Trends in developing one-pot CRISPR diagnostics strategies.

The integration of nucleic acid amplification (NAA) with the CRISPR detection system has led to significant advancements and opportunities for development in molecular diagnostics. Nevertheless, the incompatibility between CRISPR cleavage and NAA has significantly impeded the commercialization of this technology. Currently, several one-pot detection strategies based on CRISPR systems have been devised to address concerns regarding aerosol contamination risk and operational complexity associated with step-by-step detection as well as the sensitivity limitation of conventional one-pot methods. In this review, we provide a comprehensive introduction and outlook of the various solutions of the one-pot CRISPR assay for practitioners who are committed to developing better CRISPR nucleic acid detection technologies to promote the progress of molecular diagnostics.

Low-potential anodic electrochemiluminescence of terbium metal-organic frameworks for selective microRNA-155 detection.

High excitation potential is recognized as a harmful factor for the biological activity of biomacromolecules, such as proteins and nucleic acids, in electrochemiluminescence (ECL) biosensing. Developing low-potential ECL luminophores is vital for improving ECL accuracy in actual sample sensing. In this work, based on porous metal-organic framework (MOF) structure with multiple active sites and energy transfer between the excited ligands and Ln nodes, we designed a series of Ln-MOFs and observed ECL emission at low potential, providing a novel method to realize low-potential ECL. The MOF nanoemitters were prepared using 1,3,5-tri (4-carboxyphenyl)benzene ligand and several lanthanide ions as nodes through mild hydrothermal reaction. Interestingly, strong ECL emission at +0.75 V of peak potential was observed in the ECL-potential curve of Tb-based MOF using 2,2',2″-nitrilotriethanol as coreactant, which was beneficial for reducing background interference in biosensing, and this ECL emission was attributed to the energy transfer between Tb and excited ligand. This low-potential ECL was then applied to construct an ECL biosensor with newly developed Cas12a-based method for selective detection of microRNA-155 without the help of strand displacement or reverse transcription. For this ECL system, the limit of detection was 0.78 nM, and the overall detection time was 2.5 h. The Ln-MOF nanoemitter provides a robust ECL platform to selectively detect various targets by integrating new bio-related techniques.

Development of a nucleic acid detection method based on the CRISPR-Cas13 for point-of-care testing of bovine viral diarrhea virus-1b.

Bovine viral diarrhea (BVD) is a single-stranded, positive-sense ribonucleic acid (RNA) virus belonging to the genus Pestivirus of the Flaviviridae family. BVD frequently causes economic losses to farmers. Among bovine viral diarrhea virus (BVDV) strains, BVDV-1b is predominant and widespread in Hanwoo calves. Reverse-transcription polymerase chain reaction (RT-PCR) is an essential method for diagnosing BVDV-1b and has become the gold standard for diagnosis in the Republic of Korea. However, this diagnostic method is time-consuming and requires expensive equipment. Therefore, Clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) systems have been used for point-of-care (POC) testing of viruses. Developing a sensitive and specific method for POC testing of BVDV-1b would be advantageous for controlling the spread of infection. Thus, this study aimed to develop a novel nucleic acid detection method using the CRISPR-Cas13 system for POC testing of BVDV-1b. The sequence of the BVD virus was extracted from National Center for Biotechnology Information (NC_001461.1), and the 5' untranslated region, commonly used for detection, was selected. CRISPR RNA (crRNA) was designed using the Cas13 design program and optimized for the expression and purification of the LwCas13a protein. Madin Darby bovine kidney (MDBK) cells were infected with BVDV-1b, incubated, and the viral RNA was extracted. To enable POC viral detection, the compatibility of the CRISPR-Cas13 system was verified with a paper-based strip through collateral cleavage activity. Finally, a colorimetric assay was used to evaluate the detection of BVDV-1b by combining the previously obtained crRNA and Cas13a protein on a paper strip. In conclusion, the CRISPR-Cas13 system is highly sensitive, specific, and capable of nucleic acid detection, making it an optimal system for the early point-of-care testing of BVDV-1b.

A compact microfluidic platform for rapid multiplex detection of respiratory viruses via centrifugal polar-absorbance spectroscopy.

Nucleic acid detection technology has become a crucial tool in cutting-edge research within the life sciences and clinical diagnosis domains. Its significance is particularly highlighted during the respiratory virus pandemic, where nucleic acid testing plays a pivotal role in accurately detecting the virus. Isothermal amplification technologies have been developed and offer advantages such as rapidity, mild reaction conditions and excellent stability. Among these methods, recombinase polymerase amplification (RPA) has gained significant attention due to its simple primer design and resistance to multiple reaction inhibitors. However, the detection of RPA amplicons hinders the widespread adoption of this technology, leading to a research focus on cost-effective and convenient detection methods for RPA nucleic acid testing. In this study, we propose a novel computational absorption spectrum approach that utilizes the polar GelRed dye to efficiently detect RPA amplicons. By exploiting the asymmetry of GelRed molecules upon binding with DNA, polar electric dipoles are formed, leading to precipitate formation through centrifugal vibration and electrostatic interaction. The quantification of amplicon content is achieved by measuring the residual GelRed concentration in the supernatant. Our proposed portable and integrated microfluidic device successfully detected five respiratory virus genes simultaneously. The optimized linear detection was achieved and the sensitivity for all the targets reached 100 copies/µL. The total experiment could be finished in 27 min. The clinical experiments demonstrated the practicality and accuracy. This cost-effective and convenient detection scheme presents a promising biosensor for rapid virus detection, contributing to the advancement of RPA technology.

[Application of the CRISPR/Cas system in gene editing and nucleic acid detection of parasitic diseases: a review].

CRISPR/Cas system, an adaptive immune system with clustered regularly interspaced short palindromic repeats, may interfere with exogenous nucleic acids and protect prokaryotes from external damages, is an effective gene editing and nucleic acid detection tools. The CRISPR/Cas system has been widely applied in virology and bacteriology; however, there is relatively less knowledge about the application of the CRISPR/Cas system in parasitic diseases. The review summarizes the mechanisms of action of the CRISPR/Cas system and provides a comprehensive overview of their application in gene editing and nucleic acid detection of parasitic diseases, so as to provide insights into future studies on parasitic diseases.

Simultaneous Detection of Helicobacter pylori and Clarithromycin Resistance Mutations Using RAA-CRISPR/Cas13a Assay.

Background: Infection caused by Helicobacter pylori (H. pylori) affects approximately 50% of the global population. It is a major pathogenic factor for chronic gastritis and gastric cancer. Besides, the resistance to antibiotics such as clarithromycin could reduce the eradication rate. Currently, there is an urgent need for a swift, easy to perform, and highly sensitive detection method for H. pylori and clarithromycin resistance. Methods: We used FAM/Digoxin labeled primers to amplify specific H. pylori 23S rRNA fragments by Recombinase Aided Amplification (RAA), and resistance mutations were distinguished using CRISPR/Cas13a system combined with lateral flow strip. Twenty-eight saliva samples were analyzed using qPCR, gene sequencing and this method to evaluate the detection efficiency. Results: We developed a simultaneous detection method for H. pylori and clarithromycin resistance mutations named sensitive H. pylori easy-read dual detection (SHIELD). The results showed both A2142G and A2143G mutant DNAs causing clarithromycin resistance could be distinguished from the wild type with a concentration of 50 copies/µL, and no cross-reaction with other 5 common gastrointestinal bacteria was observed. For the detection of H. pylori in 28 saliva samples, the positive predictive value of this method was 100% (19/19) in comparison with qPCR. For detecting clarithromycin resistance, the positive predictive value of this method was 84.6% (11/13) compared with gene sequencing. Conclusion: SHIELD assay showed high sensitivity and specificity in detecting H. pylori and clarithromycin resistance mutations. It could be a potential measure in the rapid detection of H. pylori, large-scale screening and guiding clinical medication.

Development of an Integrated Disposable Device for SARS-CoV-2 Nucleic Acid Extraction and Detection.

Objective: To develop a highly sensitive and rapid nucleic acid detection method for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods: We designed, developed, and manufactured an integrated disposable device for SARS-CoV-2 nucleic acid extraction and detection. The precision of the liquid transfer and temperature control was tested. A comparison between our device and a commercial kit for SARS-Cov-2 nucleic acid extraction was performed using real-time fluorescence reverse transcription polymerase chain reaction (RT-PCR). The entire process, from SARS-CoV-2 nucleic acid extraction to amplification, was evaluated. Results: The precision of the syringe transfer volume was 19.2 ± 1.9 µL (set value was 20), 32.2 ± 1.6 (set value was 30), and 57.2 ± 3.5 (set value was 60). Temperature control in the amplification tube was measured at 60.0 ± 0.0 °C (set value was 60) and 95.1 ± 0.2 °C (set value was 95) respectively. SARS-Cov-2 nucleic acid extraction yield through the device was 7.10 × 10 6 copies/mL, while a commercial kit yielded 2.98 × 10 6 copies/mL. The mean time to complete the entire assay, from SARS-CoV-2 nucleic acid extraction to amplification detection, was 36 min and 45 s. The detection limit for SARS-CoV-2 nucleic acid was 250 copies/mL. Conclusion: The integrated disposable devices may be used for SARS-CoV-2 Point-of-Care test (POCT).

Evaluation of dried blood spots for Epstein-Barr virus nucleic acid testing.

Epstein-Barr virus (EBV) is a ubiquitous and oncogenic virus that is associated with various malignancies and non-malignant diseases and EBV DNA detection is widely used for the diagnosis and prognosis prediction for these diseases. The dried blood spots (DBS) sampling method holds great potential as an alternative to venous blood samples in geographically remote areas, for individuals with disabilities, or for newborn blood collection. Therefore, the objective of this study was to assess the viability of detecting EBV DNA load from DBS. Matched whole blood and DBS samples were collected for EBV DNA extraction and quantification detection. EBV DNA detection in DBS presented a specificity of 100 %. At different EBV DNA viral load in whole blood, the sensitivity of EBV DNA detection in DBS was 38.78 % (≥1 copies/mL), 43.18 % (≥500 copies/mL), 58.63 % (≥1000 copies/mL), 71.43 % (≥2000 copies/mL), 82.35 % (≥4000 copies/mL), and 92.86 % (≥5000 copies/mL), respectively. These results indicated that the sensitivity of EBV DNA detection in DBS increased with elevating viral load. Moreover, there was good correlation between EBV DNA levels measured in whole blood and DBS, and on average, the viral load measured in whole blood was about 6-fold higher than in DBS. Our research firstly demonstrated the feasibility of using DBS for qualitative and semi-quantitative detection of EBV DNA for diagnosis and surveillance of EBV-related diseases.

Dual lateral flow assay based on PdRu nanocages for human Papillomavirus detection.

Cervical cancer is one of the most common gynecological malignancies, with the vast majority of which being caused by persistent infection with Human Papillomavirus (HPV) 16 and 18. The current available HPV detection methods are sensitive and genotyped but are restricted by expensive instruments and skilled personnel. The development of an easy-to-use, rapid, and cost-friendly analysis method for HPV is of great need. Herein, hollow palladium-ruthenium nanocages modified with two oligonucleotides (PdRu capture probes) were constructed for genotyping and simultaneous detection of target nucleic acids HPV16 and HPV18 by dual lateral flow assay (DLFA). PdRu capture probes were endowed with bi-functions for the first time, which could be used to output signals and hybridize target nucleic acids. Under optimized conditions, the PdRu based-DLFA with detection limits of 0.93 nM and 0.19 nM, respectively, exhibited convenient operation, and high sensitivity. Meanwhile, the DLFA achieved excellent rapid detection within 20 min, which was attributed to capture probes that can be directly bound to amplification-free target nucleic acids. Therefore, the development of PdRu-based DLFA can be utilized for rapid, sensitive, and simultaneous genotyping detection of HPV16 and HPV18, showing great application for nucleic acid detection.

RNA extraction-free reduced graphene oxide-based RT-LAMP fluorescence assay for highly sensitive SARS-CoV-2 detection.

Infectious diseases have always been a seriously endanger for human life and health. A rapid, accurate and ultra-sensitive virus nucleic acid detection is still a challenge to deal with infectious diseases. Here, a RNA extraction-free reduced graphene oxide-based reverse transcription-loop-mediated isothermal amplification (EF-G-RT-LAMP) fluorescence assay was developed to achieve high-throughput, rapid and ultra-sensitive SARS-CoV-2 RNA detection. The whole detection process only took ∼36 min. The EF-G-RT-LAMP assay achieves a detection limit of 0.6 copies µL-1 with a wide dynamic range of aM-pM. A large number (up to 384) of samples can be detected simultaneously. Simulated detection of the COVID-19 pseudovirus and clinical samples in nasopharyngeal swabs demonstrated a high-throughput, rapid and ultra-sensitive practical detection capability of the EF-G-RT-LAMP assay. The results proved that the assay would be used as a rapid, easy-to-implement approach for epidemiologic diagnosis and could be extended to other nucleic acid detections.

Application of the TaqMan ARMS-PCR Approach for Genotyping Drug-Induced Hearing Loss Using Dried Blood Samples.

A single nucleotide variant in mitochondrial DNA (mtDNA) 1555A>G is associated with drug-induced hearing loss. For the 1555A>G mutation site, 1555A wild-type and 1555G mutant-type plasmids were constructed, respectively. In this study, a PCR method based on the TaqMan amplification refractory mutation system was proposed to detect mtDNA 1555A>G. A common upstream primer, a common TaqMan probe, and two downstream allele-specific primers with mismatched bases were designed. One-step amplification and detection of the wild-type and mutant type at the 1555 site were realized for the deafness-related gene through two reactions. Based on this detection method, the minimum detection limit of the wild-type and mutant type detection systems for plasmids was 50 copies/µL. The minimum sensitivity for the detection of nucleic acids in real dried blood spot (DBS) samples was 0.1 ng/µL. In the normal DBS DNA sample, the detection limit of the mutation abundance reached 0.78%. The specificity of the detection method was 100%, and the coefficient of variation was less than 3.36%. This approach was validated using clinical DNA extracted from 113 DBS samples of newborns. Additionally, it showed 100% agreement with bi-directional Sanger sequencing. It can be used as an optional method for the clinical detection of deafness-related genes.

Recent Advances in and Application of Fluorescent Microspheres for Multiple Nucleic Acid Detection.

Traditional single nucleic acid assays can only detect one target while multiple nucleic acid assays can detect multiple targets simultaneously, providing comprehensive and accurate information. Fluorescent microspheres in multiplexed nucleic acid detection offer high sensitivity, specificity, multiplexing, flexibility, and scalability advantages, enabling precise, real-time results and supporting clinical diagnosis and research. However, multiplexed assays face challenges like complexity, costs, and sample handling issues. The review explores the recent advancements and applications of fluorescent microspheres in multiple nucleic acid detection. It discusses the versatility of fluorescent microspheres in various fields, such as disease diagnosis, drug screening, and personalized medicine. The review highlights the possibility of adjusting the performance of fluorescent microspheres by modifying concentrations and carrier forms, allowing for tailored applications. It emphasizes the potential of fluorescent microsphere technology in revolutionizing nucleic acid detection and advancing health, disease treatment, and medical research.

Performance evaluation of a newly developed 2019-nCoV nucleic acid detection kit based on Ion Proton sequencing platform and its comparison with the MGI Tech (DNBSEQ-G99) platform.

PURPOSE: To evaluate the performance of a newly developed 2019-nCoV nucleic acid detection kit based on Ion Proton sequencing platform and make comparation with MGI Tech (DNBSEQ-G99) platform. METHODS: References and clinical samples were used to evaluate the precision, agreement rate, limit of detection (LOD), anti-interference ability and analytical specificity. Twenty-seven clinical specimens were used to make comparison between two platforms. RESULTS: The kit showed good intra-assay, inter-assay, inter-day precision between different operators and laboratories, fine agreement rate with references, a relatively low LOD of 1 × 103 copies/ml, anti-interference capability of 5 % whole blood and 1mg/ml mucin and no cross reaction with twenty-nine common clinical pathogens. Consistency of variant classification was observed between two platforms. The WGS from Ion Proton tended to have higher coverage and less missing data. CONCLUSIONS: The newly developed kit has shown satisfactory performances and excellent consistency with DNBSEQ-G99, making it a good alternative choice clinically.

A Fast and Sensitive One-Tube SARS-CoV-2 Detection Platform Based on RTX-PCR and Pyrococcus furiosus Argonaute.

Since SARS-CoV-2 is a highly transmissible virus, alternative reliable, fast, and cost-effective methods are still needed to prevent virus spread that can be applied in the laboratory and for point-of-care testing. Reverse transcription real-time fluorescence quantitative PCR (RT-qPCR) is currently the gold criteria for detecting RNA viruses, which requires reverse transcriptase to reverse transcribe viral RNA into cDNA, and fluorescence quantitative PCR detection was subsequently performed. The frequently used reverse transcriptase is thermolabile; the detection process is composed of two steps: the reverse transcription reaction at a relatively low temperature, and the qPCR performed at a relatively high temperature, moreover, the RNA to be detected needs to pretreated if they had advanced structure. Here, we develop a fast and sensitive one-tube SARS-CoV-2 detection platform based on Ultra-fast RTX-PCR and Pyrococcus furiosus Argonaute-mediated Nucleic acid Detection (PAND) technology (URPAND). URPAND was achieved ultra-fast RTX-PCR process based on a thermostable RTX (exo-) with both reverse transcriptase and DNA polymerase activity. The URPAND can be completed RT-PCR and PAND to detect nucleic acid in one tube within 30 min. This method can specifically detect SARS-CoV-2 with a low detection limit of 100 copies/mL. The diagnostic results of clinical samples with one-tube URPAND displayed 100% consistence with RT-qPCR test. Moreover, URPAND was also applied to identify SARS-CoV-2 D614G mutant due to its single-nucleotide specificity. The URPAND platform is rapid, accurate, tube closed, one-tube, easy-to-operate and free of large instruments, which provides a new strategy to the detection of SARS-CoV-2 and other RNA viruses.

Optical lateral flow assays in early diagnosis of SARS-CoV-2 infection.

So far, the 2019 novel coronavirus (COVID-19) is spreading widely worldwide. The early diagnosis of infection by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is essential to provide timely treatment and prevent its further spread. Lateral flow assays (LFAs) have the advantages of rapid detection, simple operation, low cost, ease of mass production, and no need for special devices and professional operators, which make them suitable for self-testing at home. This review focuses on the early diagnosis of SARS-CoV-2 infection based on optical LFAs including colorimetric, fluorescent (FL), chemiluminescent (CL), and surface-enhanced Raman scattering (SERS) LFAs for the detection of SARS-CoV-2 antigens and nucleic acids. The types of recognition components, detection modes used for antigen detection, labels employed in different optical LFAs, and strategies to improve the detection sensitivity of LFAs were reviewed. Meanwhile, LFAs coupled with different nucleic acid amplification techniques and CRISPR-Cas systems for the detection of SARS-CoV-2 nucleic acids were summarized. We hope this review provides research mentalities for developing highly sensitive LFAs that can be used in home self-testing for the early diagnosis of SARS-CoV-2 infection.

An Emission-Based Probe for the Detection and Quantification of DNA and RNA.

Sequence-independent detection of low concentrations of nucleic acids is important for applications in forensics and diagnostics. An emission-based probe for detecting and quantifying DNA and RNA utilizing a water-soluble dicationic tetraphenylethene (TPE) derivativewas developed. The recognition is based on the electrostatic and other non-covalent interactions between the phosphate backbone of nucleic acids and the cationic probe, which cause the restriction of rotation of the aryl units of the probe, ensuing in the enhancement of the fluorescence signal. The binding was validated by different spectroscopic techniques and also by electrophoretic mobility shift assay. The probable mode of binding with the nucleic acids was studied by blind-docking studies that correlated well with the experimental results.

Open and closed microfluidics for biosensing.

Biosensing is vital for many areas like disease diagnosis, infectious disease prevention, and point-of-care monitoring. Microfluidics has been evidenced to be a powerful tool for biosensing via integrating biological detection processes into a palm-size chip. Based on the chip structure, microfluidics has two subdivision types: open microfluidics and closed microfluidics, whose operation methods would be diverse. In this review, we summarize fundamentals, liquid control methods, and applications of open and closed microfluidics separately, point out the bottlenecks, and propose potential directions of microfluidics-based biosensing.

Rapid Nucleic Acid Diagnostic Technology for Pandemic Diseases.

The recent global pandemic of coronavirus disease 2019 (COVID-19) has enormously promoted the development of diagnostic technology. To control the spread of pandemic diseases and achieve rapid screening of the population, ensuring that patients receive timely treatment, rapid diagnosis has become the top priority in the development of clinical technology. This review article aims to summarize the current rapid nucleic acid diagnostic technologies applied to pandemic disease diagnosis, from rapid extraction and rapid amplification to rapid detection. We also discuss future prospects in the development of rapid nucleic acid diagnostic technologies.

Palm-Sized Lab-In-A-Magnetofluidic Tube Platform for Rapid and Sensitive Virus Detection.

Simple, sensitive, and accurate molecular diagnostics are critical for preventing rapid spread of infection and initiating early treatment of diseases. However, current molecular detection methods typically rely on extensive nucleic acid sample preparation and expensive instrumentation. Here, a simple, fully integrated, lab-in-a-magnetofluidic tube (LIAMT) platform is presented for "sample-to-result" molecular detection of virus. By leveraging magnetofluidic transport of micro/nano magnetic beads, the LIAMT device integrates viral lysis, nucleic acid extraction, isothermal amplification, and CRISPR detection within a single engineered microcentrifuge tube. To enable point-of-care molecular diagnostics, a palm-sized processor is developed for magnetofluidic separation, nucleic acid amplification, and visual fluorescence detection. The LIAMT platform is applied to detect SARS-CoV-2 and HIV viruses, achieving a detection sensitivity of 73.4 and 63.9 copies µL-1, respectively. Its clinical utility is further demonstrated by detecting SARS-CoV-2 and HIV in clinical samples. This simple, affordable, and portable LIAMT platform holds promise for rapid and sensitive molecular diagnostics of infectious diseases at the point-of-care.

PfAgo-Based Zika Virus Detection.

As a mosquito-borne flavivirus, Zika virus (ZIKV) has been identified as a global health threat. The virus has been linked to severe congenital disabilities, including microcephaly and other congenital malformations, resulting in fatal intrauterine death. Therefore, developing sensitive and specific methods for the early detection and accurate diagnosis of the ZIKV is essential for controlling its spread and mitigating its impact on public health. Herein, we set up a novel nucleic acid detection system based on Pyrococcus furiosus Argonaute (PfAgo)-mediated nucleic acid detection, targeting the non-structural protein 5 (NS5) region of the ZIKV genome (abbreviated ZIKV-PAND). Without preamplification with the polymerase chain reaction (PCR), the minimum detection concentration (MDC) of ZIKV-PAND was about 10 nM. When introducing an amplification step, the MDC can be dramatically decreased to the aM level (8.3 aM), which is comparable to qRT-PCR assay (1.6 aM). In addition, the diagnostic findings from the analysis of simulated clinical samples or Zika virus samples using ZIKV-PAND show a complete agreement of 100% with qRT-PCR assays. This correlation can aid in the implementation of molecular testing for clinical diagnoses and the investigation of ZIKV infection on an epidemiological scale.