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.

Development and application of a novel recombinase polymerase amplification-Pyrococcus furiosus argonaute system for rapid detection of goose parvovirus.

Rapid and accurate detection of goose parvovirus (GPV) is crucial for controlling outbreaks and mitigating their economic impact on the poultry industry. This study introduces recombinase polymerase amplification combined with the Pyrococcus furiosus argonaute (RPA-PfAgo) system, a novel diagnostic platform designed to address the limitations of traditional GPV detection methods. Capitalizing on the rapid DNA amplification of RPA and stringent nucleic acid cleavage by the PfAgo protein, the RPA-PfAgo system offers high specificity and sensitivity in detecting GPV. Our optimization efforts included primer and probe configurations, reaction parameters, and guided DNA selection, culminating in a detection threshold of 102 GPV DNA copies per microlitre. The specificity of the proposed method was rigorously validated against a spectrum of avian pathogens. Clinical application to lung tissues from GPV-infected geese yielded a detection concordance of 100%, surpassing that of qPCR and PCR in both rapidity and operational simplicity. The RPA-PfAgo system has emerged as a revolutionary diagnostic modality for managing this disease, as it is a promising rapid, economical, and onsite GPV detection method amenable to integration into broad-scale disease surveillance frameworks. Future explorations will extend the applicability of this method to diverse avian diseases and assess its field utility across various epidemiological landscapes.

Capillarity-powered and CRISPR/Cas12a-responsive DNA hydrogel distance sensor for highly sensitive visual detection of HPV DNA.

The rapid and specific identification and sensitive detection of human papillomavirus (HPV) infection is critical for preventing cervical cancer, particularly in resource-limited regions. In this work, we hope to propose a capillarity-powered and CRISPR/Cas12a-responsive DNA hydrogel distance sensor for point-of-care (POC) DNA testing. Using the thermal reversibility of DNA hydrogel and capillarity, the novel DNA hydrogel distance sensor can be rapidly and simply constructed by loading an ultra-thin CRISPR/Cas12a-responsive DNA-crosslinked hydrogel film at the end of the capillary tube. The target DNA-specific recombinase polymerase reaction (RPA) amplicons activate the trans-cleavage activity of the Cas12a enzyme, cleaving the crosslinked DNA in hydrogel film, and causing an increase of hydrogel's permeability. As a result, a sample solution containing target DNA travels into the capillary tube at a longer distance compared to the negative samples. Reading the solution traveling distance in capillary tubes, the novel sensor realizes target DNA detection without any special equipment. Benefiting from the exponential target amplification of RPA and multiple turnover response of trans-cleavage of CRISPR/Cas12a, the developed sensor can visually and specifically detect as low as 1 aM HPV 16 DNA within 30 min. These outstanding features, including exceptional sensitivity and specificity, simple and portable design, mild measurement conditions, quick turnaround time, and user-friendly read-out, make the novel distance sensor a promising option for POC diagnostic applications.

RT-RPA-PfAgo detection platform for one-tube simultaneous typing diagnosis of human respiratory syncytial virus.

Human respiratory syncytial virus (HRSV) is the most prevalent pathogen contributing to acute respiratory tract infections (ARTI) in infants and young children and can lead to significant financial and medical costs. Here, we developed a simultaneous, dual-gene and ultrasensitive detection system for typing HRSV within 60 minutes that needs only minimum laboratory support. Briefly, multiplex integrating reverse transcription-recombinase polymerase amplification (RT-RPA) was performed with viral RNA extracted from nasopharyngeal swabs as a template for the amplification of the specific regions of subtypes A (HRSVA) and B (HRSVB) of HRSV. Next, the Pyrococcus furiosus Argonaute (PfAgo) protein utilizes small 5'-phosphorylated DNA guides to cleave target sequences and produce fluorophore signals (FAM and ROX). Compared with the traditional gold standard (RT-qPCR) and direct immunofluorescence assay (DFA), this method has the additional advantages of easy operation, efficiency and sensitivity, with a limit of detection (LOD) of 1 copy/µL. In terms of clinical sample validation, the diagnostic accuracy of the method for determining the HRSVA and HRSVB infection was greater than 95%. This technique provides a reliable point-of-care (POC) testing for the diagnosis of HRSV-induced ARTI in children and for outbreak management, especially in resource-limited settings.

RT-RPA Assay Combined with a Lateral Flow Strip to Detect Soybean Mosaic Virus.

Soybean (Glycine max L.) is one of the most widely planted and used legumes in the world, being used for food, animal feed products, and industrial production. The soybean mosaic virus (SMV) is the most prevalent virus infecting soybean plants. This study developed a diagnostic method for the rapid and sensitive detection of SMV using a reverse transcription-recombinase polymerase amplification (RT-RPA) technique combined with a lateral flow strip (LFS). The RT-RPA and RT-RPA-LFS conditions to detect the SMV were optimized using the selected primer set that amplified part of the VPg protein gene. The optimized reaction temperature for the RT-RPA primer and RT-RPA-LFS primer used in this study was 38℃ for both, and the minimum reaction time was 10 min and 5 min, respectively. The RT-RPA-LFS was as sensitive as RT-PCR to detect SMV with 10 pg/µl of total RNA. The reliability of the developed RT-RPA-LFS assay was evaluated using leaves collected from soybean fields. The RT-RPA-LFS diagnostic method developed in this study will be useful as a diagnostic method that can quickly and precisely detect SMV in the epidemiological investigation of SMV, in the selection process of SMV-resistant varieties, on local farms with limited resources.

RPA-CRISPR-Cas13a-assisted detection method of transmissible gastroenteritis virus.

Background and aim: Transmissible gastroenteritis virus (TGEV) is a highly contagious gastrointestinal virus that causes diarrhea, vomiting, anorexia, dehydration, and weight loss in piglets. In clinical practice, it often occurs in mixed infections with other pathogens, and is therefore difficult to diagnose and prevent. It mainly harms piglets of about 2 weeks old, causing huge losses on farms. The clinical confirmation of TGEV usually requires a laboratory diagnosis, but traditional PCR and immunofluorescence assays have some limitations. Moreover, most farms in China are ill-equipped to accurately diagnose the disease. Therefore, a new detection method with high sensitivity and specificity and less dependence on instrumentation is required. Methods: We used recombinase polymerase amplification (RPA), combined with the nuclease characteristics of the activated Cas13a protein to establish a visual CRISPR-Cas13a-assisted detection method for TGEV by adding a reporter RNA with fluorescent and quenching moieties to the system. Result: We selected the optimal RPA primer and best CRISPR RNA (crRNA). The reaction system was optimized and its repeatability, specificity, and sensitivity verified. The TGEV detection system did not cross-react with other common diarrhea viruses, and its detection limit was 101 copies, which is similar with the sensitivity of qPCR. We successfully established an RPA-CRISPR-Cas13a-assisted detection method, and used this detection system to analyze 123 pig blood samples. qPCR was used as the gold standard method. The sensitivity, specificity, positive coincidence rate, and negative coincidence rate of the new method were 100, 98.93, 96.66, and 100%, respectively.

Point-of-care isothermal nucleic acid amplification tests: progress and bottlenecks for extraction-free sample collection and preparation.

INTRODUCTION: Suitable sample collection and preparation methods are essential to enable nucleic acid amplification testing at the point of care (POC). Strategies that allow direct isothermal nucleic acid amplification testing (iNAAT) of crude sample lysate without the need for nucleic acid extraction minimize time to result as well as the need for operator expertise and costly infrastructure. AREAS COVERED: The authors review research to understand how sample matrix and preparation affect the design and performance of POC iNAATs. They focus on approaches where samples are directly combined with liquid reagents for preparation and amplification via iNAAT strategies. They review factors related to the type and method of sample collection, storage buffers, and lysis strategies. Finally, they discuss RNA targets and relevant regulatory considerations. EXPERT OPINION: Limitations in sample preparation methods are a significant technical barrier preventing implementation of nucleic acid testing at the POC. The authors propose a framework for co-designing sample preparation and amplification steps for optimal performance with an extraction-free paradigm by considering a sample matrix and lytic strategy prior to an amplification assay and readout. In the next 5 years, the authors anticipate increasing priority on the co-design of sample preparation and iNAATs.

Recombinase polymerase amplification combined with lateral flow biosensor for rapid visual detection of Clostridium perfringens in chicken meat and milk.

Aims: Clostridium perfringens is one of the major anaerobic pathogen causing food poisoning and animal enteritis. With the rise of antibiotic resistance and the restrictions of the use of antibiotic growth promoting agents (AGPs) in farming, Clostridium enteritis and food contamination have become more common. It is time-consuming and labor-intensive to confirm the detection by standard culture methods, and it is necessary to develop on-site rapid detection tools. In this study, a combination of recombinase polymerase amplification (RPA) and lateral flow biosensor (LFB) was used to visually detect C. perfringens in chicken meat and milk. Methods and results: Two sets of primers were designed for the plc gene of C. perfringens, and the amplification efficiency and specificity of the primers. Selection of primers produces an amplified fragment on which the probe is designed. The probe was combined with the lateral flow biosensor (LFB). The reaction time and temperature of RPA-LFB assay were optimized, and the sensitivity of the assay was assessed. Several common foodborne pathogens were selected to test the specificity of the established method. Chicken and milk samples were artificially inoculated with different concentrations (1 × 102 CFU/mL to 1 × 106 CFU/mL) of C. perfringens, and the detection efficiency of RPA-LFB method and PCR method was compared. RPA-LFB can be completed in 20 min and the results can be read visually by the LFB test strips. The RPA-LFB has acceptable specificity and the lowest detection limit of 100 pg./µL for nucleic acid samples. It was able to stably detect C. perfringens contamination in chicken and milk at the lowest concentration of 1 × 104 CFU/mL and 1 × 103 CFU/mL, respectively. Conclusion: In conclusion, RPA-LFB is specific and sensitive. It is a rapid, simple and easy-to-visualize method for the detection of C. perfringens in food and is suitable for use in field testing work.

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.

Specific detection of gut pathogens for one-pot chip based on RPA-CRISPR/Cas12a.

BACKGROUND: There are billions of bacteria in the intestine, most of which are harmless and play important roles in humans. Although only a very small number of bacteria can cause diseases, once the pathogenic bacteria are ingested into the body and multiply in large quantities, it can lead to inflammatory diseases in the intestines and even other organs. Although polymerase chain reaction can specifically detect bacterial nucleic acid. However, the demand for temperature cycling limits its portability. Therefore, it is hoped to establish a high-throughput, highly specific and portable detection platform for directly detecting nucleic acid of intestinal pathogens. RESULTS: Herein, a one-pot chip based on RPA-CRCISPR/Cas12a platform was developed. The chip is the same size as a glass slide and allows detection at the same temperature. Multiple samples could be detected simultaneously on the one chip, achieved high-throughput detection and improved the integration of detection. The specific recognition of CRISPR/Cas12a avoided the influence of non-specific amplification of RPA and enhanced the specificity of the analysis. At the same time, the one-pot chip avoided secondary contamination when the lid was opened during the analysis process. And the bacterial concentration showed good linearity at 102-108 cfu mL-1. The limit of detection could be as low as 0.43 cfu mL-1. This method has been successfully used to detect pollution samples. It can provide a reliable platform for early screening of gastrointestinal and other inflammatory diseases. SIGNIFICANCE: The one-pot chip based on the RPA-CRISPR/Cas12a platform established can directly detect the nucleic acid of intestinal pathogens, with portability and specificity. It is worth noting that the platform has good programmability, can be used for other target detection by changing crRNA and RPA primers, it can achieve multi sample detection on the one chip.

Sensitive and rapid detection of three foodborne pathogens in meat by recombinase polymerase amplification with lateral flow dipstick (RPA-LFD).

Foodborne illnesses, caused by harmful microorganisms in food, are a significant global health issue. Current methods for identifying these pathogens are both labor-intensive and time-consuming. In this research, we devised a swift and precise detection technique using recombinase polymerase amplification combined with a lateral flow dipstick (RPA-LFD) for three foodborne pathogens found in meat. By employing a dedicated detection device, RPA-LFD allows for the rapid analysis of DNA from Escherichia coli O157 (E. coli O157), Salmonella, and Shigella-pathogens that are prohibited in food. The detection thresholds for E. coli O157, Salmonella, and Shigella are 0.168 fg/µl (1.04 CFU/ml), 0.72 fg/µl (27.49 CFU/ml), and 1.25 fg/µl (48.84 CFU/ml), respectively. This method provides a short detection window, operates at low temperatures, follows simple procedures, and exhibits high sensitivity. Our study establishes the RPA-LFD method for simultaneously identifying the nucleic acid of three foodborne pathogens, offering an efficient solution for quickly identifying multiple contaminants.

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.

A rapid and ultra-sensitive dual readout platform for Klebsiella pneumoniae detection based on RPA-CRISPR/Cas12a.

The bacterium Klebsiella pneumoniae (Kp) was the primary pathogen of hospital-acquired infection, but the current detection method could not rapidly and conveniently identify Kp. Recombinase polymerase amplification (RPA) was a fast and convenient isothermal amplification technology, and the clustered regularly interspaced short palindromic repeats (CRISPR) system could rapidly amplify the signal of RPA and improve its limit of detection (LOD). In this study, we designed three pairs of RPA primers for the rcsA gene of Kp, amplified the RPA signal through single-strand DNA reporter cleavage by CRISPR/Cas12a, and finally analyzed the cleavage signal using fluorescence detection (FD) and lateral flow test strips (LFTS). Our results indicated that the RPA-CRISPR/Cas12a platform could specifically identify Kp from eleven common clinical pathogens. The LOD of FD and LFTS were 1 fg/µL and 10 fg/µL, respectively. In clinical sample testing, the RPA-CRISPR/Cas12a platform was consistent with the culture method and qPCR method, and its sensitivity and specificity were 100% (16/16) and 100% (9/9), respectively. With the advantages of detection speed, simplicity, and accuracy, the RPA-CRISPR/Cas12a platform was expected to be a convenient tool for the early clinical detection of Kp.

Development of a novel Cas13a/Cas12a-mediated 'one-pot' dual detection assay for genetically modified crops.

INTRODUCTION: Genetically modified (GM) crops have been widely cultivated across the world and the development of rapid, ultrasensitive, visual multiplex detection platforms that are suitable for field deployment is critical for GM organism regulation. OBJECTIVE: In this study, we developed a novel one-pot system, termed MR-DCA (Multiplex RPA and Dual CRISPR assay), for the simultaneous detection of CaMV35S and NOS genetic targets in GM crops. This innovative approach combined Multiplex RPA (recombinase polymerase amplification) with the Dual CRISPR (clustered regularly interspaced short palindromic repeat) assay technique, to provide a streamlined and efficient method for GM crop detection. METHODS: The RPA reaction used for amplification CaMV35S and NOS targets was contained in the tube base, while the dual CRISPR enzymes were placed in the tube cap. Following centrifugation, the dual CRISPR (Cas13a/Cas12a) detection system was initiated. Fluorescence visualization was used to measure CaMV35S through the FAM channel and NOS through the HEX channel. When using lateral flow strips, CaMV35S was detected using rabbit anti-digoxin (blue line), whilst NOS was identified using anti-mouse FITC (red line). Line intensity was quantified using Image J and depicted graphically. RESULTS: Detection of the targets was completed in 35 min, with a limit of detection as low as 20 copies. In addition, two analysis systems were developed and they performed well in the MR-DCA assay. In an analysis of 24 blind samples from GM crops with a wide genomic range, MR-DCA gave consistent results with the quantitative PCR method, which indicated high accuracy, applicability and semi-quantitative ability. CONCLUSION: The development of MR-DCA represents a significant advancement in the field of GM detection, offering a rapid, sensitive and portable method for multiple target detection that can be used in resource-limited environments.

Refining the Schistosoma haematobium recombinase polymerase amplification (Sh-RPA) assay: moving towards point-of-care use in endemic settings.

BACKGROUND: Urogenital schistosomiasis is caused by the parasitic trematode Schistosoma haematobium. Sensitive and specific point-of-care diagnostics are needed for elimination of this disease. Recombinase polymerase amplification (RPA) assays meet these criteria, and an assay to diagnose S. haematobium has been developed (Sh-RPA). However, false-positive results can occur, and optimisation of reaction conditions to mitigate these is needed. Ease of use and compatibility of DNA extraction methods must also be considered. METHODS: Using synthetic DNA, S. haematobium genomic DNA (gDNA), and urine samples from clinical cases, Sh-RPA reactions incorporating different betaine concentrations (0 M, 1 M, 2.5 M, 12.5 M) and the sample-to-water ratios were tested to determine effects on assay specificity and sensitivity. In addition, five commercial DNA extraction kits suitable for use in resource-limited settings were used to obtain gDNA from single S. haematobium eggs and evaluated in terms of DNA quality, quantity, and compatibility with the Sh-RPA assay. All samples were also evaluated by quantitative polymerase chain reaction (qPCR) to confirm DNA acquisition. RESULTS: The analytical sensitivity of the Sh-RPA with all betaine concentrations was ≥ 10 copies of the synthetic Dra1 standard and 0.1 pg of S. haematobium gDNA. The addition of betaine improved Sh-RPA assay specificity in all reaction conditions, and the addition of 2.5 M of betaine together with the maximal possible sample volume of 12.7 µl proved to be the optimum reaction conditions. DNA was successfully isolated from a single S. haematobium egg using all five commercial DNA extraction kits, but the Sh-RPA performance of these kits varied, with one proving to be incompatible with RPA reactions. CONCLUSIONS: The addition of 2.5 M of betaine to Sh-RPA reactions improved reaction specificity whilst having no detrimental effect on sensitivity. This increases the robustness of the assay, advancing the feasibility of using the Sh-RPA assay in resource-limited settings. The testing of commercial extraction kits proved that crude, rapid, and simple methods are sufficient for obtaining DNA from single S. haematobium eggs, and that these extracts can be used with Sh-RPA in most cases. However, the observed incompatibility of specific kits with Sh-RPA highlights the need for each stage of a molecular diagnostic platform to be robustly tested prior to implementation.

Rapid and one-tube detection of human metapneumovirus using the RT-RPA and CRISPR/Cas12a.

Human metapneumovirus (HMPV) is a common pathogen that can cause acute respiratory tract infections and is prevalent worldwide. There is yet no effective vaccine or specific treatment for HMPV. Early, rapid, and accurate detection is essential to treat the disease and control the spread of infection. In this study, we created the One-tube assay by combining Reverse Transcription-Recombinase Polymerase Amplification (RT-RPA) with the CRISPR/Cas12a system. By targeting the nucleoprotein (N) gene of HMPV to design specific primers and CRISPR RNAs (crRNAs), combining RT-RPA and CRISPR/Cas12a, established the One-tube assay. Meanwhile, the reaction conditions of the One-tube assay were optimized to achieve rapid and visual detection of HMPV. This assay could detect HMPV at 1 copy/µL in 30 min, without cross-reactivity with nine other respiratory pathogens. We validated the detection performance using clinical specimens and showed that the coincidence rate was 98.53 %，compared to the quantitative reverse-transcription polymerase chain reaction. The One-tube assay reduced the detection time and simplified the manual operation, while maintaining the detection performance and providing a new platform for HMPV detection.

Cas12a/Guide RNA-Based Platform for Rapidly and Accurately Detecting blaKPC Gene in Carbapenem-Resistant Enterobacterales.

Purpose: Accurate detection and identification of pathogens and their associated resistance mechanisms are essential prerequisites for implementing precision medicine in the management of Carbapenem-resistant Enterobacterales (CRE). Among the various resistance mechanisms, the production of KPC carbapenemase is the most prevalent worldwide. Consequently, this study aims to develop a convenient and precise nucleic acid detection platform specifically for the blaKPC gene. Methods: The initial phase of our research methodology involved developing a CRISPR/Cas12a detection framework, which was achieved by designing highly specific single-guide RNAs (sgRNAs) targeting the blaKPC gene. To enhance the sensitivity of this system, we incorporated three distinct amplification techniques-polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and recombinase polymerase amplification (RPA)-into the CRISPR/Cas12a framework. Subsequently, we conducted a comparative analysis of the sensitivity and specificity of these three amplification methods when used in combination with the CRISPR/Cas12a system. Additionally, we assessed the clinical applicability of the methodologies by evaluating fluorescence readouts from 80 different clinical isolates. Furthermore, we employed lateral flow assay technology to provide a visual representation of the results, facilitating point-of-care testing. Results: Following a comparative analysis of the sensitivity and specificity of the three methods, we identified the RPA-Cas12a approach as the optimal detection technique. Our findings demonstrated that the limit of detection (LoD) of the RPA-Cas12a platform was 1 aM (~1 copy/µL) for plasmid DNA and 5 × 10³ fg/µL for genomic DNA. Furthermore, both the sensitivity and specificity of the platform achieved 100% upon validation with 80 clinical isolates. Conclusion: These findings suggest that the developed RPA-Cas12a platform represents a promising tool for the cost-effective, convenient, and accurate detection of the blaKPC gene.

Cas-OPRAD: a one-pot RPA/PCR CRISPR/Cas12 assay for on-site Phytophthora root rot detection.

Phytophthora sojae is a devastating plant pathogen that causes soybean Phytophthora root rot worldwide. Early on-site and accurate detection of the causal pathogen is critical for successful management. In this study, we have developed a novel and specific one-pot RPA/PCR-CRISPR/Cas12 assay for on-site detection (Cas-OPRAD) of Phytophthora root rot (P. sojae). Compared to the traditional RPA/PCR detection methods, the Cas-OPRAD assay has significant detection performance. The Cas-OPRAD platform has excellent specificity to distinguish 33 P. sojae from closely related oomycetes or fungal species. The PCR-Cas12a assay had a consistent detection limit of 100 pg. µL-1, while the RPA-Cas12a assay achieved a detection limit of 10 pg. µL-1. Furthermore, the Cas-OPRAD assay was equipped with a lateral flow assay for on-site diagnosis and enabled the visual detection of P. sojae on the infected field soybean samples. This assay provides a simple, efficient, rapid (<1 h), and visual detection platform for diagnosing Phytophthora root rot based on the one-pot CRISPR/Cas12a assay. Our work provides important methods for early and accurate on-site detection of Phytophthora root rot in the field or customs fields.

Establishment of a Nucleic Acid Detection Method for Norovirus GII.2 Genotype Based on RT-RPA and CRISPR/Cas12a-LFS.

To establish a rapid detection method for norovirus GII.2 genotype, this study employed reverse transcription recombinase polymerase amplification (RT-RPA) combined with CRISPR/Cas12a and lateral flow strip (RT-RPA-Cas12a-LFS). Here, the genome of norovirus GII.2 genotype was compared to identify highly conserved sequences, facilitating the design of RT-RPA primers and crRNA specific to the conserved regions of norovirus GII.2. Subsequently, the reaction parameters of RT-RPA were optimized and evaluated using agar-gel electrophoresis and LFS. The results indicate that the conserved sequences of norovirus GII.2 were successfully amplified through RT-RPA at 37°C for 25 minutes. Additionally, CRISPR/Cas12a-mediated cleavage detection was achieved through LFS at 37°C within 10 minutes using the amplification products as templates. Including the isothermal amplification reaction time, the total time is 35 minutes. The established RT-RPA-Cas12a-LFS method demonstrated specific detection of norovirus GII.2, yielding negative results for other viral genomes, and exhibited an excellent detection limit of 10 copies/µl. The RT-RPA-Cas12a-LFS method was further compared with qRT-PCR by analyzing 60 food-contaminated samples. The positive conformity rate was 100%, the negative conformity rate was 95.45%, and the overall conformity rate reached 98.33%. This detection method for norovirus GII.2 genotype is cost-effective, highly sensitive, specific, and easy to operate, offering a promising technical solution for field-based detection of the norovirus GII.2 genotype.

An Automated Versatile Diagnostic Workflow for Infectious Disease Detection in Low-Resource Settings.

Laboratory automation effectively increases the throughput in sample analysis, reduces human errors in sample processing, as well as simplifies and accelerates the overall logistics. Automating diagnostic testing workflows in peripheral laboratories and also in near-patient settings -like hospitals, clinics and epidemic control checkpoints- is advantageous for the simultaneous processing of multiple samples to provide rapid results to patients, minimize the possibility of contamination or error during sample handling or transport, and increase efficiency. However, most automation platforms are expensive and are not easily adaptable to new protocols. Here, we address the need for a versatile, easy-to-use, rapid and reliable diagnostic testing workflow by combining open-source modular automation (Opentrons) and automation-compatible molecular biology protocols, easily adaptable to a workflow for infectious diseases diagnosis by detection on paper-based diagnostics. We demonstrated the feasibility of automation of the method with a low-cost Neisseria meningitidis diagnostic test that utilizes magnetic beads for pathogen DNA isolation, isothermal amplification, and detection on a paper-based microarray. In summary, we integrated open-source modular automation with adaptable molecular biology protocols, which was also faster and cheaper to perform in an automated than in a manual way. This enables a versatile diagnostic workflow for infectious diseases and we demonstrated this through a low-cost N. meningitidis test on paper-based microarrays.