Reverse Transcription Recombinase Polymerase Amplification Research Articles

Establishment of a reverse transcription–recombinase polymerase amplification–lateral flow dipstick method for the dual detection of Israeli acute paralysis virus and chronic bee paralysis virus

IntroductionAs an important social insect, honey bees play crucial roles in agricultural production, sustainable development of agricultural production, and the balance of the natural environment. However, in recent years, Israeli acute paralysis virus (IAPV) and chronic bee paralysis virus (CBPV), the main pathogens of bee paralysis, have continuously harmed bee colonies and caused certain losses to the beekeeping industry. Some beekeeping farms are located in wild or remote mountainous areas, and samples from these farms cannot be sent to the laboratory for testing in a timely manner, thereby limiting the accurate and rapid diagnosis of the disease.Methods and resultsIn this study, we used a reverse transcription–recombinase polymerase amplification–lateral flow dipstick (RT–RPA–LFD) method for the dual detection of IAPV and CBPV. RPA primers and LFD detection probes were designed separately for their conserved genes. Primers and probes were screened, and the forward and reverse primer ratios, reaction times, and temperatures were optimized. According to the results of the optimization tests, the optimal reaction temperature for RT–RPA is 37°C, and when combined with LFD, detection with the naked eye requires <20 min. The developed RPA–LFD method specifically targets IAPV and CBPV and has no cross-reactivity with other common bee viruses. In addition, the minimum detection limit of the RT–RPA–LFD method is 101 copies/μL.ConclusionBased this study, this method is suitable for the detection of clinical samples and can be used for field detection of IAPV and CBPV.

Improving trans-cleavage activity of CRISPR-Cas13a using engineered crRNA with a uridinylate-rich 5′-overhang

The engieering of Cas13a crRNA to enhance its binding affinity with the Cas enzyme or target is a promising method of improving the collateral cleavage efficiency of CRISPR-Cas13a systems, thereby amplifying the sensitivity of nucleic acid detection. An examination of the top-performing engineered crRNA (24 nt 5′7U LbuCas13a crRNA, where the 5′-end was extended using 7-mer uridinylates) and optimized conditions revealed an increased rate of LbuCas13a-mediated collateral cleavage activity that was up to seven-fold higher than that of the original crRNA. Particularly, the 7-mer uridinylates extension to crRNA was determined to be spacer-independent for enhancing the LbuCas13a-mediacted collateral cleavage activity, and also benefited the LwaCas13a system. The improved trans-cleavage activity was explained by the interactions between crRNA and LbuCas13a at the molecular level, i.e. the 5′-overhangs were anchored in the cleft formed between the Helical-1 and HEPN2 domains with the consequence of more stable complex, and experimentally verified. Consequently, the improved CRISPR-Cas13a system detected the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA with a sensitivity of 2.36 fM that was 160-times higher than that of the original system. Using isothermal amplification via reverse transcription–recombinase polymerase amplification (RT-RPA), the system was capable to detect SARS-CoV-2 with attomolar sensitivity and accurately identified the SARS-CoV-2 Omicron variant (20/21 agreement) in clinical samples within 40 min.

Identification of Viruses Infecting Phalaenopsis Orchids Using Nanopore Sequencing and Development of an RT-RPA-CRISPR/Cas12a for Rapid Visual Detection of Nerine Latent Virus.

Phalaenopsis orchids are one of the most popular ornamental plants. More than thirty orchid viruses have been reported, and virus-infected Phalaenopsis orchids significantly lose their commercial value. Therefore, the development of improved viral disease detection methods could be useful for quality control in orchid cultivation. In this study, we first utilized the MinION, a portable sequencing device based on Oxford Nanopore Technologies (ONT) to rapidly detect plant viruses in Phalaenopsis orchids. Nanopore sequencing revealed the presence of three plant viruses in Phalaenopsis orchids: odontoglossum ringspot virus, cymbidium mosaic virus, and nerine latent virus (NeLV). Furthermore, for the first time, we detected NeLV infection in Phalaenopsis orchids using nanopore sequencing and developed the reverse transcription-recombinase polymerase amplification (RT-RPA)-CRISPR/Cas12a method for rapid, instrument-flexible, and accurate diagnosis. The developed RT-RPA-CRISPR/Cas12a technique can confirm NeLV infection in less than 20 min and exhibits no cross-reactivity with other viruses. To determine the sensitivity of RT-RPA-CRISPR/Cas12a for NeLV, we compared it with RT-PCR using serially diluted transcripts and found a detection limit of 10 zg/μL, which is approximately 1000-fold more sensitive. Taken together, the ONT platform offers an efficient strategy for monitoring plant viral pathogens, and the RT-RPA-CRISPR/Cas12a method has great potential as a useful tool for the rapid and sensitive diagnosis of NeLV.

Development of Simplified Recombinase Polymerase Amplification Assay for Rapid and Robust Detection of Citrus Yellow Vein Clearing Virus.

Citrus is an economically important fruit crop, belongs to family Rutaceae, cultivated commercially in over 130 countries, which holds a leading profitable position in the international market. The most important citrus varieties are mandarins, oranges, lemons, sweet limes, grapefruits and pomelos. Citrus yellow vein clearing virus (CYVCV) is an important graft transmissible plant pathogen known to reduce productivity of citrus fruits due to its predominant association and widespread occurrence. Requirement of fast, reliable, efficient & economical CYVCV indexing assay is a prerequisite for production of healthy planting material. Currently, nucleic acid isolation and thermal cycler-based assay available for CYVCV indexing is a cumbersome lab intensive method. The present study was undertaken to develop and validate reverse transcription-recombinase polymerase amplification (RT-RPA) assay requiring no tedious RNA isolation, separate cDNA synthesis and costlier instrument like thermo-cycler. Optimized RT-RPA assay was able to amplify CYVCV up to 10-7 dilution (equivalent to 0.1pg/μl) with the prepared templates of both RNA and crude saps and showed higher sensitivity in detection of CYVCV infection in field samples as compared to the conventional RT-PCR. Developed RT-RPA assay showed high specificity without any cross-reaction with other citrus pathogens (Indian citrus ringspot virus, citrus yellow mosaic virus, citrus tristeza virus, citrus exocortis viroid and huanglongbing). RT-RPA using crude leaf sap as template is quite simple, robust, highly sensitive, time and cost effective; therefore, it can be used in resource constrained laboratories as screening tool, for field surveys and on-site testing programs in farms, nurseries and biosecurity. Present study, first time reports the development, optimization and validation of crude sap-based RT-RPA assay for the detection of CYVCV infection in citrus plants namely; Kinnow mandarin, Mosambi and Grape fruit.

Ultrasensitive Detection Strategy of Norovirus Based on a Dual Enhancement Strategy: CRISPR-Responsive Self-Assembled SNA and Isothermal Amplification.

Spherical nucleic acids (SNAs) have been used to construct various nanobiosensors with gold nanoparticles (AuNPs) as nuclei. The SNAs play a critical role in biosensing due to their various physical and chemical properties, programmability, and specificity recognition ability. In this study, CRISPR-responsive self-assembled spherical nucleic acid (CRISPR-rsSNA) detection probes were constructed by conjugating fluorescein-labeled probes to the surface of AuNPs to improve the sensing performance. Also, the mechanism of ssDNA and the role of different fluorescent groups in the self-assembly process of CRISPR-rsSNA were explored. Then, CRISPR-rsSNA and reverse transcription-recombinase polymerase amplification (RT-RPA) were combined to develop an ultrasensitive fluorescence-detection strategy for norovirus. In the presence of the virus, the target RNA sequence of the virus was transformed and amplified by RT-RPA. The resulting dsDNA activated the trans-cleavage activity of CRISPR cas12a, resulting in disintegrating the outer nucleic acid structure of the CRISPR-rsSNA at a diffusible rate, which released reporter molecules. Norovirus was quantitated by fluorescence detection. This strategy facilitated the detection of the norovirus at the attomolar level. An RT-RPA kit for norovirus detected would be developed based on this method. The proposed method would be used for the detection of different viruses just by changing the target RNA and crRNA of the CRISPR cas12a system which provided a foundation for high-throughput detection of various substances.

Rapid detection of porcine sapelovirus by reverse transcription recombinase polymerase amplification assay.

Porcine Sapelovirus (PSV) infection has been confirmed in pigs worldwide, mostly asymptomatic, but in some cases, it can lead to significant issues in the gastrointestinal, respiratory, neurological, or reproductive systems. PSV is considered an emerging pathogen of porcine species. Recombinase polymerase amplification (RPA) is a simple and fast isothermal technique that uses three enzymes for amplification without the use of any sophisticated equipment. The reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed and optimized for field based detection of PSV. The assay was developed by targeting 5´UTR region of PSV genome and optimized for reaction time, temperature, primer and MgOAc concentration. The analytical sensitivity and specificity of assay was determined. The assay was evaluated on 85 porcine faecal samples collected from field. In addition to conventional format, this assay was also optimized for visual dye-based detection format and lateral flow strips based detection (in combination with probe). The developed assay works at constant temperature of 35°C for 20min with forward primer concentration 20pm, reverse primer concentration 10pm and MgOAc concentration of 14mM. This assay is highly sensitive and detects up to 28 copies of viral nucleic acid both in the conventional as well as in fluorescent dye based detection format. Using the newly developed assay 21 samples out of 85 samples were found positive, showing positivity rate of 24.7%. The positivity rate of RT-RPA assay corroborated with the gold standard RT-PCR test. This study presented the development of an RT-RPA isothermal assay for rapid and accurate detection of PSV. The assay is highly sensitive, specific, works at a low and constant temperature, does not require any high-end instrument and can be a potential diagnostics tool for pen-side testing of PSV in the field conditions. The newly developed RT-RPA assay could successfully detect PSV circulating in swine population of Haryana, India. This is a first report of this kind from the region.

Identification of an Isolate of Citrus Tristeza Virus by Nanopore Sequencing in Korea and Development of a CRISPR/Cas12a-based Assay for Rapid Visual Detection of the Virus

Citrus tristeza virus (CTV) is a highly destructive viral pathogen posing a significant threat to citrus crops worldwide. The disease management and crop protection strategies necessitate the development of rapid and accurate detection methods. In this study, we employed Oxford Nanopore sequencing (ONT) to detect CTV in Citrus unshiu samples. Subsequently, we developed a specific and sensitive detection assay combining CRISPR/Cas12a with reverse transcription-recombinase polymerase amplification. The CRISPR-Cas12a assay exhibited exceptional specificity for CTV, surpassing conventional RT-PCR by at least 10-fold in sensitivity. Remarkably, the developed assay detected CTV in field samples, with zero false negatives. This diagnostic approach is user-friendly, cost-effective, and offers tremendous potential for rapid on-site detection of CTV. Therefore, the CRISPR-Cas12a assay plays a significant role in managing and preserving citrus trees that are free from viruses in the industry.

Rapid and Visual Detection of Actinidia Chlorotic Ringspot-Associated Virus Using One-Step Reverse-Transcription Recombinase Polymerase Amplification Combined with Lateral Flow Dipstick Assay.

Actinidia chlorotic ringspot-associated virus (AcCRaV) occurs widely in major kiwifruit producing areas of China and is often accompanied by coinfecting viruses, affecting the growth, yield, and quality of kiwifruit. Therefore, a rapid and sensitive detection method is crucial for diagnosing and developing effective AcCRaV management strategies. In this study, a one-step reverse-transcription recombinase polymerase amplification combined with a lateral flow dipstick (RT-RPA-LFD) assay was developed for rapid detection of AcCRaV. Specific primers and a probe were designed based on the conserved region of the coat protein gene sequence of AcCRaV. The one-step RT-RPA reaction can be performed at 35 and 40°C within 10 to 30 min, and the amplification results can be read directly on the LFD within 5 min. The detection limit of the one-step RT-RPA-LFD assay was 10-8 ng (about 20 viral copies), which was equal with one-step RT-qPCR and 100 times more sensitive than one-step RT-PCR. Moreover, the one-step RT-RPA-LFD assay was successfully applied to detect AcCRaV from crude extracts, and the entire detection process can be completed within 40 min. These results indicate that the RT-RPA-LFD assay is a simple, rapid, and sensitive strategy that can be used for accurate diagnosis of AcCRaV-infected kiwifruit plants in the field. To our knowledge, this is the first study applying the one-step RT-RPA-LFD assay to detect a kiwifruit virus.

Rapid detection of avian influenza virus based on CRISPR-Cas12a

BackgroundAvian influenza (AI) is a disease caused by the avian influenza virus (AIV). These viruses spread naturally among wild aquatic birds worldwide and infect domestic poultry, other birds, and other animal species. Currently, real-time reverse transcription polymerase chain reaction (rRT-PCR) is mainly used to detect the presence of pathogens and has good sensitivity and specificity. However, the diagnosis requires sophisticated instruments under laboratory conditions, which significantly limits point-of-care testing (POCT). Rapid, reliable, non-lab-equipment-reliant, sensitive, and specific diagnostic tests are urgently needed for rapid clinical detection and diagnosis. Our study aimed to develop a reverse transcription recombinase polymerase amplification (RT-RPA)/CRISPR method which improves on these limitations.MethodsThe Cas12a protein was purified by affinity chromatography with Ni-agarose resin and observed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Specific CRISPR RNA (crRNA) and primers targeting the M and NP genes of the AIV were designed and screened. By combining RT-RPA with the Cas12a/crRNA trans-cleavage system, a detection system that uses fluorescence readouts under blue light or lateral flow strips was established. Sensitivity assays were performed using a tenfold dilution series of plasmids and RNA of the M and NP genes as templates. The specificity of this method was determined using H1–H16 subtype AIVs and other avian pathogens, such as newcastle disease virus (NDV), infectious bursal disease virus (IBDV), and infectious bronchitis virus (IBV).ResultsThe results showed that the method was able to detect AIV and that the detection limit can reach 6.7 copies/μL and 12 copies/μL for the M and NP gene, respectively. In addition, this assay showed no cross-reactivity with other avian-derived RNA viruses such as NDV, IBDV, and IBV. Moreover, the detection system presented 97.5% consistency and agreement with rRT-PCR and virus isolation for detecting samples from poultry. This portable and accurate method has great potential for AIV detection in the field.ConclusionAn RT-RPA/CRISPR method was developed for rapid, sensitive detection of AIV. The new system presents a good potential as an accurate, user-friendly, and inexpensive platform for point-of-care testing applications.

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.

Rapid detection of goose astrovirus genotypes 2 using real-time reverse transcription recombinase polymerase amplification

BackgroundGoose astrovirus (GoAstV) is an important pathogen that causes joint and visceral gout in goslings. It has been circulating in many provinces of China since 2017. Goose astrovirus genotypes 2 (GoAstV-2) is the main epidemic strain, and its high morbidity and mortality have caused huge economic losses to the goose industry. An accurate point-of-care detection for GoAstV-2 is of great significance. In this study, we developed a real-time reverse transcription recombinase polymerase amplification (RT-RPA) method for the on-site detection of GoAstV-2 infection.ResultsThe real-time RT-RPA reaction was carried out at a constant temperature of 39 °C, and the entire detection time from nucleic acid preparation to the end of amplification was only 25 min using the portable device. The results of a specificity analysis showed that no cross-reaction was observed with other related pathogens. The detection limit of the assay was 100 RNA copies/μL. The low coefficient of variation value indicated excellent repeatability. We used 270 clinical samples to evaluate the performance of our established method, the positive concordance rates with RT-qPCR were 99.6%, and the linear regression analysis revealed a strong correlation.ConclusionsThe established real-time RT-RPA assay showed high rapidity, specificity and sensitivity, which can be widely applied in the laboratory, field and especially in the resource-limited settings for GoAstV-2 point-of-care diagnosis.

Optimization of a simple, low-cost one-step reverse transcription recombinase polymerase amplification method for real-time detection of potato virus A in potato leaves and tubers.

Vegetative propagation of potatoes makes it possible for potato viruses to be transmitted through tubers. Potato virus A (PVA) is one of these viruses, which belongs to the Potyvirus genus in the Potyviridae family. Potato tuber yield can be reduced by 30-40% by PVA alone. Losses can be further exacerbated by potato virus X and/or potato virus Y infection. PVA is transmitted primarily by several species of aphids in non-persistent manner. With the aim of resolving this problem, we developed one-step reverse transcription-recombinase polymerase amplification (RT-RPA), a highly sensitive and cost-effective method for detecting PVA in both potato tubers and leaves. Detection and amplification are performed using isothermal conditions in this method. There was good amplification of the coat protein gene in PVA with all three primers tested. To conduct this study, a primer set that can amplify specific 185 base pair (bp) product was selected. PVA detection was optimized by 30-min amplification reactions, which showed no cross-reactivity with other potato viruses. A simple heating block or water bath was used to amplify PVA product using RT-RPA at a temperature range of 38-42°C. In comparison to conventional reverse transcription-polymerase chain reaction (RT-PCR), the newly developed RT-RPA protocol exhibited high sensitivity for both potato leaves and tuber tissues. Using cellular paper-based simple RNAextraction procedure, the virus was detected in leaf samples as efficiently as purified total RNA. We also found that combining LiCl-based RNA precipitation with cellular paper discs allowed us to successfully optimize RNA extraction for one-step RT-RPA for detecting PVA in tubers. Tests using this simplified one-step RT-RPA method were successfully applied to 300 samples of both leaves and tubers from various potato cultivars. In our knowledge, this is the first report of an RT-RPA assay utilizing simple RNA obtained from either cellular disc paper or LiCl coupled with cellular disc paper to detect PVA. As a result, this method was equally sensitive and specific for detecting PVA in potatoes. The developed RT-RPA assay is more versatile, durable, and do not require highly purified RNA templates, thus providing an effective alternative to RT-PCR assays for screening of germplasm, certifying planting materials, breeding for virus resistance, and real-time monitoring of PVA.

A smartphone-controllable molecular diagnostic platform for SARS-CoV-2 detection by reverse-transcription recombinase polymerase amplification

We constructed a portable molecular diagnostic microsystem, enabling the whole processes of genetic analysis for SARS-CoV-2. The proposed platform was a miniaturized size and was comprised of three main parts: a microfluidic device with a 3D-printed solution loading cartridge, an operating system including a tailor-made heater board, a syringe pump and an optical fluorescence detector, and a smartphone with a customized app for the remote and automatic control. The microfluidic device was designed to control the complicated fluidic flow through only the passive valves, and could perform the RNA extraction from the viral lysate via glass membranes and the target gene amplification by reverse-transcription recombinase polymerase amplification (RT-RPA). The 3D-printed cartridge allowed the facile chemical solution storage and loaded a designated solution into the microfluidic device. A smartphone with a browser-based app could control the on-and-off status of solenoid valves, the workflow of a syringe pump to automatically introduce solutions on a chip, the heater board to supply the constant temperature for RT-RPA, and the periodic activation of the Pi camera and a 488 nm laser to measure the fluorescence signal in the reaction chambers that showed the amplification profiles on the smartphone display in real-time. Using the proposed smartphone-controllable diagnostic platform, the N gene and S gene of SARS-CoV-2 were successfully detected at a limit-of-detection (LOD) of 10 copies/μL within 1 hr in a sample-to-answer-out format. Ten out of 22 clinical samples were successfully analyzed for SARS-CoV-2, demonstrating the practical applications for the on-site diagnostics of patient samples.

Rapid and visual detection of grapevine fleck virus using recombinase polymerase amplification combined with lateral flow strips

AbstractGrapevine flack virus (GFkV) causes fleck disease and leads to severe symptoms when the infection occurs with multiple viruses. Restricting the propagation of GFkV‐infected grapevines is the ideal management strategy to control the spread of this virus. Therefore, the development of rapid detection methods for GFkV is urgently required. In the present study, we developed a rapid and reliable assay based on the reverse transcription‐recombinase polymerase amplification (RT‐RPA) combined with lateral flow strips (LFS) using sequence‐specific primers and a probe‐based coat protein sequences for the GFkV detection in grapevines. The GFkV RT‐RPA‐LFS assay was optimized at 38°C for 10 min and an extra 5 min LFS incubation time. The optimized RT‐RPA‐LFS assay had similar sensitivity to RT‐PCR and showed no cross‐reaction with major viruses infecting grapevines in Korea. The assay was successfully validated for GFkV detection in grapevine field samples. This study provides a reliable technique for rapidly detecting GFkV as an alternative diagnostic approach for producing virus‐free grapevine nurseries.

SARS-CoV-2 and Its Omicron Variants Detection with RT-RPA -CRISPR/Cas13a-Based Method at Room Temperature.

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a global health crisis, with genetic mutations and evolution further creating uncertainty about epidemic risk. It is imperative to rapidly determine the nucleic acid sequence of SARS-CoV-2 and its variants to combat the coronavirus pandemic. Our goal was to develop a rapid, room-temperature, point-of-care (POC) detection system to determine the nucleic acid sequences of SARS-CoV-2 isolates, especially omicron variants. Based on the conserved nucleotide sequence of SARS-CoV-2, bioinformatics software was used to analyze, design, and screen optimal enzymatic isothermal amplification primers and efficient CRISPR RNAs (crRNAs) of CRISPR/Cas13a to the target sequences. Reverse transcription-recombinase polymerase amplification (RT-RPA) was used to amplify the virus, and CRISPR/Cas13a-crRNA was used to cleave the SARS-CoV-2 target sequence. The sensitivity of nucleic acid detection was assessed by serial dilution of plasmid templates. All reactions were performed at room temperature. RT-RPA, combined with CRISPR/Cas13a, can detect the SARS-CoV-2 with a minimum content of 102 copies/μL, and can effectively distinguish between the original strain and the Omicron variant with a minimum limit of detection (LOD) of 103 copies/μL. The method developed in this study has potential application in clinical detection of SARS-CoV-2 and its omicron variants.

Development of Recombinase Polymerase Amplification Combined with Lateral Flow Strips for Rapid Detection of Cowpea Mild Mottle Virus.

Cowpea mild mottle virus (CPMMV) is a global plant virus that poses a threat to the production and quality of legume crops. Early and accurate diagnosis is essential for effective managing CPMMV outbreaks. With the advancement in isothermal recombinase polymerase amplification and lateral flow strips technologies, more rapid and sensitive methods have become available for detecting this pathogen. In this study, we have developed a reverse transcription recombinase polymerase amplification combined with lateral flow strips (RT-RPA-LFS) method for the detection of CPMMV, specifically targeting the CPMMV coat protein (CP) gene. The RT-RPA-LFS assay only requires 20 min at 40°C and demonstrates high specificity. Its detection limit was 10 copies/μl, which is approximately up to 100 times more sensitive than RT-PCR on agarose gel electrophoresis. The developed RT-RPA-LFS method offers a rapid, convenient, and sensitive approach for field detection of CPMMV, which contribute to controlling the spread of the virus.

(Invited) liquid Biopsy Tool: Integrated Aiot Digital Bead-Based Sensor for Molecular-Fingerprint Profiling of Extracellular Vesicles

Development of liquid biopsies for non-invasive tumor characterization techniques shows great promise for real-time monitoring of tumor recurrence, progression, and treatment response. To better assess the various tumor evolution, cellular heterogeneity, consequent drug-resistance mechanisms, it is critical to screen proteins and nucleic acids of extracellular vesicles (EVs) in biofluids. We are developing an integrated sensitive digital bead-based sensor enabling evaluation of molecular profiling of extracellular vesicles in glioblastoma. The tumor relevant genetic information in EVs is enriched by antibody-specific magnetic beads and amplified by reverse transcription recombinase polymerase amplification with CRISPR/Cas13a system in twenty-five thousand droplets, and results are analyzed by a miniaturized imaging device. The niches of our developing technique include a) the streamlined sample preparation process and miniaturized detection system enable the ultrasensitive detection of mRNA in extracellular vesicles within one and half hour, b) the fluorescent signal-to-noise ratio in each 40-µm sized droplet is significantly enhanced via using a simple Cas13a assay and triggering greater than 104 turnovers of fluorescent reporters on a chip, c) real-time image data are transferred to a cloud-based server to be classified with trained YOLOv5 models and RNAomic results are displayed on the Raspberry Pi touchscreen. This system allows use by lower skill health care workers. This approach can provide inexpensive, rapid, and treatment-informative diagnostics for underserved regions, and underfunded health care systems. It could be further developed for other cancers, leading up the speed of diagnosis and treatment decision-making in using liquid biopsy, thus find widespread use in global health. Figure 1

Triplex visual detection of tobacco potyviruses using reverse transcription recombinase polymerase amplification assay combined with lateral flow dipstick

Currently, several analytical methods have been developed for identifying tobacco viral species. However, conventional methods are based on labor-intensive, time-consuming, and instrument-required approaches. A novel, rapid and visual method based on the combination of reverse transcription recombinase polymerase amplification assay (RT-RPA) and multiplex lateral flow dipstick (LFD) was established specifically for the rapid on-site detection of three tobacco viruses and evaluated. The biosensor used coat protein (CP) genes of potyviruses potato Y virus, chilli veinal mottle virus, and tobacco vein banding mosaic virus as detection markers. A triplex RT-RPA reaction for both targets was constructed and three chemical labels of the amplification products were carefully tested for effective and accurate visualization on the strip. This method demonstrated reasonable specificity and achieved a sensitivity of 103 copies per reaction. Validation with clinical samples showed results consistent with a real-time polymerase chain reaction. The detection process was simple, with little dependence on laboratory settings, and rapid, with a 30-min reaction at 39 °C and visualization on the strip within 5 min. This newly developed triplex RT-RPA-LFD assay was sensitive, specific, and suitable for the on-site detection of three tobacco viruses simultaneously.

Trouble-free detection of grapevine leafroll-associated virus-3 employing reverse transcription-recombinase polymerase amplification assay

Trouble-free detection of grapevine leafroll-associated virus-3 employing reverse transcription-recombinase polymerase amplification assay

A CRISPR-Cas12a-Based Diagnostic Method for Japanese Encephalitis Virus Genotypes I, III, and V.

The Japanese encephalitis virus (JEV) is prevalent in Asian countries, including Korea, Japan, China, Vietnam, and India. JEV is transmitted to humans by Culex mosquitoes. Despite extensive research efforts, no approved antiviral agents are currently available, although JE can be prevented by vaccination. DNA endonuclease-targeted CRISPR trans reporter (DETECTR) is a newly emerging CRISPR-Cas12a-based molecular diagnostic method combined with isothermal nucleic acid amplification. In this study, DETECTR with reverse transcription-recombinase polymerase amplification (RT-RPA) was effectively utilized for JEV diagnosis and detected down to 10 RNA copies for JEV genotype I (GI) and 1 × 102 copies for both GIII and GV, achieving similar sensitivity to RT-PCR while displaying no cross-reaction with other viruses. A one-tube, one-temperature format of DETECTR was further developed, and its efficiency compared with that of conventional DETECTR.