Rapid detection of Impatiens necrotic spot virus from thrips vectors using reverse transcription-recombinase polymerase amplification.

The plant virus, Impatiens necrotic spot virus (INSV), is an economically important pathogen of vegetables, fruits, and ornamental crops. INSV is vectored by the western flower thrips, Frankliniella occidentalis, a small insect pest that is globally distributed. In recent years, INSV outbreaks have reached epidemic levels in the Salinas Valley of California-an agriculturally rich region where most of the lettuce (Lactuca sativa) is produced in the United States. Due to the obligate nature in which virus transmission occurs, new tools that could rapidly detect INSV from thrips vectors would enhance our ability to predict where virus outbreaks may occur. Here, we report on the development of a reverse transcription-recombinase polymerase amplification (RT-RPA) assay that can detect INSV from individual thrips. The assay uses crude extraction methods, is performed at a single temperature of 42 °C, can be completed in 25 min, and provides sensitivity levels that are comparable to other available detection methods. When the assay was used on field populations of thrips, INSV was successfully identified and quantified from individual larvae and adults. The work provides a new cost-effective surveillance tool that can rapidly detect INSV from its insect vector and from plants.

The development of RT-RPA and CRISPR-Cas12a based assay for sensitive detection of Hirame novirhabdovirus.

Hirame novirhabdovirus (HIRRV) is a highly pathogenic fish virus that poses a significant threat to the farming of a variety of economic fish. Due to no commercial vaccines and effective drugs available, sensitive and rapid detection of HIRRV at latent and early stages is important and critical for the control of disease outbreaks. However, most of the current methods for HIRRV detection have a large dependence on instruments and operations. For better detection of HIRRV, we have established a detection technology based on the reverse transcription and recombinase polymerase amplification (RT-RPA) and CRISPR/Cas12a to detect the N gene of HIRRV in two steps. Following the screening of primer pairs, the reaction temperature and time for RPA were optimized to be 40 °C and 32min, respectively, and the CRISPR/Cas12a reaction was performed at 37 °C for 15min. The whole detection procedure including can be accomplished within 1 h, with a detection sensitivity of about 8.7 copies/µl. The detection method exhibited high specificity with no cross-reaction to the other Novirhabdoviruses IHNV and VHSV, allowing naked-eye color-based interpretation of the detection results through lateral flow (LF) strip or fluorescence under violet light. Furthermore, the proliferation dynamic of HIRRV in the spleen of flounder were comparatively detected by LF- and fluorescence-based RPA-CRISPR/Cas12a assay in comparison to qRT-PCR at the early infection stage, and the results showed that the viral positive signal could be firstly detected by the two RPA-CRISPR/Cas12a based methods at 6 hpi, and then by qRT-PCR at 12 hpi. Overall, our results demonstrated that the developed RPA-CRISPR/Cas12a method is a stable, specific, sensitive and more suitable in the field, which has a significant effect on the prevention of HIRRV. RT-RPA-Cas12a-mediated assay is a rapid, specific and sensitive detection method for visual and on-site detection of HIRRV, which shows a great application promise for the prevention of HIRRV infections.

Rapid detection of blood using a novel application of RT-RPA integrated with CRISPR-Cas: ALAS2 detection as a model.

A rapid, sensitive and specific test for blood is reported based on a novel application of recombinase polymerase amplification integrated with CRISPR-Cas and lateral flow assay (LFA). The blood specific marker ALAS2 was used as the target to record the presence of blood. The assay used either RNA extracted from a body fluid as a template, or omitting this extraction step and using a direct approach where the questioned body fluid was added directly to the assay. The assay only detected blood (all peripheral blood and some menstrual blood samples) and no other body fluid (semen, saliva, or vaginal fluid). The limit of detection varied from an initial template of 0.195 ng extracted RNA (27 dilution) or 0.0218 µL (26 dilution) liquid peripheral blood. The assay gave the expected result when peripheral blood was mixed with saliva: ratios of peripheral blood/saliva at 19:1, 3:1, 1:1, 1:3 and 1:19 all gave a positive result using extracted RNA. By contrast, only three ratios of peripheral blood and saliva gave a positive result for blood (19:1, 3:1 and 1:1) when adding these two body fluids directly. When peripheral blood was mixed with semen there was a strong inhibition of the assay and ALAS2 could only be detected at ratio of 19:1 using RNA. Using reconstituted peripheral bloodstains gave comparable results to liquid peripheral blood. This is the first application of RT-RPA integrated CRISPR and combined with a LFA assay to detect body fluid-specific RNA. The proposed method opens up the potential to perform this method remote from laboratories such as at crime scenes.

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.

Development of reverse transcriptase-recombinase polymerase amplification (RT-RPA) assay for rapid detection of large cardamom chirke virus.

Large cardamom chirke virus (LCCV) causing chirke disease of large cardamom is a major production constraint of this crop. Rapid and accurate detection of LCCV is important for managing the disease. In the present study an isothermal assay namely, reverse transcriptase-recombinase polymerase amplification (RT-RPA) was developed for the detection of LCCV. Total RNA isolated by two different methods and crude extracts isolated using five different methods as templates were assessed for their ability to detect LCCV. Of these, only the total RNA isolated by both methods gave consistent and repeatable results while all the crude extracts used as templates gave non-specific amplification. RT-RPA was up to 1000 times more sensitive than conventional RT-PCR for the detection of LCCV. The detection limit of RPA was 10 fg when recombinant plasmid was used as the template. The RT-RPA assay was validated using field samples and found suitable for large-scale screening of large cardamom plants against LCCV for the selection of virus-free plants. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-024-00861-2.

On-Site and Visual Detection of the H5 Subtype Avian Influenza Virus Based on RT-RPA and CRISPR/Cas12a.

Avian influenza viruses (AIVs) of the H5 subtype rank among the most serious pathogens, leading to significant economic losses in the global poultry industry and posing risks to human health. Therefore, rapid and accurate virus detection is crucial for the prevention and control of H5 AIVs. In this study, we established a novel detection method for H5 viruses by utilizing the precision of CRISPR/Cas12a and the efficiency of RT-RPA technologies. This assay facilitates the direct visualization of detection results through blue light and lateral flow strips, accurately identifying H5 viruses with high specificity and without cross-reactivity against other AIV subtypes, NDV, IBV, and IBDV. With detection thresholds of 1.9 copies/µL (blue light) and 1.9 × 103 copies/µL (lateral flow strips), our method not only competes with but also slightly surpasses RT-qPCR, demonstrating an 80.70% positive detection rate across 81 clinical samples. The RT-RPA/CRISPR-based detection method is characterized by high sensitivity, specificity, and independence from specialized equipment. The immediate field applicability of the RT-RPA/CRISPR approach underscores its importance as an effective tool for the early detection and management of outbreaks caused by the H5 subtype of AIVs.

Real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay for detection of cassava brown streak viruses.

Cassava brown streak disease (CBSD) caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) is the most economically important viral disease of cassava. As cassava is a vegetatively propagated crop, the development of rapid and sensitive diagnostics would aid in the identification of virus-free planting material and development of effective management strategies. In this study, a rapid, specific and sensitive real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed for real-time detection of CBSV and UCBSV. The RT-RPA was able to detect as little as 2 pg/µl of purified RNA obtained from infected cassava leaves, a sensitivity equivalent to that obtained by quantitative real-time reverse transcription PCR (qRT-PCR), within 20 min at 37 °C. Further, the RT-RPA detected each target virus directly from crude leaf and stem extracts, avoiding the tedious and costly isolation of high-quality RNA. The developed RT-RPA assay provides a valuable diagnostic tool that can be adopted by cassava seed certification and virus resistance breeding programs to ensure distribution of virus-free cassava planting materials to farmers. This is the first report on the development and validation of crude sap-based RT-RPA assay for the detection of cassava brown streak viruses (UCBSV and CBSV) infection in cassava plants.

Development and Application of a Duplex RT-RPA Assay for the Simultaneous Detection of Cymbidium mosaic virus and Odontoglossum ringspot virus.

Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) are among the world's most serious and widespread orchid viruses; they often infect orchids, causing devastating losses to the orchid industry. Therefore, it is critical to establish a method that can rapidly and accurately detect viruses in the field using simple instruments, which will largely reduce the further spread of viruses and improve the quality of the orchid industry and is suitable for mass promotion and application at grassroots agrotechnical service points. In this investigation, we established a rapid amplification method for virus detection at 39 °C for 35 min to detect the presence of CymMV and ORSV simultaneously, sensitively, and specifically in orchids. Primers for the capsid protein (CP)-encoding genes of both viruses were designed and screened, and the reaction conditions were optimized. The experimental amplification process was completed in just 35 min at 39 °C. There were no instances of nonspecific amplification observed when nine other viruses were present. The RPA approach had detection limits of 104 and 103 copies for pMD19T-CymMV and pMD19T-ORSV, respectively. Moreover, the duplex RT-RPA investigation confirmed sensitivity and accuracy via a comparison of detection results from 20 field samples with those of a gene chip. This study presents a precise and reliable detection method for CymMV and ORSV using RT-RPA. The results demonstrate the potential of this method for rapid virus detection. It is evident that this method could have practical applications in virus detection processes.

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.

The development of RT-RPA and CRISPR-Cas12a based assay for sensitive detection of infectious hematopoietic necrosis virus (IHNV).

Infectious hematopoietic necrosis virus (IHNV) is an economically important virus causing significant mortalities among wild and cultured salmonid fish worldwide. Rapid and sensitive diagnostic methods of IHNV are crucial for timely controlling infections. For better detection of IHNV, we have established a detection technology based on the reverse transcription and recombinase polymerase amplification (RT-RPA) and CRISPR/Cas12a to detect the N gene of IHNV in two steps. Following the screening of primer pairs, the reaction temperature and time for RPA were optimized to be 41 °C and 35 min, respectively, and the CRISPR/Cas12a reaction was performed at 37 °C for 15 min. The whole detection procedure including can be accomplished within one hour, with a detection sensitivity of about 9.5 copies/µL. The detection method exhibited high specificity with no cross-reaction to the other Novirhabdoviruses HIRRV and VHSV, allowing naked-eye interpretation of the results through lateral flow or fluorescence under ultraviolet light. Overall, our results demonstrated that the developed RT-RPA-Cas12a-mediated assay is a rapid, specific and sensitive detection method for routine and on-site detection of IHNV, which shows a great application promise for the prevention of IHNV infections.

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

BACKGROUND: 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. METHODS AND RESULTS: 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 20 min 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. CONCLUSIONS: 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.

Rapid detection of avian influenza virus based on CRISPR-Cas12a.

BACKGROUND: Avian 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. METHODS: The 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). RESULTS: The 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. CONCLUSION: An 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.

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 RNA extraction 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.

RT-RPA-PfAgo System: A Rapid, Sensitive, and Specific Multiplex Detection Method for Rice-Infecting Viruses.

The advancement in CRISPR-Cas biosensors has transmuted the detection of plant viruses owing to their rapid and higher sensitivity. However, false positives and restricted multiplexing capabilities are still the challenges faced by this technology, demanding the exploration of novel methodologies. In this study, a novel detection system was developed by integrating reverse transcriptome (RT) techniques with recombinase polymerase isothermal amplification (RPA) and Pyrococcus furiosus Argonaute (PfAgo). The RT-RPA-PfAgo system enabled the simultaneous detection of rice ragged stunt virus (RRSV), rice grassy stunt virus (RGSV), and rice black streaked dwarf virus (RBSDV). Identifying targets via guide DNA without being hindered by protospacer adjacent motif sequences is the inherent merit of PfAgo, with the additional advantage of it being simple, cost-effective, and exceptionally sensitive, with detection limits between 3.13 and 5.13 copies/µL, in addition to it effectively differentiating between the three distinct viruses. The field evaluations were also in accordance with RT-PCR methods. The RT-RPA-PfAgo system proved to be a robust, versatile, highly specific, and sensitive method with great potential for practicality in future plant virus diagnostics.

Rapid isothermal point-of-care test for screening of SARS-CoV-2 (COVID-19).

Rapid on-site diagnosis of emerging pathogens is key for early identification of infected individuals and for prevention of further spreading in a population. Currently available molecular diagnostic tests are instrument-based whereas rapid antibody and antigen tests are often not sufficiently sensitive for detection in pre-symptomatic subjects. There is a need for rapid point of care molecular screening tests that can be easily adapted to emerging pathogens and are selective, sensitive, reliable in different settings around the world. We have developed a simple, rapid (<30 â€‹min), and inexpensive test for SARS-CoV-2 that is based on combination of isothermal reverse transcription recombinase polymerase amplification (RT-RPA) using modified primers and visual detection with paper-based microfluidics. Our test (CoRapID) is specific for SARS-CoV-2 (alpha to omicron variants) and does not detect other coronaviruses and pathogens by in silico and in vitro analysis. A two-step test protocol was developed with stable lyophilized reagents that reduces handling by using portable and disposable components (droppers, microapplicators/swabs, paper-strips). After optimization of assay components and conditions, we have achieved a limit of detection (LoD) of 1 copy/reaction by adding a blocking primer to the lateral flow assay. Using a set of 138 clinical samples, a sensitivity of 88.1% (P â€‹< â€‹0.05, CI: 78.2-93.8%) and specificity of 93.9% (P â€‹< â€‹0.05, CI: 85.4-97.6%) was determined. The lack of need for instrumentation for our CoRapID makes it an ideal on-site primary screening tool for local hospitals, doctors' offices, senior homes, workplaces, and in remote settings around the world that often do not have access to clinical laboratories.

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.

Development of a reverse transcription recombinase polymerase amplification combined with lateral flow assay for equipment-free on-site field detection of tomato chlorotic spot virus.

BACKGROUND: Tomato chlorotic spot virus (TCSV) is an economically important, thrips-transmitted, emerging member of the Orthotospovirus genus that causes significant yield loss mainly in tomatoes, but also in other vegetable and ornamental crops. Disease management of this pathogen is often challenging due to the limited availability of natural host resistance genes, the broad host range of TCSV, and the wide distribution of its thrips vector. Point-of-care detection of TCSV with a rapid, equipment-free, portable, sensitive, and species-specific diagnostic technique can provide prompt response outside the laboratory, which is critical for preventing disease progression and further spread of the pathogen. Current diagnostic techniques require either laboratory-dependent or portable electronic equipment and are relatively time-consuming and costly. RESULTS: In this study, we developed a novel technique for reverse-transcription recombinase polymerase amplification combined with lateral flow assay (RT-RPA-LFA) to achieve a faster and equipment-free point-of-care detection of TCSV. The RPA reaction tubes containing crude RNA are incubated in the hand palm to obtain sufficient heat (∼36 °C) for the amplification without the need for equipment. Body-heat mediated RT-RPA-LFA is highly TCSV-specific with a detection limit as low as ∼6 pg/µl of total RNA from TCSV-infected tomato plants. The assay can be performed in 15 min in the field. CONCLUSION: To the best of our knowledge, this is the first equipment-free, body-heat-mediated RT-RPA-LFA technique developed to detect TCSV. Our new system offers a time-saving advantage for the sensitive and specific diagnostic of TCSV that local growers and small nurseries in low-resource settings can use without skilled personnel.

A CRISPR-based approach using dead Cas9-sgRNA to detect SARS-CoV-2.

Rapid, highly specific, and robust diagnostic kits to detect viruses and pathogens are needed to control disease spread and transmission globally. Of the many different methods proposed to diagnose COVID-19 infection, CRISPR-based detection of nucleic acids tests are among the most prominent. Here, we describe a new way of using CRISPR/Cas systems as a rapid and highly specific tool to detect the SARS-CoV-2 virus using the in vitro dCas9-sgRNA-based technique. As a proof of concept, we used a synthetic DNA of the M gene, one of the SARS-CoV-2 virus genes, and demonstrated that we can specifically inactivate unique restriction enzyme sites on this gene using CRISPR/Cas multiplexing of dCas9-sgRNA-BbsI and dCas9-sgRNA-XbaI. These complexes recognize and bind to the target sequence spanning the BbsI and XbaI restriction enzyme sites, respectively, and protect the M gene from digestion by BbsI and/or XbaI. We further demonstrated that this approach can be used to detect the M gene when expressed in human cells and from individuals infected with SARS-CoV-2. We refer to this approach as dead Cas9 Protects Restriction Enzyme Sites, and believe that it has the potential to be applied as a diagnostic tool for many DNA/RNA pathogens.

A fully-enclosed prototype 'pen' for rapid detection of SARS-CoV-2 based on RT-RPA with dipstick assay at point-of-care testing.

A fully-enclosed prototype 'pen' for rapid detection of SARS-CoV-2 based on reverse transcriptase isothermal recombinase polymerase amplification (RT-RPA) with dipstick assay was developed. The integrated handheld device, consisting of amplification, detection and sealing modules, was developed to perform rapid nucleic acid amplification and detection under a fully enclosed condition. After RT-RPA amplification with a metal bath or a normal PCR instrument, the amplicons were mixed with dilution buffer prior to being detected on a lateral flow strip. To avoid aerosol contamination causing false-positive, from amplification to final detection, the detection 'pen' had been enclosed to isolate from the environment. With colloidal gold strip-based detection, the detection results could be directly observed by eyes. By cooperating with other inexpensive and rapid methods for POC nucleic acid extraction, the developed 'pen' could detect COVID-19 or other infectious diseases in a convenient, simple and reliable way.