Reverse Transcription Recombinase Polymerase Amplification Assay Research Articles

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.

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.

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.

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

Simplified extraction protocol for plant tissues and reverse transcription RPA assay for quick and reliable diagnosis and its application in resistance screening of chilli veinal mottle virus

The widespread occurrence and predominant association of chilli veinal mottle virus (ChiVMV) with the valuable chilli landraces of North East Region (NER) of India and associated threat to chilli production necessitated the development of simplified and robust detection procedure. Present study reports a simplified reverse transcription-recombinase polymerase amplification (RT-RPA) method for detection of ChiVMV in crude leaf extract prepared through simplified extraction protocol using NaOH:EDTA (1:1) based tissue lysis, without the need of conventional RNA extraction and sample processing. Developed RT-RPA assay could be performed at an isothermal temperature (38 °C) in 30 min and exhibited no cross-reactivity with other prevalent chilli viruses (potato virus Y, cucumber mosaic virus, large cardamom chirke virus, capsicum chlorosis orthotospovirus, pepper mild mottle virus and chilli leaf curl virus), indicating high specificity. It could detect ChiVMV up to 10−9 dilution (100 ag/μl) of crude leaf extract, 10 ag/μl of RNA of ChiVMV infected leaf tissues and plasmid DNA containing viral gene insert, thus exhibiting sufficient sensitivity which is at par or better than the standard RT-PCR. Developed RT-RPA assay was employed to reveal the widespread occurrence of ChiVMV in NER India with 50.81% (1545 samples tested) of symptomatic and 7.85% (1593 samples tested) of asymptomatic samples tested positive. RT-RPA exhibited higher sensitivity in detection of ChiVMV infection in field samples as compared to the standard RT-PCR which gave false negative in 2.07% of symptomatic and 2.01% of asymptomatic samples. The developed assay was further applied in screening of 49 chilli germplasm for ChiVMV infection. The pooled results of field screening based on the disease score indicated that none of the chilli germplasm had immune or complete resistance, while 29 chilli germplasm (bird's eye chilli: C. frutescens; Morokatekpa: C. annuum, Leihao morok, Morok masingkha; Chingpimorok: Capsicum sp.) exhibited tolerant reaction with a disease score of 1.27–2.00. All the tested bird's eye chilli germplasm showed tolerance to the ChiVMV. The artificial screening to the severe isolate and qRT-PCR based viral titre determination confirmed the results of field screening wherein the germplasm RCMC6, RCMC7, RCMC17, RCMC27 and RCMC42 exhibited either asymptomatic or mild mosaic symptoms and decrease in viral titre at 20 DAI thus categorized as tolerant. The simplified RT-RPA assay developed and validated in the present study can be applied in routine detection of ChiVMV in field samples, disease-free seedling production and resistance screening under resource poor laboratory setup with limited technical expertise. Chilli germplasm with tolerance to ChiVMV identified in the present study can be used as donors in resistance breeding.

453 Rapid SARS-CoV-2 testing with duplexed recombinase polymerase amplification and a bacteriophage internal control

OBJECTIVES/GOALS: Current COVID-19 rapid molecular tests require cartridge-reader detection, expensive circuitry, and complex microfluidics making the most accurate tests unavailable to the masses. Here we present a rapid molecular diagnostic leveraging isothermal amplification and paper-based microfluidics for a low-cost ultra-sensitive COVID-19 assay. METHODS/STUDY POPULATION: We designed a reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of SARS-CoV-2 and bacteriophage MS2 RNA. RT-RPA is a sequence specific, ultrasensitive, rapid isothermal DNA amplification technique that is well suited to home based testing due to its rapid assay time, robustness, ease of use, and readout options. RT-RPA reagents are added to a tube and incubated at 39°C in a fluorometer. Realtime fluorometer data gives results in under 15 minutes. This assay also provides visual detection via lateral flow readout with results in 23 minutes. RESULTS/ANTICIPATED RESULTS: We have developed a rapid multiplexed nucleic acid amplification assay with an internal process control for SARS-CoV-2 using single-pot RT-RPA. We screened 21 primer combinations to select primers that demonstrated excellent performance and target specificity against common respiratory viruses. We demonstrate the ability to multiplex SARS-CoV-2 and MS2 detection, utilizing MS2 as an internal process control for lysis, reverse transcription, amplification, and readout. We show duplexed detection using both fluorescence readout and visual readout using lateral flow strips. Duplexed fluorescence detection shows a limit of detection of 25 copies per reaction. Duplexed lateral flow readout shows a limit of detection of 50 copies per reaction DISCUSSION/SIGNIFICANCE: We developed a duplexed RT-RPA assay for SARS-CoV-2 with fluorescence or lateral flow readout. Our assay does not re-quire expensive reader, circuity, or fluid handling. The low material cost, temperature, and robustness make it ideal for a more accurate home-based COVID-19 diagnostic.

Sperm mRNA screening by reverse transcription-recombinase polymerase amplification (RT-RPA) assay: Decision-making for differential extraction

The presence of sperm cells is an indicator for differential extraction on sexual assault samples. In general, sperm cells are identified by microscopic analysis; however, this conventional method takes time and effort, even for trained personnel. Here, we present a reverse transcription-recombinase polymerase amplification (RT-RPA) assay targeting sperm mRNA marker (PRM1). The RT-RPA assay requires only 40 min for PRM1 detection and demonstrates a sensitivity of 0.1 μL of semen. Our results indicate that the RT-RPA assay may be a rapid, simple, and specific strategy for screening sperm cells in sexual assault samples.

Development of Reverse Transcription Recombinase Polymerase Amplification (RT-RPA): A Methodology for Quick Diagnosis of Potato Leafroll Viral Disease in Potato.

Potatoes are developed vegetatively from tubers, and therefore potato virus transmission is always a possibility. The potato leafroll virus (PLRV) is a highly devastating virus of the genus Polerovirus and family Luteoviridae and is regarded as the second-most destructive virus after Potato virus Y. Multiple species of aphids are responsible for the persistent and non-propagating transmission of PLRV. Due to intrinsic tuber damage (net necrosis), the yield and quality are drastically diminished. PLRV is mostly found in phloem cells and in extremely low amounts. Therefore, we have attempted to detect PLRV in both potato tuber and leaves using a highly sensitive, reliable and cheap method of one-step reverse transcription-recombinase polymerase amplification (RT-RPA). In this study, an isothermal amplification and detection approach was used for efficient results. Out of the three tested primer sets, one efficiently amplified a 153-bp product based on the coat protein gene. In the present study, there was no cross-reactivity with other potato viruses and the optimal amplification reaction time was thirty minutes. The products of RT-RPA were amplified at a temperature between 38 and 42 °C using a simple heating block/water bath. The present developed protocol of one-step RT-RPA was reported to be highly sensitive for both leaves and tuber tissues equally in comparison to the conventional reverse transcription-polymerase chain reaction (RT-PCR) method. By using template RNA extracted employing a cellular disc paper-based extraction procedure, the method was not only simplified but it detected the virus as effectively as purified total RNA. The simplified one-step RT-RPA test was proven to be successful by detecting PLRV in 129 samples of various potato cultivars (each consisting of leaves and tubers). According to our knowledge, this is the first report of a one-step RT-RPA performed using simple RNA extracted from cellular disc paper that is equally sensitive and specific for detecting PLRV in potatoes. In terms of versatility, durability and the freedom of a highly purified RNA template, the one-step RT-RPA assay exceeds the RT-PCR assay, making it an effective alternative for the certification of planting materials, breeding for virus resistance and disease monitoring.

Application of Rapid and Reliable Detection of Cymbidium Mosaic Virus by Reverse Transcription Recombinase Polymerase Amplification Combined with Lateral Flow Immunoassay

Cymbidium mosaic virus (CymMV) is one of economically important viruses that cause significant losses of orchids in the world. In the present study, a reverse transcription recombinase polymerase amplification (RT-RPA) assay combined with a lateral flow immunostrip (LFI) assay was developed for the detection of CymMV in orchid plants. A pair of primers containing fluorescent probes at each terminus that amplifies highly specifically a part of the coat protein gene of CymMV was determined for RT-RPA assay. The RT-RPA assay involved incubation at an isothermal temperature (39°C) and could be performed rapidly within 30 min. In addition, no cross-reactivity was observed to occur with odontoglossum ringspot virus and cymbidium chlorotic mosaic virus. The RT-RPA with LFI assay (RT-RPA-LFI) for CymMV showed 100 times more sensitivity than conventional reverse transcription polymerase chain reaction (RT-PCR). Furthermore, the RT-PCR-LFI assay demonstrated the simplicity and the rapidity of CymMV detection since the assay did not require any equipment, by comparing results with those of conventional RT-PCR. On-site application of the RT-RPA-LFI assay was validated for the detection of CymMV in field-collected orchids, indicating a simple, rapid, sensitive, and reliable method for detecting CymMV in orchids.

Improved reverse transcription-recombinase polymerase amplification assay for blood mRNA screening: comparison with one-step RT-qPCR assay

mRNA profiling is effective for body fluid identification because of its sensitivity, specificity, and multiplexing capability. Body fluid mRNA markers can typically be detected using RT-qPCR, RT-PCR followed by capillary electrophoresis, or targeted RNA sequencing. However, due to the multiple handling steps involved, the analysis of many forensic samples using these methods requires time and effort. Here, we describe a rapid and simple method for detecting the blood mRNA marker hemoglobin β (HBB), intended for use in screening before definitive blood identification. We employed a reverse transcription-recombinase polymerase amplification (RT-RPA) assay that can detect target mRNA within 20 min in a single tube. For comparison, we used a one-step RT-qPCR assay. We optimized the RT-RPA assay and found that it could detect HBB from 10−3–10−4 ng of leukocyte RNA and approximately 10−3 µL of blood. The sensitivity was 10-fold lower than that of the one-step RT-qPCR assay but higher than that of the comprehensive analysis methods for definitive blood identification. Thus, the rapidity and sensitivity of the RT-RPA assay support its use as a screening tool. We also found that the RT-RPA assay was highly tolerant to common inhibitors such as humic acid, hematin, tannic acid, and melanin. Considering the inhibitor tolerability, we integrated a simple lysis method (addition of TCEP/EDTA and heating at 95 °C for 5 min) without the RNA purification process into the RT-RPA assay. This direct assay successfully detected HBB in crude blood samples. Our findings suggest that the RT-RPA assay for HBB is a promising strategy for mRNA-based blood screening.

Sensitive and rapid RT-RPA-Cas12a-mediated detection method capable of human rhinovirus A and/or C species by targeting VP4

Human rhinovirus (HRV), the main etiologic agent of the common cold, is responsible for significant morbidity, medical costs, and the loss of productivity in the workplace and school. To prevent the spread of HRV, accurate, low-cost and rapid diagnostics of HRV is crucial for identifying those at-risk for the illness associated with HRV, with the most frequently detected species, including HRV species A (HRV-A) and C (HRV-C). Here, a novel HRV-A and/or HRV-C molecular diagnostic assay that integrates reverse-transcription recombinase polymerase amplification assay (RT-RPA) amplification with CRISPR/Cas12a detection, with the result readout using a fluorescence detector or lateral flow strip (LFS). The established assay could be completed within 50 min without complex instruments and skilled technicians. The limit of detection of the RT-RPA-Cas12a-mediated real-time fluorescence or LFS assay could reach 0.1 copy/μl, and 0.5 copy/μl for the end-point fluorescence assay with a UV light illuminator readout, respectively. Meanwhile, the assay demonstrates excellent specificity without cross-reactivity to non-target viruses. Furthermore, they were appraised using 80 clinical samples, and RT-RPA-Cas12a-mediated fluorescence or LFS assay displayed high-accuracy with positive and negative predictive agreement of 96.7%, 95% and 100%, respectively. Taken together, the RT-RPA-Cas12a-mediated assay is a rapid, sensitive, and specific detection tool for routine and on-site detection method for HRV-A and/or HRV-C infections, and shows great promise for use in resource-poor or constrained settings.

Rapid detection of hepatitis C virus using recombinase polymerase amplification.

Over 71 million people are infected with hepatitis C virus (HCV) worldwide, and approximately 400,000 global deaths result from complications of untreated chronic HCV. Pan-genomic direct-acting antivirals (DAAs) have recently become widely available and feature high cure rates in less than 12 weeks of treatment. The rollout of DAAs is reliant on diagnostic tests for HCV RNA to identify eligible patients with viremic HCV infections. Current PCR-based HCV RNA assays are restricted to well-resourced central laboratories, and there remains a prevailing clinical need for expanded access to decentralized HCV RNA testing to provide rapid chronic HCV diagnosis and linkage to DAAs in outpatient clinics. This paper reports a rapid, highly accurate, and minimally instrumented assay for HCV RNA detection using reverse transcription recombinase polymerase amplification (RT-RPA). The assay detects all HCV genotypes with a limit of detection of 25 copies per reaction for genotype 1, the most prevalent in the United States and worldwide. The clinical sensitivity and specificity of the RT-RPA assay were both 100% when evaluated using 78 diverse clinical serum specimens. The accuracy, short runtime, and low heating demands of RT-RPA may enable implementation in a point-of-care HCV test to expand global access to effective treatment via rapid chronic HCV diagnosis.

Rapid detection of human coronavirus NL63 by isothermal reverse transcription recombinase polymerase amplification

BackgroundHuman coronaviruses are one of the leading causes for respiratory tract infections and for frequent primary care consultation. The human coronavirus NL63 (HCoV..µNL63) is one representative of the seasonal coronaviruses and capable of infecting the upper and lower respiratory tract and causative agent for croup in children. ObjectivesFor fast detection of HCoV-NL63, we developed an isothermal reverse transcription recombinase polymerase amplification (RT-RPA) assay. Study designThe analytical sensitivities of the RT-RPA assay were identified for in vitro transcribed ribonucleic acid (RNA) and for genomic viral RNA from cell culture supernatant. Moreover, specificity was tested with nucleic acids from other human coronaviruses and a variety of clinically relevant respiratory viruses. Finally, a clinical nasopharyngeal swab sample with spiked genomic viral HCoV-NL63 RNA was analyzed. ResultsOur HCoV-NL63 RT-RPA assay is highly specific and has an analytical sensitivity of 13 RNA molecules/reaction for in vitro transcribed RNA. For genomic viral RNA from cell culture supernatant spiked into a clinical nasopharyngeal swab sample the assay...s analytical sensitivity is 170 RNA molecules/reaction. The assay shows amplification of the lowest detectable target copy number after 8 minutes and 7 minutes, respectively. ConclusionsWe were able to design a sensitive and specific RT-RPA assay for the detection of HCoV-NL63. Additionally, the assay is characterized by short duration, isothermal amplification, and simple instrumentation.

Clinical Validation of a Rapid Variant-Proof RT-RPA Assay for the Detection of SARS-CoV-2.

The COVID-19 pandemic has unveiled a pressing need to expand the diagnostic landscape to permit high-volume testing in peak demand. Rapid nucleic acid testing based on isothermal amplification is a viable alternative to real-time reverse transcription polymerase chain reaction (RT-PCR) and can help close this gap. With the emergence of SARS-CoV-2 variants of concern, clinical validation of rapid molecular tests needs to demonstrate their ability to detect known variants, an essential requirement for a robust pan-SARS-CoV-2 assay. To date, there has been no clinical validation of reverse transcription recombinase polymerase amplification (RT-RPA) assays for SARS-CoV-2 variants. We performed a clinical validation of a one-pot multi-gene RT-RPA assay with the E and RdRP genes of SARS-CoV-2 as targets. The assay was validated with 91 nasopharyngeal samples, with a full range of viral loads, collected at University College London Hospitals. Moreover, the assay was tested with previously sequenced clinical samples, including eleven lineages of SARS-CoV-2. The rapid (20 min) RT-RPA assay showed high sensitivity and specificity, equal to 96% and 97%, respectively, compared to gold standard real-time RT-PCR. The assay did not show cross-reactivity with the panel of respiratory pathogens tested. We also report on a semi-quantitative analysis of the RT-RPA results with correlation to viral load equivalents. Furthermore, the assay could detect all eleven SARS-CoV-2 lineages tested, including four variants of concern (Alpha, Beta, Delta, and Omicron). This variant-proof SARS-CoV-2 assay offers a significantly faster and simpler alternative to RT-PCR, delivering sensitive and specific results with clinical samples.

Rapid detection of blood and semen mRNA markers by reverse transcription-recombinase polymerase amplification

Body fluid identification is crucial for crime scene reconstruction. Recently, messenger RNA (mRNA) profiling has been an effective approach for body fluid identification. In general, mRNA is detected by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) or end-point RT-PCR; however, these conventional methods are time-consuming and require extensive sample processing. Therefore, we developed a rapid and simple method for the detection of blood and semen mRNA markers by reverse transcription-recombinase polymerase amplification (RT-RPA). First, we screened mRNA markers for blood and semen and selected hemoglobin beta (HBB) and protamine 1 (PRM1), respectively, based on amplification specificity. Under optimized conditions, our RT-RPA assay detected HBB and PRM1 mRNAs within 20 min at a constant temperature of 42 °C. The detection limits for the assay were 0.01 ng/µL leukocyte RNA for HBB and 0.2 ng/µL semen RNA for PRM1. In addition, our RT-RPA assay exhibited high specificity and accuracy for HBB and PRM1 mRNA detection from mixed samples. Furthermore, as RPA has been reported to possess inhibitor tolerance, we evaluated the feasibility of direct RT-RPA for HBB mRNA detection. This direct approach reduced the number of processing steps and time required for template preparation and enabled the successful detection of HBB mRNA within 45 min from sample preparation. These findings suggest that RT-RPA is a useful method for mRNA-based blood and semen identification.

Rapid diagnostic detection of tomato apical stunt viroid based on isothermal reverse transcription-recombinase polymerase amplification

Tomato apical stunt viroid (TASVd) is a serious threat to tomato plants that can cause a considerable yield loss. In the present study, two isothermal molecular diagnostic assays based on reverse transcription-recombinase polymerase amplification (RT-RPA) utilizing the AmplifyRP® platform for plant pathogen detection were developed. The results of this research demonstrated distinct specificity of both developed assays, AmplifyRP® Acceler8™ and AmplifyRP® XRT, expressed in the absence of any cross-reaction activity to all total RNA extracts obtained from plants infected with other pospiviroids. The RT-RPA assays detected viroid RNA in 81- and 27-fold dilutions of the original TASVd-infected crude extract for AmplifyRP® Acceler8™ and AmplifyRP® XRT, respectively. The sensitivity tests in serial water dilutions showed the ability of AmplifyRP® Acceler8™ and AmplifyRP® XRT to detect 8 and 80 fg of pure TASVd RNA transcript, respectively. The influence of crude extract on viroid RNA transcript detection was also examined and a decrease of sensitivity of approximately 100-fold for both RT-RPA assays was revealed. To our knowledge, this is the first report describing development of RT-RPA assays to detect TASVd in plants using the AmplifyRP® platform that can be further employed both in laboratory conditions and in the field for on-site diagnosis.

Characterization of an Isolate of Citrus Concave Gum-Associated Virus from Apples in China and Development of an RT-RPA Assay for the Rapid Detection of the Virus.

Apple (Malus domestica) fruits exhibiting bright stripe symptoms were identified in Weihai City, Shandong Province, China. To investigate the virome in the apple samples, the method of high throughput sequencing (HTS) was used to identify the viruses. It was found that the sequence of citrus concave gum-associated virus (CCGaV) was involved in the apple transcriptome dataset. The full-length genome of the CCGaV-Weihai isolate contained two segments, the RNA1 was 6674 nt in size containing a conserved RNA-dependent RNA polymerase (RdRp), and the RNA2 was ambisense, 2706 nt in length, encoding a movement protein (MP) and a coat protein (CP). Sequence alignment and phylogenetic analyses indicated that CCGaV-Weihai was more closely related to CCGaV-H2799 isolated from the apple host in the United States and distantly related to CCGaV-CGW2 from Citrus sinensis in Italy, indicating a possibly geographical and host differentiation of CCGaV isolates. This was the first identification and characterization of CCGaV infecting apples in China. Additionally, a rapid and sensitive reverse transcription recombinase polymerase amplification (RT-RPA) assay technique was established for CCGaV detection in apple plants. The RT-RPA of CCGaV was not affected by other common viruses in apple plants and is about 10-fold more sensitive than the conventional reverse transcription polymerase chain reaction method, which can be used in large-scale testing.

Reverse Transcription Recombinase Polymerase Amplification Assay for Rapid Detection of Avian Influenza Virus H9N2 HA Gene

The H9N2 subtype of avian influenza A virus (aIAV) is circulating among birds worldwide, leading to severe economic losses. H9N2 cocirculation with other highly pathogenic aIAVs has the potential to contribute to the rise of new strains with pandemic potential. Therefore, rapid detection of H9 aIAVs infection is crucial to control virus spread. A qualitative reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of aIAV subtype H9N2 was developed. All results were compared to the gold standard (real-time reverse transcription polymerase chain reaction (RT-PCR)). The RT-RPA assay was designed to detect the hemagglutinin (HA) gene of H9N2 by testing three pairs of primers and a probe. A serial concentration between 106 and 100 EID50 (50% embryo infective dose)/mL was applied to calculate the analytical sensitivity. The H9 RT-RPA assay was highly sensitive as the lowest concentration point of a standard range at one EID50/mL was detected after 5 to 8 min. The H9N2 RT-RPA assay was highly specific as nucleic acid extracted from H9 negative samples and from other avian pathogens were not cross detected. The diagnostic sensitivity when testing clinical samples was 100% for RT-RPA and RT-PCR. In conclusion, H9N2 RT-RPA is a rapid sensitive and specific assay that easily operable in a portable device for field diagnosis of aIAV H9N2.

Rapid and sensitive detection of potato virus X by one-step reverse transcription-recombinase polymerase amplification method in potato leaves and dormant tubers

Potato virus X (PVX), is a serious threat to global potato production. A simple and rapid detection method is imperative for PVX diagnosis and early management. In this study, an isothermal one-step reverse transcription-recombinase polymerase amplification (RT-RPA) method was optimized for the quick and convenient detection of PVX in potato leaves and tubers. Our results revealed that this one-step RT-RPA method was highly efficient than the conventional reverse transcription-polymerase chain reaction (RT-PCR). The amplification reaction was free from cross-reactivity with other common potato viruses and completed within 30 min. Moreover, this RT-RPA assay did not require a thermocycler based specific temperature phase amplification and can be easily performed using a simple heating block or water bath at a temperature range of 39–42 °C. The sensitivity assay demonstrated that the developed one-step RT-RPA method was 100 times more sensitive than a routine one-step RT-PCR. Initially, the purified total RNA as the template isolated from infected leaves of potato was used for the detection of PVX. One-step RT-RPA was later performed using cellular disc paper-based simple RNA extract as a template that could detect the virus more efficiently than purified total RNA. The performance of the one-step RT-RPA assay was further evaluated using 500 field samples of leaves and tubers representing different cultivars and geographical regions. To our knowledge, this is the first report of rapid, sensitive, and reliable detection of PVX infection by one-step RT-RPA using cellular disc paper-based simple RNA extract from leaves and dormant tubers of potato. It is superior to the common RT-PCR assay in terms of its versatility, quickness, and independence of highly purified RNA template and can be adopted as a substitute to RT-PCR as an effective technique for seed potato certification, quarantine, breeding, and field surveys.