Droplet magnetic-controlled microfluidic chip integrated nucleic acid extraction and amplification for the detection of pathogens and tumor mutation sites.

Traditional nucleic acid extraction and detection is based on open operation, which may cause cross-contamination and aerosol formation. This study developed a droplet magnetic-controlled microfluidic chip integrated nucleic acid extraction, purification and amplification. The reagent is sealed in oil to form a droplet, and the nucleic acid is extracted and purified by controlling the movement of the magnetic beads (MBs) through a permanent magnet, ensuring a closed environment. This chip can automatically extract nucleic acid from multiple samples within 20 min, and can be directly placed in the in situ amplification instrument for amplification without further transfer of nucleic acid, characterized by simple, fast, time-saving and labor-saving. The results showed that the chip was able to detect <10 copies/test SARS-CoV-2 RNA, and EGFR exon 21 L858R mutations were detected in H1975 cells as low as 4 cells. In addition, on the basis of the droplet magnetic-controlled microfluidic chip, we further developed a multi-target detection chip, which used MBs to divide the nucleic acid of the sample into three parts. And the macrolides resistance mutations A2063G and A2064G, and the P1 gene of mycoplasma pneumoniae (MP) were successfully detected in clinical samples by the multi-target detection chip, providing the possibility for future application in the detection of multiple pathogens.

Engineering highly thermostable Cas12b via de novo structural analyses for one-pot detection of nucleic acids.

CRISPR-Cas-based diagnostics have the potential to elevate nucleic acid detection. CRISPR-Cas systems can be combined with a pre-amplification step in a one-pot reaction to simplify the workflow and reduce carryover contamination. Here, we report an engineered Cas12b with improved thermostability that falls within the optimal temperature range (60°C-65°C) of reverse transcription-loop-mediated isothermal amplification (RT-LAMP). Using de novo structural analyses, we introduce mutations to wild-type BrCas12b to tighten its hydrophobic cores, thereby enhancing thermostability. The one-pot detection assay utilizing the engineered BrCas12b, called SPLENDID (single-pot LAMP-mediated engineered BrCas12b for nucleic acid detection of infectious diseases), exhibits robust trans-cleavage activity up to 67°C in a one-pot setting. We validate SPLENDID clinically in 80 serum samples for hepatitis C virus (HCV) and 66 saliva samples for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with high specificity and accuracy. We obtain results in as little as 20 min, and with the extraction process, the entire assay can be performed within an hour.

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.

Comparison of CRISPR/Cas and Argonaute for nucleic acid tests.

Guided, programmable, and target-activated nucleases, exemplified by Cas in the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system and Argonaute (Ago), are emerging as a new generation of nucleic acid tests (NATs). A specific approach for comparison of these two nucleases side by side in terms of similarities, differences, and complementarities is instrumental for the sensible design of novel NATs.

Development of nucleic acid based lateral flow assays for SARS-CoV-2 detection.

SARS-CoV-2 is still threat for humanity and its detection is crucial. Although real time reverse transcriptase polymerase chain reaction is the most reliable method for detection of N protein genes, alternative methods for molecular detection are still needed. Thus, lateral flow assay models for 2019-nCoV_ N3 were developed for molecular detection. Briefly, gold nanoparticles were used as label and three sandwich models (1A, 1B, and 1.2) were designed. Prob concentrations on gold nanoparticles, types of sandwich model and membrane, limit of detection of target gene and buffer efficiency were studied. Model 1B has shown the best results with M170 membrane. Lower limit of detection was achieved by model 1.2 as 5 pM. All parameters have significant role for molecular detection of SARS-CoV-2 by lateral flow assays, and these results will be useful for nucleic acid based lateral flow assays for viral detection or multiple detection of mutated forms in various detection systems.

Capturing nucleic acid variants with precision using CRISPR diagnostics.

CRISPR/Cas systems have the ability to precisely target nucleotide sequences and enable their rapid identification and modification. While nucleotide modification has enabled the therapeutic correction of diseases, the process of identifying the target DNA or RNA has greatly expanded the field of molecular diagnostics in recent times. CRISPR-based DNA/RNA detection through programmable nucleic acid binding or cleavage has been demonstrated for a large number of pathogenic and non-pathogenic targets. Combining CRISPR detection with nucleic acid amplification and a terminal signal readout step allowed the development of numerous rapid and robust nucleic acid platforms. Wherever the Cas effector can faithfully distinguish nucleobase variants in the target, the platform can also be extended for sequencing-free rapid variant detection. Some initial PAM disruption-based SNV detection reports were limited to finding or integrating mutated/mismatched nucleotides within the PAM sequences. In this review, we try to summarize the developments made in CRISPR diagnostics (CRISPRDx) to date emphasizing CRISPR-based SNV detection. We also discuss the applications where such diagnostic modalities can be put to use, covering various fields of clinical research, SNV screens, disease genotyping, primary surveillance during microbial infections, agriculture, food safety, and industrial biotechnology. The ease of rapid design and implementation of such multiplexable assays can potentially expand the applications of CRISPRDx in the domain of affinity-based target sequencing, with immense possibilities for low-cost, quick, and widespread usage. In the end, in combination with proximity assays and a suicidal gene approach, CRISPR-based in vivo SNV detection and cancer cell targeting can be formulated as personalized gene therapy.

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.

Functional nucleic acids as modular components against SARS-CoV-2: From diagnosis to therapeutics.

Coronavirus Disease 2019 (COVID-19), which poses an extremely serious global impact on human public healthcare, represents a high transmission and disease-causing viral infection caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) that is expanding at a rapid pace. Therefore, it is urgent for researchers to establish effective platforms for the assay and treatment of SARS-CoV-2. Functional nucleic acids (FNAs), comprising aptamers and nucleases, are of primary concern within the biological and medical communities owing of the distinctive properties of their target recognition and catalysis. This review will concentrate on the essential aspects of insights regarding FNAs and their technological expertise for the diagnostic and therapeutic utilization against COVID-19. We first offer a historical perspective of the COVID-19 pandemics, its clinical characteristics and potential biomarkers. Then, we briefly discuss the current diagnostic and therapeutic methodology towards COVID-19, highlighting the superiorities and existing shortcomings. After that, we introduce the key features of FNAs, and summarize recent progress of in vitro selection of FNAs for SARS-CoV-2 specific proteins and RNAs, followed by highlighting the general concept of translating FNAs into functional probes for diagnostic and therapeutic purposes. Then, we critically review the emerging FNAs-based diagnostic and therapeutic strategies that are fast, precise, efficient, and highly specific to fight COVID-19. Finally, we identify remaining challenges and offer future outlook of this emerging field.

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.

Droplet Microarray Based on Nanosensing Probe Patterns for Simultaneous Detection of Multiple HIV Retroviral Nucleic Acids.

Multiplexed detection of viral nucleic acids is important for rapid screening of viral infection. In this study, we present a molybdenum disulfide (MoS2) nanosheet-modified dendrimer droplet microarray (DMA) for rapid and sensitive detection of retroviral nucleic acids of human immunodeficiency virus-1 (HIV-1) and human immunodeficiency virus-2 (HIV-2) simultaneously. The DMA platform was fabricated by omniphobic-omniphilic patterning on a surface-grafted dendrimer substrate. Functionalized MoS2 nanosheets modified with fluorescent dye-labeled oligomer probes were prepatterned on positively charged amino-modified omniphilic spots to form a fluorescence resonance energy transfer (FRET) sensing microarray. With the formation of separated microdroplets of sample on the hydrophobic-hydrophilic micropattern, prepatterned oligomer probes specifically hybridized with the target HIV genes and detached from the MoS2 nanosheet surface due to weakening of the adsorption force, leading to fluorescence signal recovery. As a proof of concept, we used this microarray with a small sample size (<150 nL) for simultaneous detection of HIV-1 and HIV-2 nucleic acids with a limit of detection (LOD) of 50 pM. The multiplex detection capability was further demonstrated for simultaneous detection of five viral genes (HIV-1, HIV-2, ORFlab, and N genes of SARS-COV-2 and M gene of Influenza A). This work demonstrated the potential of this novel MoS2-DMA FRET sensing platform for high-throughput multiplexed viral nucleic acid screening.

Current state of commercial point-of-care nucleic acid tests for infectious diseases.

The COVID-19 pandemic has put the spotlight on the urgent need for integrated nucleic acid tests (NATs) for infectious diseases, especially those that can be used near patient ("point-of-care", POC), with rapid results and low cost, but without sacrificing sensitivity or specificity of gold standard PCR tests. In the US, the Clinical Laboratory Improvement Amendments Certificate of Waiver (CLIA-waiver) is mandated by the Food and Drug Administration (FDA) and designated to any laboratory testing with high simplicity and low risk for error, suitable for application in the POC. Since the first issuance of CLIA-waiver to Abbot's ID NOW Influenza A&B in 2015, many more NAT systems have been developed, received the CLIA-waiver in the US or World Health Organization (WHO)'s pre-qualification, and deployed to the front line of infectious disease detection. This review highlights the regulatory process for FDA and WHO in evaluating these NATs and the technology innovation of existing CLIA-waived systems. Understanding the technical advancement and challenges, unmet needs, and the trends of commercialization facilitated through the regulatory processes will help pave the foundation for future development and technology transfer from research to the market place.

Open-Source Miniature Fluorimeter to Monitor Real-Time Isothermal Nucleic Acid Amplification Reactions in Resource-Limited Settings.

Traditional methods to detect and quantify nucleic acids rely on polymerase chain reaction (PCR) and require the use of expensive thermocyclers with integrated fluorescence detection of amplicons. Isothermal nucleic acid amplification technologies eliminate the need for thermal cycling; however, fluorescence-based detection of products is still required for real-time, quantitative results. Several portable isothermal heaters with integrated fluorescence detection are now commercially available; however, the cost of these devices remains a significant barrier to widespread adoption in resource-limited settings. Described here is a protocol for the design and assembly of a modular, low-cost fluorimeter constructed from off-the-shelf components. Enclosed in a compact 3D printed housing, the fluorimeter is designed to be placed atop a commercially available heat block holding a PCR tube. The fluorimeter described here was optimized to detect fluorescein isothiocyanate (FITC) dye, but the system can be modified for use with dyes commonly used as reporters in real-time nucleic acid amplification reactions. Clinical applicability of the system is demonstrated by performing real-time nucleic acid detection with two isothermal amplification technologies: recombinase polymerase amplification (RPA) for detection of positive control DNA provided in a commercial kit and reverse transcription loop-mediated isothermal amplification (RT-LAMP) for detection of clinically meaningful levels of SARS-CoV-2 RNA.

Pathogenesis of COVID-19 and the quality control of nucleic acid detection.

The new coronavirus pneumonia (COVID-19) epidemic spread rapidly throughout the world. Considering the strong infectivity and clustering of COVID-19, early detection of infectious cases is of great significance to control the epidemic. Nucleic acid testing (NAT) plays an important role in rapid laboratory diagnosis, treatment assessment, epidemic prevention and control of COVID-19. However, since COVID-19 is caused by a new emerging virus and NAT for COVID-19 has not been clinically applied before, false negative results inconsistent with clinical diagnosis have appeared in clinical practice. Therefore, it is urgent to improve the sensitivity of NAT for COVID-19. This study aimed to summarize the current situation and prospect of NAT based on the latest findings on COVID-19 infection. Also, the quality control of sample collection was discussed. Hopefully, this study could help to improve the effectiveness of NAT for COVID-19.

Positive detection of SARS-CoV-2 combined HSV1 and HHV6B virus nucleic acid in tear and conjunctival secretions of a non-conjunctivitis COVID-19 patient with obstruction of common lacrimal duct.

BACKGROUND: The current outbreak of COVID-19 has spread rapidly all over the world. Respiratory droplets and contaction with infected patients are the two major transmission routes. However, the value of tear virus nucleic acid is still not clear. We dynamic detected the SARS-CoV-2 in eye sample of one COVID-19 patient with obstruction of common lacrimal ducts. METHODS: Besides the routine examination, nasopharyngeal and eye swab were continuously measured by polymerase chain reaction assay and next-generation sequencing (NGS). Gene detection was performed for drug use guidance, and flow cytometry was performed to analyse the lymphocyte subsets. RESULTS: Nasopharyngeal swabs were positive for 22 days, but eye swabs were still continuously positive for 2 weeks after nasopharyngeal swabs turned negative. The low level of lymphocyte and the high level IL-6 lasted for almost 4 weeks, then became near normal. Next-generation sequencing (NGS) confirmed the existing of SARS-CoV-2, HSV1 and HHV6B virus nucleic acid. The gene detection for drug use guidance showed the genetic locus ABCB1 (3435T>C) rs1045642 belonged to type CC and it mean the efficiency of lopinavir-ritonavir would be significantly decreased. The flow cytometry of lymphocyte subsets showed PD-1+  CD95+ cells was accounting for 94.8% in CD3+  CD8+ T subset and for 94.8% in CD3+  TCRγδ+ T subset. CONCLUSIONS: As obstruction of common lacrimal duct, positively detection in one eye for 2 weeks more after nasopharyngeal swab became negative. More eye swabs should be collected from COVID-19 patients, especially from those immunocompromised, those with eye symptoms and those had a history of ocular diseases.