Nucleic Acid Detection Research Articles

Development and evaluation of a multi-target droplet digital PCR assay for highly sensitive and specific detection of Yersinia pestis.

Plague, caused by the bacterium Yersinia pestis, is a zoonotic disease that poses considerable threats to human health. Nucleic acid tests are crucial for plague surveillance and the rapid detection of Y. pestis. However, inhibitors in complex samples such as soil and animal tissues often hamper nucleic acid detection, leading to a reduced rate of identifying low concentrations of Y. pestis. To address this challenge, we developed a sensitive and specific droplet digital polymerase chain reaction (ddPCR) assay for detecting Y. pestis DNA from soil and animal tissue samples. Three genes (ypo2088, caf1, and pla) from Y. pestis were used to develop a multi-target ddPCR assay. The limits of detection (LoD), reproducibility, and specificity were assessed for bacterial genomic DNA samples. The ability of the assay to detect low concentrations of Y. pestis DNA from simulated soil and mouse liver tissue samples was respectively evaluated and compared with that of quantitative real-time PCR (qPCR). The results showed that the ddPCR LoDs ranged from 6.2 to 15.4 copies/reaction for the target genes, with good reproducibility and high specificity for Y. pestis. By testing 130 soil and mouse liver tissue samples spiked with Y. pestis, the ddPCR assay exhibited a better sensitivity than that of the qPCR assay used in the study, with LoDs of 102 colony forming units (CFU)/100 mg soil and 103 CFU/20 mg liver. Moreover, the assay presented good quantitative linearity (R2 = 0.99) for Y. pestis at 103-106 CFU/sample for soil and liver samples. The ddPCR assay presented good performance for detecting Y. pestis DNA from soil and mouse tissue samples, showing great potential for improving the detection rate of low concentrations of Y. pestis in plague surveillance and facilitating the early diagnosis of plague cases.

An integrated low-cost automatic pipetting used in nucleic acid detection PCR instrument and clinical detection

PurposeThis paper aims to introduce a custom-designed integrated nucleic acid detection polymerase chain reaction (PCR) instrument for clinical detection applications.Design/methodology/approachThe PCR instrument can make rapid, sensitive, low-cost and quantitative molecular diagnosis compared with the current routine test flow from the pipette, series reagent to RT-PCR by manual manipulation. By integrating the multichannel automatic pipetting module, heat amplification module and real-time fluorescence detection module for the first time, the custom-designed integrated nucleic acid detection PCR instrument can achieve sample collection, subpackage, mixing, extracting, measuring and result presentation.FindingsThe multichannel automatic pipetting module was assembled with an accuracy of 0.4% (2 microliters) for accuracy measurement. Besides, the accuracy and sensitivity of nucleic acid using integrated low-cost nucleic acid detection PCR instruments were checked with COV-2019 virus (staining method) and African swine fever virus (probe method) under different concentrations.Practical implicationsBecause of its high cost, complex system and bulky laboratory settings, including sample subpackage, mixing, extracting, measuring and finally result in presentation, the current nucleic acid detection system is not suitable for field operation and disease diagnosis in remote areas. The group independently designed and assembled an integrated low-cost multichannel nucleic acid detection PCR instrument, including a multichannel automatic pipetting module, a heat amplification module and a real-time fluorescence detection module.Originality/valueThe above equipment showed better reliability compared with commercial qPCR. These results can lay the foundation for functional, fast and low-cost PCR equipment for trace measurements.

Performance verification and clinical evaluation of the NAP-Fluo Cycler system for detecting five genital tract pathogens based on microfluidic technology

IntroductionSexually transmitted infections (STIs) are among the most common infectious diseases worldwide, often leading to coinfections. Timely detection of genital tract pathogens in at-risk populations is crucial for preventing STIs. We evaluated the NAP-Fluo Cycler System, an innovative microfluidic nucleic acid detection platform, for its ability to simultaneously identify Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Ureaplasma urealyticum (UU), Mycoplasma genitalium (MG), and Mycoplasma hominis (MH) in urethral or cervical secretions. Materials and methodsThe limits of detection (LODs), repeatability, specificity, and interference resistance of the system were evaluated using standard strains, a panel of 24 pathogens, and seven interferents. We used the system to analyze 302 clinical samples and compared the results with those of five approved commercial reference kits. ResultsThe system achieved LODs of 500 IFU/mL, 500 CFU/mL, and 500 CCU/mL for CT, NG, and UU/MG/MH, respectively, demonstrating high stability (coefficient of variation <1.1 %), specificity, and resistance to interference. Among 302 clinical samples, 237 tested positive with single, dual, and triple infection rates of 35.6 %, 16.2 %, and 3.0 %, respectively. The reference kits detected 138 positive samples. The concordance rates with commercial reference kits were 100 % for UU, NG, and MH; 94.85 % for CT; and 80.00 % for MG. ConclusionsThis system offers a streamlined, rapid, and multiplex detection method that reduces testing time and complexity. Although it performs well with pure strains, it has limitations when using clinical samples of CT and MG, suggesting the need for further refinement before its widespread use in the clinic.

Detection of sgRNA via SHERLOCK as Potential CRISPR Related Gene Doping Control Strategy.

Apprehensions about gene doping have grown consistently due to advancements in gene engineering techniques, particularly with the emergence of clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas)-based tools. These tools not only provide unprecedented possibilities for illicit performance enhancement by athletes but also offer new avenues for the detection of gene doping through biosensing of nucleic acids. Hence, pursuing on a previous study, an analytical method based on reverse transcriptase-recombinase polymerase amplification (RT-RPA) and subsequent qualitative nucleic acid detection by means of Specific High Sensitive Enzymatic Reporter UnLOCKing (SHERLOCK) was optimized for the direct detection of sgRNA associated with Streptococcus pyogenes in serum. Detection device, assay parameters, and sample handling were adjusted, to overcome previously determined assay limitations. The conducted method characterization confirmed the methods' specificity and increased detection sensitivity from 100 pM to 1 fM sgRNA in 100 μL of serum. Furthermore, reanalysis of in vivo mouse administration samples collected in a previous proof-of-concept study was conducted with successful identification of sgRNA in all anticipated postadministration samples within the 24-h collection period. Those findings support the applicability of the refined analytical procedure for the detection of illegal doping attempts via ribonucleoprotein-based CRISPR/Cas application through sgRNA identification, offering a new potential doping control strategy for CRISPR related gene doping.

Enhanced CRISPR/Cas12a-based quantitative detection of nucleic acids using double emulsion droplets

Pairing droplet microfluidics and CRISPR/Cas12a techniques creates a powerful solution for the detection and quantification of nucleic acids at the single-molecule level, due to its specificity, sensitivity, and simplicity. However, traditional water-in-oil (W/O) single emulsion (SE) droplets often present stability issues, affecting the accuracy and reproducibility of assay results. As an alternative, water-in-oil-in-water (W/O/W) double emulsion (DE) droplets offer superior stability and uniformity for droplet digital assays. Moreover, unlike SE droplets, DE droplets are compatible with commercially available flow cytometry instruments for high-throughput analysis. Despite these advantages, no study has demonstrated the use of DE droplets for CRISPR-based nucleic acid detection. In our study, we conducted a comparative analysis to assess the performance of SE and DE droplets in quantitative detection of human papillomavirus type 18 (HPV18) DNA based on CRISPR/Cas12a. We evaluated the stability of SEs and DEs by examining size variation, merging extent, and content interaction before and after incubation at different temperatures and time points. By integrating DE droplets with flow cytometry, we achieved high-throughput and high-accuracy CRISPR/Cas12a-based quantification of target HPV18 DNA. The DE platform, when paired with CRISPR/Cas12a and flow cytometry techniques, emerges as a reliable tool for absolute quantification of nucleic acid biomarkers.

Biominerals and Bioinspired Materials in Biosensing: Recent Advancements and Applications.

Inspired by nature's remarkable ability to form intricate minerals, researchers have unlocked transformative strategies for creating next-generation biosensors with exceptional sensitivity, selectivity, and biocompatibility. By mimicking how organisms orchestrate mineral growth, biomimetic and bioinspired materials are significantly impacting biosensor design. Engineered bioinspired materials offer distinct advantages over their natural counterparts, boasting superior tunability, precise controllability, and the ability to integrate specific functionalities for enhanced sensing capabilities. This remarkable versatility enables the construction of various biosensing platforms, including optical sensors, electrochemical sensors, magnetic biosensors, and nucleic acid detection platforms, for diverse applications. Additionally, bioinspired materials facilitate the development of smartphone-assisted biosensing platforms, offering user-friendly and portable diagnostic tools for point-of-care applications. This review comprehensively explores the utilization of naturally occurring and engineered biominerals and materials for diverse biosensing applications. We highlight the fabrication and design strategies that tailor their functionalities to address specific biosensing needs. This in-depth exploration underscores the transformative potential of biominerals and materials in revolutionizing biosensing, paving the way for advancements in healthcare, environmental monitoring, and other critical fields.

In situ quantitative mapping of coding single nucleotide polymorphism on mRNA inside cells by SERS-fluorescence dual-mode probe

Surface Enhanced Raman Scattering (SERS) has potential in nucleic acid detection, but its ability on single nucleotide polymorphisms (SNPs) detection in the early diagnosis of diseases is still limited. In fact, there is few research about in situ detection of coding SNPs which are carried on mRNA. In this paper, we developed SERS-Fluorescence Dual-mode probes to achieve in situ quantitative mapping of intracellular cSNP. The probes were based on Au@Ag core-shell structure that possessed both excellent biocompatibility and localized surface plasmon resonance. Fluorescence can provide a rapid visualization of intracellular cSNP, while SERS can achieve its in situ quantification. The quantitation was conducted by comparing the SERS signals of the reporting molecule p-mercaptobenzoic acid and fluorescent molecule FAM, the detecting limitation of which can be achieved as low as 1.4 fM. This ratiometric detection is more accurate than traditional analysis based on signal intensity directly. It successfully achieved intracellular imaging of the G12D mutation site of KRAS gene in A549 cells, which can help to get the distribution of cSNP in single cell more clearly. And it also demonstrated the good mapping ability for biomolecules (1003 cm−1 for phenylalanine) simultaneously. In conclusion, this kind of probe could be used as a potential tool to monitor cSNP as well as biomolecules in multiple biological processes, which could provide reference for disease prediction.

A Poisson-Independent Approach to Precision Nucleic Acid Quantification in Microdroplets.

Digital PCR (dPCR) has become indispensable in nucleic acid (NA) detection across various fields, including viral diagnostics and mutant detection. However, misclassification of partitions in dPCR can significantly impact accuracy. Despite existing methods to minimize misclassification bias, accurate classification remains elusive, especially for nonamplified target partitions. To address these challenges, this study introduces an innovative microdroplet-based competitive PCR platform for nucleic acid quantification in microfluidic devices independent of Poisson statistics. In this approach, the target concentration (T) is determined from the concentration of competitor DNA (C) at the equivalence point (E.P.), where C/T is 1. Competitive PCR ensures that the ratio of target to competitor DNA remains constant during amplification, reflected in the resultant fluorescence intensity, allowing the quantification of target DNA concentration at the equivalence point. The unique amplification technique eliminates Poisson distribution, addressing misclassification challenges. Additionally, our approach reduces the need for post-PCR procedures and shortens analytical time. We envision this platform as versatile, reproducible, and easily adaptable for driving significant progress in molecular biology and diagnostics.

Bimetallic interface on an optical microfiber nucleic acid sensor: Early detection in body fluids

Nucleic acid detection, which is not limited by the “window period”, is an efficacious approach for detecting human immunodeficiency virus (HIV) infection at an early time. Here, method with a short turn-around time, portability, connectivity and cost-effectiveness is developed to suppress HIV disease in the early stage, especially in remote areas. Specifically, an optical microfiber sensor enhanced by a bimetallic interface is developed for the HIV-related DNA detection in body fluids. A bimetallic interface is constructed on the optical microfiber surface to ensure that target nucleic acid hybridization occurs between two gold nanostructures where the evanescent field is strongest. The selective enhancement of the evanescent field at the binding site prevents nonspecific amplification of the whole fiber surface, which may lead to false-positive results. With the enhancement in the bimetallic interface, the sensor can detect HIV-related DNA at concentrations from 1 aM to 0.1 pM, with a limit of detection (LOD) of 0.3 aM. The portability, flexibility and selectivity of the sensor enable its application in detecting in several body fluids, such as whole serum and artificial saliva, with LODs of 1.15 aM and 1.64 aM, respectively. This work provides a powerful tool for diagnosing HIV in early stages. Ultrasensitive nucleic acid detection in artificial saliva also provides a possibility for noninvasive detection.

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

PurposeTo 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. MethodsReferences 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. ResultsThe 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. ConclusionsThe newly developed kit has shown satisfactory performances and excellent consistency with DNBSEQ-G99, making it a good alternative choice clinically.

Concomitant and sequential administration of nirmatrelvir‐ritonavir and azvudine in patients with COVID‐19 caused by the Omicron variant: Safety and efficacy

AbstractBackgroundThis study assessed the safety and efficacy of nirmatrelvir‐ritonavir (Paxlovid®) and azvudine when administered sequentially or concomitantly in patients with coronavirus 2019 (COVID‐19) caused by the Omicron variant.MethodsNinety‐three patients confirmed to be infected with the Omicron variant by nucleic acid detection were retrospectively investigated. Information was collected on general health status, medication, and adverse drug reactions (ADRs) according to whether nirmatrelvir‐ritonavir and azvudine were administered sequentially or concomitantly. Data on times of onset, clinical manifestations, and outcomes of ADRs and on conversion to a negative nucleic acid test were also recorded.ResultsPossible ADRs were recorded in 41 patients (44.1%). There were 22 gastrointestinal reactions in 18 patients and 18 hematological abnormalities in 16 after sequential or concomitant treatment with nirmatrelvir‐ritonavir and azvudine. Liver enzyme levels increased in nine cases and creatinine clearance decreased in two. Cases of atrial fibrillation (n = 1), sleep disorder (n = 2), rash (n = 2), dizziness (n = 1), and weakness (n = 5) were also documented. Only vomiting, poor appetite, diarrhea, xerostomia, bitter taste, and rash were considered probable ADRs; others were thought to be possible ADRs. In all cases, the nucleic acid test did not turn negative after the first antiviral was applied. The nucleic acid test of 28 patients did not turn negative before discharge. The remaining 65 patients (69.9%) returned a negative nucleic acid test after receiving the second antiviral agent.ConclusionsTreatment with nirmatrelvir‐ritonavir and azvudine is safe and effective whether administered sequentially or concomitantly in patients with COVID‐19 caused by the Omicron variant.

In Situ Detection of Nucleic Acids in Extracellular Vesicles via Membrane Fusion.

Extracellular vesicles (EVs) carry diverse biomolecules (e. g., nucleic acids, proteins) for intercellular communication, serving as important markers for diseases. Analyzing nucleic acids derived from EVs enables non-invasive disease diagnosis and prognosis evaluation. Membrane fusion, a fundamental cellular process wherein two lipid membranes merge, facilitates cell communication and cargo transport. Building on this natural phenomenon, recent years have witnessed the emergence of membrane fusion-based strategies for the detection of nucleic acids within EVs. These strategies entail the encapsulation of detection probes within either artificial or natural vesicles, followed by the induction of membrane fusion with EVs to deliver probes. This innovative approach not only enables in situ detection of nucleic acids within EVs but also ensures the maintenance of structural integrity of EVs, thus preventing nucleic acid degradation and minimizing the interference from free nucleic acids. This concept categorizes approaches into universal and targeted membrane fusion strategies, and discusses their application potential, and challenges and future prospects.

Study on the applicability of novel molecular targets of Vibrio species by pan-genome mining in the field of nucleic acid detection

Vibrio spp. are ubiquitous in marine areas worldwide and can cause diseases in humans. Aqueous environments favor the transfer of genes between strains, resulting in the loss of strain-specific target genes. This study aimed to explore new molecular targets for the detection of Vibrio strains at the species level using pan-genome analysis for nucleic acid detection and differentiation of major pathogenic Vibrio in aquatic products. Pan-genome analysis and PCR validation revealed a total of 10 specific targets: three for Vibrio parahaemolyticus, one for Vibrio alginolyticus, three for Vibrio vulnificus, three for Vibrio fluvialis. We constructed primers for the selected genes, and we verified their specificity using various Vibrio species and non-Vibrio strains. After systematic optimization, a novel molecular target-based 4-multiplex PCR (4-mPCR) method was developed, exhibiting a detection limit of 103 CFU/mL for pure cultures and 104 CFU/g for fish samples. The initial inoculum was approximately 1 Log10 CFU/g, and all samples were positive after 4 h of enrichment. Not only that, the feasibility of the mined new targets in the field of nucleic acid detection was further validated by the 4-mqPCR and LAMP techniques. These results suggest that novel targets mined by pan-genomic have good generalizability for use in a variety of nucleic acid detection techniques and can help improve the accuracy of nucleic acid-based detection techniques in the detection of pathogenic Vibrio species in aquatic samples, with potential applications for food safety investigations and disease diagnosis.

Droplet digital PCR: An effective method for monitoring and prognostic evaluation of minimal residual disease in JMML.

Juvenile myelomonocytic leukaemia (JMML) is a rare myeloproliferative neoplasm requiring haematopoietic stem cell transplantation (HSCT) for potential cure. Relapse poses a significant obstacle to JMML HSCT treatment, as the lack of effective minimal residual disease (MRD)-monitoring methods leads to delayed interventions. This retrospective study utilized the droplet digital PCR (ddPCR) technique, a highly sensitive nucleic acid detection and quantification technique, to monitor MRD in 32 JMML patients. The results demonstrated that ddPCR detected relapse manifestations earlier than traditional methods and uncovered molecular insights into JMML MRD dynamics. The findings emphasized a critical 1- to 3-month window post-HSCT for detecting molecular relapse, with 66.7% (8/12) of relapses occurring within this period. Slow MRD clearance post-HSCT was observed, as 65% (13/20) of non-relapse patients took over 6 months to achieve ddPCR-MRD negativity. Furthermore, bone marrow ddPCR-MRD levels at 1-month post-HSCT proved to be prognostically significant. Relapsed patients exhibited significantly elevated ddPCR-MRD levels at this time point (p = 0.026), with a cut-off of 0.465% effectively stratifying overall survival (p = 0.007), event-free survival (p = 0.035) and cumulative incidence of relapse (p = 0.035). In conclusion, this study underscored ddPCR's superiority in JMML MRD monitoring post-HSCT. It provided valuable insights into JMML MRD dynamics, offering guidance for the effective management of JMML.

Rapid, multiplexed, and enzyme-free nucleic acid detection using programmable aptamer-based RNA switches

Rapid, simple, and low-cost diagnostic technologies are crucial tools for combatting infectious disease. We describe a class of aptamer-based RNA switches or aptaswitches that recognize target nucleic acid molecules and initiate folding of a reporter aptamer. Aptaswitches can detect virtually any sequence and provide an intense fluorescent readout without intervening enzymes, generating signals in as little as 5 minutes and enabling detection by eye with minimal equipment. Aptaswitches can be used to regulate folding of seven fluorogenic aptamers, providing a general means of controlling aptamers and an array of multiplexable reporter colors. Coupling isothermal amplification reactions with aptaswitches, we reach sensitivities down to 1 RNA copy/μL in one-pot reactions. Application of multiplexed all-in-one reactions against RNA from clinical saliva samples yields an overall accuracy of 96.67% for detection of SARS-CoV-2 in 30 minutes. Aptaswitches are thus versatile tools for nucleic acid detection that are readily integrated into rapid diagnostic assays.

Intrinsic RNA Targeting Triggers Indiscriminate DNase Activity of CRISPR‐Cas12a

AbstractThe CRISPR‐Cas12a system has emerged as a powerful tool for next‐generation nucleic acid‐based molecular diagnostics. However, it has long been believed to be effective only on DNA targets. Here, we investigate the intrinsic RNA‐enabled trans‐cleavage activity of AsCas12a and LbCas12a and discover that they can be directly activated by full‐size RNA targets, although LbCas12a exhibits weaker trans‐cleavage activity than AsCas12a on both single‐stranded DNA and RNA substrates. Remarkably, we find that the RNA‐activated Cas12a possesses higher specificity in recognizing mutated target sequences compared to DNA activation. Based on these findings, we develop the “Universal Nuclease for Identification of Virus Empowered by RNA‐Sensing” (UNIVERSE) assay for nucleic acid testing. We incorporate a T7 transcription step into this assay, thereby eliminating the requirement for a protospacer adjacent motif (PAM) sequence in the target. Additionally, we successfully detect multiple PAM‐less targets in HIV clinical samples that are undetectable by the conventional Cas12a assay based on double‐stranded DNA activation, demonstrating unrestricted target selection with the UNIVERSE assay. We further validate the clinical utility of the UNIVERSE assay by testing both HIV RNA and HPV 16 DNA in clinical samples. We envision that the intrinsic RNA targeting capability may bring a paradigm shift in Cas12a‐based nucleic acid detection and further enhance the understanding of CRISPR‐Cas biochemistry.

Intrinsic RNA Targeting Triggers Indiscriminate DNase Activity of CRISPR-Cas12a.

The CRISPR-Cas12a system has emerged as a powerful tool for next-generation nucleic acid-based molecular diagnostics. However, it has long been believed to be effective only on DNA targets. Here, we investigate the intrinsic RNA-enabled trans-cleavage activity of AsCas12a and LbCas12a and discover that they can be directly activated by full-size RNA targets, although LbCas12a exhibits weaker trans-cleavage activity than AsCas12a on both single-stranded DNA and RNA substrates. Remarkably, we find that the RNA-activated Cas12a possesses higher specificity in recognizing mutated target sequences compared to DNA activation. Based on these findings, we develop the "Universal Nuclease for Identification of Virus Empowered by RNA-Sensing" (UNIVERSE) assay for nucleic acid testing. We incorporate a T7 transcription step into this assay, thereby eliminating the requirement for a protospacer adjacent motif (PAM) sequence in the target. Additionally, we successfully detect multiple PAM-less targets in HIV clinical samples that are undetectable by the conventional Cas12a assay based on double-stranded DNA activation, demonstrating unrestricted target selection with the UNIVERSE assay. We further validate the clinical utility of the UNIVERSE assay by testing both HIV RNA and HPV 16 DNA in clinical samples. We envision that the intrinsic RNA targeting capability may bring a paradigm shift in Cas12a-based nucleic acid detection and further enhance the understanding of CRISPR-Cas biochemistry.

Use of FTA card for the detection of two RNA (CSFV and SV-A) and two DNA viruses (ASFVand SuHV-1) of importance in veterinary medicine.

The FTA card has emerged as a promising alternative for nucleic acid extraction. The FTA card is a filter paper impregnated with chemicals that preserve and stabilize the genetic material present in the sample, allowing for its storage and transport at room temperature. The aim of this study was to test the card for the detection of RNA and DNA nucleic acids. Two RNA viruses (Senecavirus A and classical swine fever virus) and two DNA viruses (African swine fever virus and suid alphaherpesvirus 1) were tested, and in all cases, there was a decrease in sensitivity. The methods exhibited good repeatability and demonstrated a rapid and practical use for sample transport and nucleic acid extraction.

Soft interface confined DNA walker for sensitive and specific detection of SARS-CoV-2 variants

Nucleic acid detection is conducive to preventing the spread of COVID-19 pandemic. In this work, we successfully designed a soft interface confined DNA walker by anchoring hairpin reporter probes on cell membranes for the detection of SARS-CoV-2 variants. In the presence of target RNA, the cyclic self-assembly reaction occurred between hairpin probes H1 and H2, and the continuous walking of target RNA on cell membranes led to the gradual amplification of fluorescence signal. The enrichment of H1 on membranes and the unique fluidity of membranes promoted the collision efficiency between DNA strands in the reaction process, endowing this method with high sensitivity. In addition, the double-blind test of synthetic RNA in 5% normal human serum demonstrated the good stability and anti-interference in complex environment of this method, which exhibited great potential in clinical diagnostics.

Application of CRISPR/Cas12a for SARS-CoV-2 Nucleic Acid Detection

SARS-CoV-2 spans 3 years, and although the virus has been adjusted from "Category B, Tube A" to "Category B, Tube B" it is still present in the environment, causing widespread epidemic infections and great impact on human health and the environment. Currently, there are various nucleic acid detection methods for this virus, and CRISPR methods are widely used to construct biosensors for accurate and rapid specific detection of nucleic acid sequences due to their powerful gene-editing capabilities. In this article, we introduce the application of different forms of sensors such as fluorescent paper strips built with CRISPR/Cas12a for SARS-CoV-2 nucleic acid detection.