A Lateral Flow Assay Based on Streptavidin-biotin Amplification System with Recombinase Polymerase Amplification for Rapid and Quantitative Detection of Salmonella enteritidis.

Salmonella constitutes a prevalent alimentary pathogen, instigating zoonotic afflictions. Consequently, the prompt discernment of Salmonella in sustenance is of cardinal significance. Lateral flow assays utilizing colorimetric methodologies adequately fulfill the prerequisites of point-of-care diagnostics, however, their detection threshold remains elevated, generally permitting only qualitative discernment, an impediment to the preliminary screening of nascent pathogens. In response to this conundrum, we propose a lateral flow diagnostic predicated upon a streptavidin-biotin amplification system with recombinase polymerase amplification engineered for the expeditious and quantitative discernment of Salmonella enteritidis. Trace nucleic acids within a sample undergo exponential amplification via recombinase polymerase amplification to a level discernable, constituting the initial signal amplification. Subsequently, along the test line (T-line) of the lateral flow strip, the chromatic signal undergoes augmentation by securing a greater quantity of AuNPs through the magnification capacity of the streptavidin-biotin mechanism, affecting the second signal amplification. Quantitative results are procured via smartphone capture and transferred to computer software for precise calculation of the targeted quantity. The lateral flow strip exhibits a LOD at 19.41 CFU/mL for cultured S. enteritidis. The RSD of three varying concentrations were respectively 3.74 %, 5.96 %, and 4.25 %.

Simple, sensitive, and visual detection of 12 respiratory pathogens with one-pot-RPA-CRISPR/Cas12a assay.

Respiratory infections pose a serious threat to global public health, underscoring the urgent need for rapid, accurate, and large-scale diagnostic tools. In recent years, the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system, combined with isothermal amplification methods, has seen widespread application in nucleic acid testing (NAT). However, achieving a single-tube reaction system containing all necessary components is challenging due to the competitive effects between recombinase polymerase amplification (RPA) and CRISPR/Cas reagents. Furthermore, to enable precision medicine, distinguishing between bacterial and viral infections is essential. Here, we have developed a novel NAT method, termed one-pot-RPA-CRISPR/Cas12a, which combines RPA with CRISPR molecular diagnostic technology, enabling simultaneous detection of 12 common respiratory pathogens, including six bacteria and six viruses. RPA and CRISPR/Cas12a reactions are separated by paraffin, providing an independent platform for RPA reactions to generate sufficient target products before being mixed with the CRISPR/Cas12a system. Results can be visually observed under LED blue light. The sensitivity of the one-pot-RPA-CRISPR/Cas12a method is 2.5 × 100 copies/µL plasmids, with no cross-reaction with other bacteria or viruses. Additionally, the clinical utility was evaluated by testing clinical isolates of bacteria and virus throat swab samples, demonstrating favorable performance. Thus, our one-pot-RPA-CRISPR/Cas12a method shows immense potential for accurate and large-scale detection of 12 common respiratory pathogens in point-of-care testing.

Dual-RPA assay for rapid detection and differentiation of E.granulosus and E.multilocularis.

Echinococcus granulosus (Eg) and Echinococcus multilocularis (Em) are the two most widely prevalent types of echinococcosis. Several diagnostic methods have been developed for detecting Eg and Em. However, some limitations, such as being time-consuming, needing expensive instruments, or exhibiting low sensitivity, make these methods unsuitable for on-site detection. In this study, a dual-RPA assay was established to detect and differentiate Eg and Em. The primer concentration ratio, reaction time, and reaction temperature of the dual-RPA were optimized. The result showed that the primer concentration ratio of Eg:Em was 400 nM:400 nM, and the best amplification efficiency was obtained by reacting at 38 °C for 20 min. The sensitivity, specificity, and repeatability of the assay were also tested. The assay's detection limit for both Eg and Em was 10 copies/µL. The assay showed reasonable specificity by testing ten parasitic nucleic acids. The assay's intra- and inter-batch coefficients of variation were below 10%, which indicates robust reproducibility of the assay. Finally, to validate the performance of the dual-RPA assay, it was compared with real-time PCR by using 86 clinical nucleic acid samples. The coincidence rate of Eg between dual-RPA and TaqMan real-time PCR was 96.51%, and the coincidence rate of Em between dual-RPA and TaqMan real-time PCR was 98.84%, indicating its potential for accurate clinical diagnosis. Therefore, this study established a rapid and sensitive dual-RPA assay that can rapidly detect and differentiate Eg and Em in one reaction tube and provided a new assay for the detection of echinococcosis in the field.

A method for Porphyromonas gingivalis based on recombinase polymerase amplification and lateral flow strip technology.

OBJECTIVE: A practical visual detection method was established to detect Porphyromonas gingivalis (P. gingivalis) by employing a combination of recombinase polymerase amplification and lateral flow strips (RPA-LF) assay, designed for conducting point-of-care testing in clinical settings. METHODS: Primers and probes targeting the P. gingivalis pepO gene were designed. The RPA-LF assay was established by optimising reaction temperature and time, determining the limit of detection (LOD). The specificity of the method was determined by assessing its cross-reactivity with deoxyribonucleic acid from 23 pathogenic bacteria. Finally, the clinical samples from healthy controls (n = 30) and individuals with periodontitis (n = 31) were analysed. The results were compared with those obtained using real-time polymerase chain reaction (PCR). RESULTS: The optimal reaction temperature and time were 39 °C and 12 min. The method exhibited a LOD at 6.40 × 10-4 µg/mL and demonstrated high specificity and sensitivity during cross-reactivity assessment. The RPA-LF assay achieved a P. gingivalis detection rate of 84 % in individuals with periodontitis and 3 % in healthy controls. The results were consistent with those obtained through real-time PCR. CONCLUSION: An RPA-LF assay was developed for detecting P. gingivalis, characterised by its high sensitivity, high specificity, simple operational procedure, and rapid reaction time.

Detection of P. malariae using a new rapid isothermal amplification lateral flow assay.

BACKGROUND: While Plasmodium falciparum and Plasmodium vivax cause the majority of malaria cases and deaths, infection by Plasmodium malariae and other Plasmodium species also causes morbidity and mortality. Current understanding of these infections is limited in part by existing point-of-care diagnostics that fail to differentiate them and have poor sensitivity for low-density infections. Accurate diagnosis currently requires molecular assays performed in well-resourced laboratories. This report describes the development of a P. malariae diagnostic assay that uses rapid, isothermal recombinase polymerase amplification (RPA) and lateral-flow-strip detection. METHODS: Multiple combinations of custom RPA primers and probes were designed using publicly available P. malariae genomic sequences, and by modifying published primer sets. Based on manufacturer RPA reaction conditions (TwistDx nfo kit), an isothermal assay was optimized targeting the multicopy P. malariae 18S rRNA gene with 39 °C incubation and 30-min run time. RPA product was visualized using lateral strips (FAM-labeled, biotinylated amplicon detected by a sandwich immunoassay, visualized using gold nanoparticles). Analytical sensitivity was evaluated using 18S rRNA plasmid DNA, and clinical sensitivity determined using qPCR-confirmed samples collected from Tanzania, Ethiopia, and the Democratic Republic of the Congo. RESULTS: Using 18S rRNA plasmid DNA, the assay demonstrates a detection limit of 10 copies/µL (~ 1.7 genome equivalents) and 100% analytical specificity. Testing in field samples showed 95% clinical sensitivity and 88% specificity compared to qPCR. Total assay time was less than 40 min. CONCLUSION: Combined with simplified DNA extraction methods, the assay has potential for future field-deployable, point-of-care use to detect P. malariae infection, which remains largely undiagnosed but a neglected cause of chronic malaria. The assay provides a rapid, simple readout on a lateral flow strip without the need for expensive laboratory equipment.

Rapid detection of human adenovirus subgroup B using recombinase polymerase amplification assay.

Human adenovirus subgroup B (HAdV B) is one of the major pathogens of human respiratory virus infections, which has considerable transmission and morbidity in a variety of populations. Therefore, rapid and specific detection of HAdV B in clinical samples is essential for diagnosis. This study aimed to develop a product for rapid nucleic acid detection of HAdV B using recombinase polymerase amplification assay (RPA) and validate the performance of this method by using clinical samples. Results showed that this method achieved a lower limit of detection (LOD) of 10 copies/µL and had no cross-reactivity with other adenovirus subgroups or respiratory pathogens. In addition to high sensitivity, it can be completed within 30 min at 40 °C. There is no need to perform nucleic acid extraction on clinical samples. Taking qPCR as the gold standard, the RPA assay possessed a high concordance (Cohen's kappa, 0.896; 95% CI 0.808-0.984; P < 0.001), with a sensitivity of 87.80% and a specificity of 100.00%. The RPA assay developed in this study provided a simple and highly specific method, making it an important tool for rapid adenovirus nucleic acid detection and facilitating large-scale population screening in resource-limited settings.

Rapid, sensitive, and user-friendly detection of Pseudomonas aeruginosa using the RPA/CRISPR/Cas12a system.

BACKGROUND: Pseudomonas aeruginosa (P. aeruginosa) is a life-threatening bacterium known for its rapid development of antibiotic resistance, posing significant challenges in clinical treatment, biosecurity, food safety, and environmental monitoring. Early and accurate identification of P. aeruginosa is crucial for effective intervention. METHODS: The lasB gene of P. aeruginosa was selected as the target for the detection. RPA primers for recombinase polymerase amplification (RPA) and crRNA for CRISPR/Cas12a detection were meticulously designed to target specific regions within the lasB gene. The specificity of the RPA/CRISPR/Cas12a detection platform was assessed using 15 strains. The detection limit of RPA/CRISPR/Cas12a detection platform was determined by utilizing a pseudo-dilution series of the P. aeruginosa DNA. The practical applicability of the RPA/CRISPR/Cas12a detection platform was validated by comparing it with qPCR on 150 samples (35 processed meat product samples, 55 cold seasoned vegetable dishes, 60 bottled water samples). RESULTS: The RPA/CRISPR/Cas12a detection platform demonstrates high specificity, with no cross-reactivity with non-P. aeruginosa strains. This assay exhibits remarkable sensitivity, with a limit of detection (LOD) of 100 copies/µL for fluorescence assay and 101 copies/µL for the LFTS method. Furthermore, the performance of the RPA/CRISPR/Cas12a detection platform is comparable to that of the well-established qPCR method, while offering advantages such as shorter reaction time, simplified operation, and reduced equipment requirements. CONCLUSIONS: The RPA/CRISPR/Cas12a detection platform presents a straightforward, accurate, and sensitive approach for early P. aeruginosa detection and holds great promise for diverse applications requiring rapid and reliable identification.

Development and evaluation of a CRISPR-Cas13a system-based diagnostic for hepatitis E virus.

OBJECTIVES: Hepatitis E virus (HEV) is the leading cause of acute viral hepatitis worldwide. HEV RNA detection is the gold standard for HEV infection diagnosis and PCR methods are commonly used but are usually time-consuming and expensive, resulting in low detection efficiency and coverage, especially in low-income areas. Here, we developed a simpler and more accessible HEV RNA detection method based on CRISPR-Cas13a system. METHODS: A total of 265 samples of different types and sources, including 89 positive samples and 176 negative samples, were enrolled for evaluations. The sensitivity and specificity of the Cas13a-crRNA detection system were evaluated. The World Health Organization reference panel for HEV genotypes was used to evaluate the capability for detecting different HEV genotypes. The validity of the assay was compared with RT-qPCR. RESULTS: The 95 % limits of detection (LOD) of Cas13a-crRNA-based fluorescence assay and strip assay were 12.5 and 200 IU/mL, respectively. They did not show cross-reactivity with samples positive for hepatitis A virus, hepatitis B virus, hepatitis C virus, coxsackievirus A16, rotavirus, enterovirus 71, norovirus or enteropathic Escherichia coli. Different HEV genotypes (HEV1-4) can be detected by the assay. Compared to RT-qPCR, the positive predictive agreements of Cas13a-crRNA-based fluorescence and strip assay were 98.9 % (95 % CI: 93.9-99.8 %) and 91.0 % (95 % CI: 83.3-95.4 %), respectively. The negative predictive agreements were both 100 % (95 % CI: 97.8-100 %). CONCLUSIONS: In conclusion, we established a rapid and convenient HEV RNA detection method with good sensitivity and specificity based on CRISPR-Cas13a system, providing a new option for HEV infection diagnosis.

Increase in the solubility of uvsY using a site saturation mutagenesis library for application in a lyophilized reagent for recombinase polymerase amplification.

BACKGROUND: Recombinase uvsY from bacteriophage T4, along with uvsX, is a key enzyme for recombinase polymerase amplification (RPA), which is used to amplify a target DNA sequence at a constant temperature. uvsY, though essential, poses solubility challenges, complicating the lyophilization of RPA reagents. This study aimed to enhance uvsY solubility. METHODS: Our hypothesis centered on the C-terminal region of uvsY influencing solubility. To test this, we generated a site-saturation mutagenesis library for amino acid residues Lys91-Glu134 of the N-terminal (His)6-tagged uvsY. RESULTS: Screening 480 clones identified A116H as the variant with superior solubility. Lyophilized RPA reagents featuring the uvsY variant A116H demonstrated enhanced performance compared to those with wild-type uvsY. CONCLUSIONS: The uvsY variant A116H emerges as an appealing choice for RPA applications, offering improved solubility and heightened lyophilization feasibility.

Rapid and sensitive detection of large yellow croaker iridovirus by real-time RPA and RPA-LFD.

Large yellow croaker (Larimichthys crocea) is a vital marine-cultured species in China. Large yellow croaker iridovirus (LYCIV) can cause a high mortality rate in L. crocea. Rapid and convenient detection of LYCIV is an urgent demand for diagnosis. In this study, rapid and simple recombinase polymerase amplification (RPA), real-time RPA and RPA combined with lateral flow dipstick (RPA-LFD) methods were developed for the detection of LYCIV based on the conserved sequence of the LYCIV major capsid protein (MCP) gene. With these optimized RPA analyses, LYCIV detection could be completed within 20 min at 40°C. Both RPA and real-time RPA could detect viral DNA as low as 102 copies/µL, while the detection limit of RPA-LFD was 101 copies/µL, and there was no cross-reaction with other aquatic pathogens (KHV, CyHV-2, GCRV-JX01, SVCV, LCDV and LMBV). In practical evaluation of RPA, real-time RPA and RPA-LFD methods, the results showed consistency with the general PCR detection. In short, the developed RPA, real-time RPA and RPA-LFD analyses could be simple, rapid, sensitive and reliable methods for field diagnosis of LYCIV infection and have significant potential in the protection of LYCIV infection.

Rapid visual detection of Giardia duodenalis in faecal samples using an RPA-CRISPR/Cas12a system.

Giardiasis is a common intestinal infection caused by Giardia duodenalis, which is a major economic and health burden for humans and livestock. Currently, a convenient and effective detection method is urgently needed. CRISPR/Cas12a-based diagnostic methods have been widely used for nucleic acid-based detection of pathogens due to their high efficiency and sensitivity. In this study, a technique combining CRISPR/Cas12a and RPA was established that allows the detection of G. duodenalis in faecal samples by the naked eye with high sensitivity (10-1 copies/µL) and specificity (no cross-reactivity with nine common pathogens). In clinical evaluations, the RPA-CRISPR/Cas12a-based detection assay detected Giardia positivity in 2% (1/50) of human faecal samples and 47% (33/70) of cattle faecal samples, respectively, which was consistent with the results of nested PCR. Our study demonstrated that the RPA-CRISPR/Cas12a technique for G. duodenalis is stable, efficient, sensitive, specific and has low equipment requirements. This technique offers new opportunities for on-site detection in remote and poor areas.

RPA-CRISPR/Cas12a-mediated isothermal amplification for rapid detection of Phytopythium helicoides.

Phytopythium helicoides, which belongs to the algae (Chromista), Oomycota, Pythiales, Pythiaceae and Phytophthora, is a quarantine pathogen that causes brown rot of fruits, stem rot and root rot, along with other symptoms that can damage several tree species in urban landscaping. Therefore, disease management requires rapid and accurate diagnosis. The present study used recombinase polymerase amplification (RPA) in conjunction with the CRISPR/Cas12a system to identify P. helicoides. The test exhibited high specificity and sensitivity and could detect 10 pg.µL-1 of P. helicoides genomic DNA at 37 ℃ within 20 minutes. The test results were visible by excitation of fluorophores by blue light. This groundbreaking test is able to detect P. helicoides in artificially inoculated Rhododendron leaves. The RPA-CRISPR/Cas12a detection assay developed in this study is characterized by its sensitivity, efficiency, and convenience. Early detection and control of P. helicoides is crucial for the protection of urban green cover species.

Isothermal Amplification Using Temperature-Controlled Frequency Mixing Magnetic Detection-Based Portable Field-Testing Platform.

Sensitive magnetic nucleic acid (NA) detection via frequency mixing magnetic detection (FMMD) requires amplified NA samples for which a reliable temperature control is necessary. The feasibility of recombinase polymerase amplification (RPA) was studied within a newly integrated temperature-controlled sensor unit of a mobile FMMD based setup. It has been demonstrated that the inherently generated heat of the low frequency (LF) excitation signal of FMMD can be utilized and controlled by means of pulse width modulation (PWM). To test control performance in a point of care (PoC) setting with changing ambient conditions, a steady state and dynamic response model for the thermal behavior at the sample position of the sensor were developed. We confirmed that in the sensor unit of the FMMD device, RPA performs similar as in a temperature-controlled water bath. For narrow steady state temperature regions, a linear extrapolation suffices for estimation of the sample position temperature, based on the temperature feedback sensor for PWM control. For any other ambient conditions, we identified and validated a lumped parameter model (LPM) performing with high estimation accuracy. We expect that the method can be used for NA amplification and magnetic detection using FMMD in resource-limited settings.

Real-Time Monitoring on the Chinese Giant Salamander Using RPA-LFD.

The Chinese giant salamander (Andrias davidianus), listed as an endangered species under "secondary protection" in China, faces significant threats due to ecological deterioration and the expansion of human activity. Extensive field investigations are crucial to ascertain the current status in the wild and to implement effective habitat protection measures to safeguard this species and support its population development. Traditional survey methods often fall short due to the elusive nature of the A. davidianus, presenting challenges that are time-consuming and generally ineffective. To overcome these obstacles, this study developed a real-time monitoring method that uses environmental DNA (eDNA) coupled with recombinase polymerase amplification and lateral flow strip (RPA-LFD). We designed five sets of species-specific primers and probes based on mitochondrial genome sequence alignments of A. davidianus and its close relatives. Our results indicated that four of these primer/probe sets accurately identified A. davidianus, distinguishing it from other tested caudata species using both extracted DNA samples and water samples from a tank housing an individual. This method enables the specific detection of A. davidianus genomic DNA at concentrations as low as 0.1 ng/mL within 50 min, without requiring extensive laboratory equipment. Applied in a field survey across four sites in Huangshan City, Anhui Province, where A. davidianus is known to be distributed, the method successfully detected the species at three of the four sites. The development of these primer/probe sets offers a practical tool for field surveying and monitoring, facilitating efforts in population recovery and resource conservation for A. davidianus.

Recombinase Polymerase Amplification Combined with Lateral Flow Dipstick Assay for the Rapid and Sensitive Detection of Pseudo-nitzschia multiseries.

The harmful algal bloom (HAB) species Pseudo-nitzschia multiseries is widely distributed worldwide and is known to produce the neurotoxin domoic acid, which harms marine wildlife and humans. Early detection and preventative measures are more critical than late management. However, the major challenge related to early detection is the accurate and sensitive detection of microalgae present in low abundance. Therefore, developing a sensitive and specific method that can rapidly detect P. multiseries is critical for expediting the monitoring and prediction of HABs. In this study, a novel assay method, recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD), is first developed for the detection of P. multiseries. To obtain the best test results, several important factors that affected the amplification effect were optimized. The internal transcribed spacer sequence of the nuclear ribosomal DNA from P. multiseries was selected as the target region. The results showed that the optimal amplification temperature and time for the recombinase polymerase amplification (RPA) of P. multiseries were 37 °C and 15 min. The RPA products could be visualized directly using the lateral flow dipstick after only 3 min. The RPA-LFD assay sensitivity for detection of recombinant plasmid DNA (1.9 × 100 pg/µL) was 100 times more sensitive than that of RPA, and the RPA-LFD assay sensitivity for detection of genomic DNA (2.0 × 102 pg/µL) was 10 times more sensitive than that of RPA. Its feasibility in the detection of environmental samples was also verified. In conclusion, these results indicated that the RPA-LFD detection of P. multiseries that was established in this study has high efficiency, sensitivity, specificity, and practicability. Management measures made based on information gained from early detection methods may be able to prevent certain blooms. The use of a highly sensitive approach for early warning detection of P. multiseries is essential to alleviate the harmful impacts of HABs on the environment, aquaculture, and human health.

Comparative Evaluation of PCR-Based, LAMP and RPA-CRISPR/Cas12a Assays for the Rapid Detection of Diaporthe aspalathi.

Southern stem canker (SSC) of soybean, attributable to the fungal pathogen Diaporthe aspalathi, results in considerable losses of soybean in the field and has damaged production in several of the main soybean-producing countries worldwide. Early and precise identification of the causal pathogen is imperative for effective disease management. In this study, we performed an RPA-CRISPR/Cas12a, as well as LAMP, PCR and real-time PCR assays to verify and compare their sensitivity, specificity and simplicity and the practicality of the reactions. We screened crRNAs targeting a specific single-copy gene, and optimized the reagent concentrations, incubation temperatures and times for the conventional PCR, real-time PCR, LAMP, RPA and Cas12a cleavage stages for the detection of D. aspalathi. In comparison with the PCR-based assays, two thermostatic detection technologies, LAMP and RPA-CRISPR/Cas12a, led to higher specificity and sensitivity. The sensitivity of the LAMP assay could reach 0.01 ng µL-1 genomic DNA, and was 10 times more sensitive than real-time PCR (0.1 ng µL-1) and 100 times more sensitive than conventional PCR assay (1.0 ng µL-1); the reaction was completed within 1 h. The sensitivity of the RPA-CRISPR/Cas12a assay reached 0.1 ng µL-1 genomic DNA, and was 10 times more sensitive than conventional PCR (1.0 ng µL-1), with a 30 min reaction time. Furthermore, the feasibility of the two thermostatic methods was validated using infected soybean leaf and seeding samples. The rapid, visual one-pot detection assay developed could be operated by non-expert personnel without specialized equipment. This study provides a valuable diagnostic platform for the on-site detection of SSC or for use in resource-limited areas.

An Integrated Paper Microfluidic Device Based on Isothermal Amplification for Simple Sample-to-Answer Detection of Campylobacter jejuni.

Campylobacter jejuni is recognized as the most common species in the genus Campylobacter that causes foodborne diseases. The main reservoirs harboring C. jejuni are poultry products, which are associated with most illnesses, creating a demand for effective detection methods to achieve point-of-need diagnostics. We developed an easy-to-use, hybrid paper/polymer-based microfluidic device that integrates paper-based DNA extraction, isothermal nucleic acid amplification, and lateral flow detection. Overall, the recombinase polymerase amplification (RPA) reaction was completed in 20 min and demonstrated 100% specificity to C. jejuni, including 2 reference strains and 6 wild strains isolated from the agroecosystem, 9 other Campylobacter subspecies strains, and 11 non-Campylobacter strains. The limit of detection (LOD) was 46 CFU/mL with DNA extracted on the cellulose paper. The sensitivity was reduced to 460 CFU/mL on the integrated hybrid paper/polymer-based microfluidic device. This device could detect C. jejuni spiked at concentrations ranging from 101 to 102 CFU/g in chicken meat after an enrichment of 5 to 10 h. For C. jejuni levels of >102 CFU/g, it managed to confirm positive results immediately, without bacterial enrichment. RPA reagents and primers remained stable on the paper platform at 22°C for 12 h. After lyophilization and storage on paper, the RPA reaction showed consistent sensitivity for 3 days, and the LOD was reduced to 103 CFU/mL when storage was extended to 25 days. The use of this hybrid paper/polymer-based microfluidic device enabled detection of Campylobacter in foods with high specificity and sensitivity, demonstrating its potential as a reliable point-of-need diagnostic platform for on-site conditions due to its low cost, portability, and simplicity. IMPORTANCE The global health and economic burden of Campylobacter prompts the development of novel detection techniques that can be implemented in resource-limited and on-site settings. This study described point-of-need identification of C. jejuni using a hybrid paper/polymer-based microfluidic device that is easy to operate. This device had high specificity and sensitivity toward C. jejuni and significantly reduced the total analysis time compared to conventional culture-based methods. Nucleic acid extraction was simplified from intensive pipetting to a paper dipstick, making it more convenient for use in the field as a promising tool for future routine surveillance and outbreak investigation.

Establishment of RT-RPA-Cas12a assay for rapid and sensitive detection of human rhinovirus B.

Human rhinovirus B (HRV-B) is a major human viral pathogen that can be responsible for various kinds of infections. Due to the health risks associated with HRV-B, it is therefore crucial to explore a rapid, specific, and sensitive method for surveillance. Herein, we exploited a novel detection method for HRV-B by combining reverse-transcription recombinase polymerase amplification (RT-RPA) of nucleic acids isothermal amplification and the trans-cleavage activity of Cas12a. Our RT-RPA-Cas12a-based fluorescent assay can be completed within 35-45 min and obtain a lower detection threshold to 0.5 copies/µL of target RNA. Meanwhile, crRNA sequences without a specific protospacer adjacent motif can effectively activate the trans-cleavage activity of Cas12a. Moreover, our RT-RPA-Cas12a-based fluorescent method was examined using 30 clinical samples, and exhibited high accuracy with positive and negative predictive agreement of 90% and 100%, respectively. Taken together, a novel promising, rapid and effective RT-RPA-Cas12a-based detection method was explored and shows promising potential for on-site HRV-B infection in resource-limited settings.

CRISPR-Cas12-based field-deployable system for rapid detection of synthetic DNA sequence of the monkeypox virus genome.

The global outbreak of the monkeypox virus (MPXV) highlights the need for rapid and cost-effective MPXV detection tools to effectively monitor and control the monkeypox disease. Herein, we demonstrated a portable CRISPR-Cas-based system for naked-eye detection of MPXV. The system harnesses the high selectivity of CRISPR-Cas12 and the isothermal nucleic acid amplification potential of recombinase polymerase amplification. It can detect both the current circulating MPXV clade and the original clades. We reached a limit of detection (LoD) of 22.4 aM (13.5 copies/µl) using a microtiter plate reader, while the visual LoD of the system is 75 aM (45 copies/µl) in a two-step assay, which is further reduced to 25 aM (15 copies/µl) in a one-pot system. We compared our results with quantitative polymerase chain reaction and obtained satisfactory consistency. For clinical application, we demonstrated a sensitive and precise visual detection method with attomolar sensitivity and a sample-to-answer time of 35 min.

Creating an ultra-sensitive detection platform for monkeypox virus DNA based on CRISPR technology.

The recent major worldwide outbreak of monkeypox virus (MPXV) has highlighted the urgent need for accurate MPXV detection methods. Although quantitative PCR (qPCR) technique is currently the gold standard for MPXV diagnosis, the high costs associated with the technique and the need for complex instrumentation, limits its application in resource-poor settings. CRISPR technology has developed rapidly in recent years and provides an effective tool for point-of-care testing pathogen identification. Here, we exploited the cleavage properties of the Cas12a enzyme and Cas13a enzyme, to detect the MPXV specific genes, F3L gene and B6R gene, respectively. We developed two detection protocols: a 2-step method in which the CRISPR Dual System reaction and the multiplex recombinase polymerase amplification reaction were carried out in separate tubes and a single-tube method in which both reactions were carried out in one tube. Evaluation of the two methods showed that our protocol can detect the MPXV genome down to 10° copies/µL with good specificity and no cross-reactivity with other poxviruses pseudoviruses, and bacteria. Mock positive samples were used to assess clinical applicability, with the results showing satisfactory concordance with the qPCR method for parallel testing. In conclusion, our study provides a reliable molecular diagnostic strategy for detection of MPXV.