoms. CONCLUSION: It is still possible for convalescent patients to show positive for SARS-CoV-2 nucleic acid detection, but most of the re-positive patients showed no deterioration in pulmonary CT findings. Continuous quarantine and close follow-up for convalescent patients are necessary to prevent possible relapse and spread of the disease to some extent.

Recent advances in lab-on-a-chip technologies for viral diagnosis.

The global risk of viral disease outbreaks emphasizes the need for rapid, accurate, and sensitive detection techniques to speed up diagnostics allowing early intervention. An emerging field of microfluidics also known as the lab-on-a-chip (LOC) or micro total analysis system includes a wide range of diagnostic devices. This review briefly covers both conventional and microfluidics-based techniques for rapid viral detection. We first describe conventional detection methods such as cell culturing, immunofluorescence or enzyme-linked immunosorbent assay (ELISA), or reverse transcription polymerase chain reaction (RT-PCR). These methods often have limited speed, sensitivity, or specificity and are performed with typically bulky equipment. Here, we discuss some of the LOC technologies that can overcome these demerits, highlighting the latest advances in LOC devices for viral disease diagnosis. We also discuss the fabrication of LOC systems to produce devices for performing either individual steps or virus detection in samples with the sample to answer method. The complete system consists of sample preparation, and ELISA and RT-PCR for viral-antibody and nucleic acid detection, respectively. Finally, we formulate our opinions on these areas for the future development of LOC systems for viral diagnostics.