CRISPR-based diagnostics of different biomolecules from nucleic acids, proteins, and small molecules to exosomes.

CRISPR-based detection technologies have been widely explored for molecular diagnostics. However, the challenge lies in converting the signal of different biomolecules, such as nucleic acids, proteins, small molecules, exosomes, and ions, into a CRISPR-based nucleic acid detection signal. Understanding the detection of different biomolecules using CRISPR technology can aid in the development of practical and promising detection approaches. Unfortunately, existing reviews rarely provide an overview of CRISPR-based molecular diagnostics from the perspective of different biomolecules. Herein, we first introduce the principles and characteristics of various CRISPR nucleases for molecular diagnostics. Then, we focus on summarizing and evaluating the latest advancements in CRISPR-based detection of different biomolecules. Through a comparison of different methods of amplification and signal readout, we discuss how general detection methods can be integrated with CRISPR. Finally, we conclude by identifying opportunities for the improvement of CRISPR in quantitative, amplification-free, multiplex, all-in-one, and point-of-care testing (POCT) purposes.

Rapid and Accurate Detection of SARS-CoV-2 Using an iPad-Controlled, High-Throughput, Portable, and Multiplex Hive-Chip Platform (HiCube).

Rapid and accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is one of the most effective measures to control the coronavirus disease 2019 (COVID-19) pandemic. However, there is still lack of an ideal detection platform capable of high sample throughput, portability, and multiplicity. Herein, by combining Hive-Chip (capillary microarray) and reverse transcriptional loop-mediated isothermal amplification (RT-LAMP), we developed an iPad-controlled, high-throughput (48 samples at one run), portable (smaller than a backpack), multiplex (monitoring 8 gene fragments in one reaction), and real-time detection platform for SARS-CoV-2 detection. This platform is composed of a portable Hive-Chip device (HiCube; 32.7 × 29.7 × 20 cm, 5 kg), custom-designed software, and optimized Hive-Chips. RT-LAMP primers targeting seven SARS-CoV-2 genes (S, E, M, N, ORF1ab, ORF3a, and ORF7a) and one positive control (human RNase P) were designed and prefixed in the Hive-Chip. On-chip RT-LAMP showed that the limit of detection (LOD) of SARS-CoV-2 synthetic RNAs is 1 copy/µL, and there is no cross-reaction among different target genes. The platform was validated by 100 clinical samples of SARS-CoV-2, and the results were highly consistent with those of the traditional real-time PCR assay. In addition, on-chip detection of 6 other respiratory pathogens showed no cross-reactivity. Overall, our platform has great potential for fast, accurate, and on-site detection of SARS-CoV-2.

Nucleic acid testing of SARS-CoV-2: A review of current methods, challenges, and prospects.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has brought a huge threat to public health and the global economy. Rapid identification and isolation of SARS-CoV-2-infected individuals are regarded as one of the most effective measures to control the pandemic. Because of its high sensitivity and specificity, nucleic acid testing has become the major method of SARS-CoV-2 detection. A deep understanding of different diagnosis methods for COVID-19 could help researchers make an optimal choice in detecting COVID-19 at different symptom stages. In this review, we summarize and evaluate the latest developments in current nucleic acid detection methods for SARS-CoV-2. In particular, we discuss biosensors and CRISPR-based diagnostic systems and their characteristics and challenges. Furthermore, the emerging COVID-19 variants and their impact on SARS-CoV-2 diagnosis are systematically introduced and discussed. Considering the disease dynamics, we also recommend optional diagnostic tests for different symptom stages. From sample preparation to results readout, we conclude by pointing out the pain points and future directions of COVID-19 detection.

Multiplex and visual detection of African Swine Fever Virus (ASFV) based on Hive-Chip and direct loop-mediated isothermal amplification.

African swine fever is caused by African swine fever virus (ASFV), and has a mortality rate approaching 100%. It has already caused tremendous economy lost around the world. Without effective vaccine, rapid and accurate on-site detection plays an indispensable role in controlling outbreaks. Herein, by combining Hive-Chip and direct loop-mediated isothermal amplification (LAMP), we establish a multiplex and visual detection platform. LAMP primers targeting five ASFV genes (B646L, B962L, C717R, D1133L, and G1340L) were designed and pre-fixed in Hive-Chip. On-chip LAMP showed the limits of detection (LOD) of ASFV synthetic DNAs and mock samples are 30 and 50 copies per microliter, respectively, and there is no cross-reaction among the target genes. The overall performance of our platform is comparable to that of the commercial kits. From sample preparation to results readout, the entire process takes less than 70 min. Multiplex detection of real samples of ASFV and other swine viruses further demonstrates the high sensitivity and specificity of Hive-Chip. Overall, our platform provides a promising option for on-site, fast and accurate detection of ASFV.

Multiplex sample-to-answer detection of bacteria using a pipette-actuated capillary array comb with integrated DNA extraction, isothermal amplification, and smartphone detection.

A pipette-actuated capillary array comb (PAAC) system operated on a smartphone-based hand-held device has been successfully developed for the multiplex detection of bacteria in a "sample-to-answer" manner. The PAAC consists of eight open capillaries inserted into a cylindrical plastic base with a piece of chitosan-modified glass filter paper embedded in each capillary. During the sample preparation, a PAAC was mounted into a 1 mL pipette tip with an enlarged opening and was operated with a 1 mL pipette for liquid handling. The cell lysate was drawn and expelled through the capillaries three times to facilitate the DNA capture on the embedded filter discs. Following washes with water, the loop-mediated isothermal amplification (LAMP) reagents were aspirated into the capillaries, in which the primers were pre-fixed with chitosan. After that, the PAAC was loaded into the smartphone-based device for a one-hour amplification at 65 °C and end-point detection of calcein fluorescence in the capillaries. The DNA capture efficiency of a 1.1 mm-diameter filter disc was determined to be 97% of λ-DNA and the coefficient of variation among the eight capillaries in the PAAC was only 2.2%. The multiplex detection of genomic DNA extracted from Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus provided limits of detection of 200, 500, and 500 copies, respectively, without any cross-contamination and cross reactions. "Sample-to-answer" detection of E. coli samples was successfully completed in 85 minutes, demonstrating a sensitivity of 200 cfu per capillary. The multiplex "sample-to-answer" detection, the streamlined operation, and the compact device should facilitate a broad range of applications of our PAAC system in point-of-care testing.

Visual Detection of Multiple Nucleic Acids in a Capillary Array.

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

A visual multiplex PCR microchip with easy sample loading.

There is an urgent demand for affordable, rapid and easy-to-use technology to simultaneously detect many different DNA targets within one reaction. Conventional multiplex PCR is an effective methodology to simultaneously amplify different DNA targets. However, its multiplicity is limited due to the intrinsic interference and competition among primer pairs within one tube. Here, we present an easy multiplex PCR microchip system, which can simultaneously detect 54 targets. The design of the microchip is quite simple. There is a microchannel connected with multiple underlying parallel microwells. And every microchannel has an inlet/outlet for loading PCRmix. The surface of the microchannel is hydrophobic and the inner surface of the microwell is hydrophilic, which enables us to load and separate the PCRmix into different microwells simultaneously. Different primer pairs and low melting agarose are pre-fixed in different microwells, and the microchip is assembled with top glass. The PCRmix is loaded into inlets and then mineral oil is sequentially pipetted into channels to push the PCRmix into all microwells and subsequently mineral oil fills the channels to avoid cross contaminations. After the PCRmix is loaded, it would be placed on a plat thermal cycler for PCR. During PCR, the low melting gel in the well is liquid and after PCR it would be solidified due to temperature changes. When PCR is completed, a nucleic acid dye is introduced into channels and then results are visualized by a home-made, potable UV detector. In our platform we successfully detected seven frequently used targets of genetically modified (GM) organisms. The results demonstrate that our platform has high flexibility and specificity. Due to the excellent performance of this technology, we believe that it can be applied to multiple nucleic acid detection fields including GM organisms.