ICTV Virus Taxonomy Profile: Fimoviridae 2024.

Members of the family Fimoviridae are plant viruses with a multipartite negative-sense enveloped RNA genome (-ssRNA), composed of 4-10 segments comprising 12.3-18.5 kb in total, within quasi-spherical virions. Fimoviruses are transmitted to plants by eriophyid mites and induce characteristic cytopathologies in their host plants, including double membrane-bound bodies in the cytoplasm of virus-infected cells. Most fimoviruses infect dicotyledonous plants, and many cause serious disease epidemics. This is a summary of the ICTV Report on the family Fimoviridae, which is available at ictv.global/report/fimoviridae.

PCR-based detection for the quarantine fungus Colletotrichum kahawae, a biosecurity threat to the coffee (Coffea arabica) industry worldwide.

Coffee berry disease (CBD) is caused by Colletotrichum kahawae, a quarantine fungus still absent from most coffee-producing countries. Given the potential adverse effects on coffee berry production, it is a severe worldwide threat to farmers and industry. Current biosecurity management focuses on exclusion by applying quarantine measures, including certification of coffee plants and their products. However, methods for detecting C. kahawae by the NPPO (National Plant Protection Organization) laboratories still need approval. This research aims to functionally demonstrate, standardize, and validate a method for detecting and discriminating C. kahawae from other Colletotrichum species that may be present in coffee plant samples. The method proposes to use an end-point PCR marker for the mating type gene (MAT1-2-1) and a confirmatory test with a qPCR marker developed on the glutamine synthetase (GS) gene. The C. kahawae amplicons for the Cen-CkM10 marker exhibited specific melting temperature (Tm) values that could be readily differentiated from other tested species, including their relatives. Given the fungus's quarantine status, specificity was tested using artificial mixtures of DNA of C. kahawae with other Colletotrichum species and coffee plant DNA. The described method will enable NPPOs in coffee producing and exporting countries, especially Colombia, to prevent this pathogen's entry, establishment, and spread.

In-silico prediction of RT-qPCR-high resolution melting for broad detection of emaraviruses.

Twenty-four species of RNA viruses contain members infecting economically important crops that are classified within the genus Emaravirus, family Fimoviridae. There are at least two other non-classified species that may be added. Some of these viruses are spreading rapidly and cause economically important diseases on several crops, raising a need for a sensitive diagnostic technique for taxonomic and quarantine purposes. High-resolution melting (HRM) has shown to be reliable for the detection, discrimination, and diagnosis of several diseases of plants, animals, and humans. This research aimed to explore the ability to predict HRM outputs coupled to reverse transcription-quantitative polymerase chain reaction (RT-qPCR). To approach this goal a pair of degenerate genus-specific primers were designed for endpoint RT-PCR and RT-qPCR-HRM and the species in the genus Emaravirus were selected to framework the development of the assays. Both nucleic acid amplification methods were able to detect in-vitro several members of seven Emaravirus species with sensitivity up to one fg of cDNA. Specific parameters for in-silico prediction of the melting temperatures of each expected emaravirus amplicon are compared to the data obtained in-vitro. A very distinct isolate of the High Plains wheat mosaic virus was also detected. The high-resolution DNA melting curves of the RT-PCR products predicted in-silico using uMeltSM allowed saving time while designing and developing the RT-qPCR-HRM assay since the approach avoided extensive searching for optimal HRM assay regions and rounds of HRM tests in-vitro for optimization. The resultant assay provides sensitive detection and reliable diagnosis for potentially any emaravirus, including new species or strains.

Loop-mediated isothermal amplification (LAMP) assay for specific and rapid detection of Dickeya fangzhongdai targeting a unique genomic region.

Dickeya fangzhongdai, a bacterial pathogen of taro (Colocasia esculenta), onion (Allium sp.), and several species in the orchid family (Orchidaceae) causes soft rot and bleeding canker diseases. No field-deployable diagnostic tool is available for specific detection of this pathogen in different plant tissues. Therefore, we developed a field-deployable loop-mediated isothermal amplification (LAMP) assay using a unique genomic region, present exclusively in D. fangzhongdai. Multiple genomes of D. fangzhongdai, and other species of Dickeya, Pectobacterium and unrelated genera were used for comparative genomic analyses to identify an exclusive and conserved target sequence from the major facilitator superfamily (MFS) transporter gene region. This gene region had broad detection capability for D. fangzhongdai and thus was used to design primers for endpoint PCR and LAMP assays. In-silico validation showed high specificity with D. fangzhongdai genome sequences available in the NCBI GenBank genome database as well as the in-house sequenced genome. The specificity of the LAMP assay was determined with 96 strains that included all Dickeya species and Pectobacterium species as well as other closely related genera and 5 hosts; no false positives or false negatives were detected. The detection limit of the assay was determined by performing four sensitivity assays with tenfold serially diluted purified genomic DNA of D. fangzhongdai with and without the presence of crude host extract (taro, orchid, and onion). The detection limit for all sensitivity assays was 100 fg (18-20 genome copies) with no negative interference by host crude extracts. The assays were performed by five independent operators (blind test) and on three instruments (Rotor-Gene, thermocycler and dry bath); the assay results were concordant. The assay consistently detected the target pathogen from artificially inoculated and naturally infected host samples. The developed assay is highly specific for D. fangzhongdai and has applications in routine diagnostics, phytosanitary and seed certification programs, and epidemiological studies.

Molecular and biological characterization of an isolate of the potyvirus passiflora virus Y naturally infecting soybean (Glycine max) in Brazil.

Passiflora virus Y was detected naturally infecting soybean (Glycine max) for the first time in Brazil. Here, we report the nearly complete genome sequence and molecular and biological properties of the PaVY-Br isolate. The nearly complete genome sequence is 9679 nt long and shares 84.4% nt sequence identity with a previously reported PaVY isolate from Passiflora sp. PaVY-Br induced chlorotic spots and systemic mosaic on soybean and chlorotic local lesions on yellow passion fruit (Passiflora edulis) and sesame (Sesamum indicum). The virus was successfully transmitted by Myzus persicae, indicating that this aphid vector can contribute to the spread of PaYV from passion fruit to soybean plants. Additional epidemiological research is in progress to investigate the distribution of PaVY in soybean production areas in Brazil.

An elution independent collection device (EICD) for rapid collection of Anaplasma marginale DNA from blood samples.

Currently utilized molecular detection methods are based mainly on nucleic acid extraction, amplification, and detection procedures that may require costly equipment, numerous reagents, and highly trained personnel. These requirements make diagnostic tests expensive, time-consuming, and not suitable for point-of-care applications. There is an increasing demand for simple, low-cost portable technologies. To overcome these challenges, a paper-based elution independent collection device (EICD) was designed to collect microorganisms and recover nucleic acids for molecular biology applications with minimal steps. In this study, we demonstrate a simpler Anaplasma marginale detection that uses an EICD for nucleic acid collection combined with recombinase polymerase amplification (RPA), and a lateral flow dipstick for detection of the specified target. A pre-lysis blood treatment was optimized that uses Triton X-100 lysis buffer and bovine serum album in wash buffer. Blood samples were incubated for 5 min at room temperature and run through the EICD. Four 1-mm diameter discs excised from EICD were used as template in basic RPA and lateral flow (nfo) (endonuclease IV) RPA assays. Each disc of soluble central membrane (SCM) carried circa 0.249 pg/µl of Anaplasma DNA. The percentage of nucleic acid recoverable from the SCM ranged between 60% - 70%. Blood samples infected with A. marginale were treated with Triton X-100 pre-lysis protocol. All samples tested positive by PCR and RPA methods. EICD-driven collection of blood samples is a practical method successfully adapted to detect Anaplasma spp. or blood-borne pathogen DNA and has potential for point-of-care detection in resource-limited settings.

Exploring in-silico prediction for the development of a RT-qPCR-high resolution melting assay for the broad detection of emaraviruses.

High-resolution melting (HRM) has shown to be reliable for the detection, discrimination, and diagnosis of several diseases of plants, animals, and humans. The aim of this research was to explore the ability to predict HRM outputs when coupled to reverse transcription quantitative polymerase chain reaction (RT-qPCR). This research used the species in the Emaravirus genus as model to framework the development of genus-specific RT-qPCR-HRM assays. A pair of degenerate genus-specific primers were designed for use in endpoint RT-PCR and RT-qPCR-HRM detection of emaraviruses. Eleven species of RNA viruses infecting economically important crops are classified within the genus Emaravirus, family Fimoviridae. There are at least fifteen other non-classified species that may be added. Some of these viruses are spreading rapidly and cause economically important diseases on several crops, raising a need for a sensitive diagnostic technique for taxonomic and quarantine purposes. RT-PCR and RT-qPCR-HRM were able to detect seven emaravirus species in-vitro with sensitivity up to one fg of cDNA. Specific parameters for prediction in-silico of the melting temperatures of each expected emaravirus amplicon are provided and compared to the data obtained in-vitro. A very distinct isolate of the High Plains wheat mosaic virus was also detected. The prediction in-silico of fluorescence of high-resolution DNA melting curves of predicted RT-PCR products using uMeltSM speeded the design and development of RT-qPCR-HRM assay. This approach avoided rounds of HRM tests in-vitro when searching for the optimal regions that provides accurate diagnosis. The resultant assay provided sensitive detection and reliable diagnosis for potentially any emaravirus, including new species or strains.

Probing Loop-Mediated Isothermal Amplification (LAMP) targeting two gene-fragments of rose rosette virus.

This study explores the development of Loop-mediated isothermal amplification (LAMP) for detection of rose rosette virus (RRV), a technique with the potential to be translated to rose nurseries. RRV is a negative-sense, single-stranded RNA virus which is a member of the genus Emaravirus (Family Fimoviridae) and the causal agent of the rose rosette disease (RRD). Although RRV symptoms are characteristics, early visual diagnosis of RRD can be misleading and confusing since it may appear like herbicide damage. Moreover, it may take incubation time for symptoms to appear after virus infection. Two sets of RRV gene sequences RNA3 and RNA4 were analyzed and two sets of four LAMP primers were designed. The direct antigen-capture method for direct trapping of RRV in plastic was used for RNA extraction followed by cDNA synthesis. RT-LAMP reactions were for 1 hour at 64°C (RRV-P3) and 66.5°C (RRV-P4) using either a thermocycler or a portable dry bath. RT-qLAMP was also optimized using DNA polymerase GspSSD LD using the same RRV sets of primers. RRV was detected in symptomatic and non-symptomatic RRD tissue from Oklahoma. The limit of detection (LoD) was 1pg/µL and 1 fg/µL using Bst 2.0 LAMP and GspSSD LD quantitative LAMP, respectively. In visual colorimetric pre- and post-reactions, the LoD was 10 pg/µL and 0.1 pg/µL using hydroxy naphthol blue (HNB, 120 µM) and SYBR green I (1:10 dilution), respectively. No cross-reactivity was detected in the RT-LAMP reaction testing cDNAs of eight commonly co-infecting rose viruses and one virus taxonomically related to RRV. Four different dyes were tested, and visible colorimetric reactions were obtained with RT-LAMP Bst 2.0 combined with SYBR I or HNB. RT-qLAMP with GspSSD2.0 offers LoD equal to RT-PCR and it is faster since it works with RNA directly.

Genome-informed loop-mediated isothermal amplification assay for specific detection of Pectobacterium parmentieri in infected potato tissues and soil.

Pectobacterium parmentieri (formerly Pectobacterium wasabiae), which causes soft rot disease in potatoes, is a newly established species of pectinolytic bacteria within the family Pectobacteriaceae. Despite serious damage caused to the potato industry worldwide, no field-deployable diagnostic tests are available to detect the pathogen in plant samples. In this study, we aimed to develop a reliable, rapid, field-deployable loop-mediated isothermal amplification (LAMP) assay for the specific detection of P. parmentieri. Specific LAMP primers targeting the petF1 gene region, found in P. parmentieri but no other Pectobacterium spp., were designed and validated in silico and in vitro using extensive inclusivity (15 strains of P. parmentieri) and exclusivity (94 strains including all other species in the genus Pectobacterium and host DNA) panels. No false positives or negatives were detected when the assay was tested directly with bacterial colonies, and with infected plant and soil samples. Sensitivity (analytical) assays using serially diluted bacterial cell lysate and purified genomic DNA established the detection limit at 10 CFU/mL and 100 fg (18-20 genome copies), respectively, even in the presence of host crude DNA. Consistent results obtained by multiple users/operators and field tests suggest the assay's applicability to routine diagnostics, seed certification programs, biosecurity, and epidemiological studies.

Recombinase polymerase amplification (RPA) with lateral flow detection for three Anaplasma species of importance to livestock health.

Anaplasma marginale, A. ovis, and A. phagocytophilum are the causative agents of bovine anaplasmosis, ovine anaplasmosis, and granulocytic anaplasmosis, respectively. The gold standard for diagnosis of post-acute and long-term persistent infections is the serological cELISA, which does not discriminate between Anaplasma species and requires highly equipped laboratories and trained personnel. This study addresses the development of a rapid, isothermal, sensitive, species-specific RPA assays to detect three Anaplasma species in blood and cELISA A. marginale-positive serum samples. Three RPA primer and probe sets were designed targeting msp4 genes of each Anaplasma species and the internal control (GAPDH gene) for each assay. The limit of detection of gel-based or RPA-basic assays is 8.99 × 104 copies/µl = A. marginale, 5.04 × 106 copies/µl = A. ovis, and 4.58 × 103 copies/µl = A. phagocytophilum, and for each multiplex lateral flow or RPA-nfo assays is 8.99 × 103 copies/µl of A. marginale, 5.04 × 103 copies/µl of A. ovis, 4.58 × 103 copies/µl of A. phagocytophilum, and 5.51 × 103 copies/µl of internal control (GAPDH). Although none of the 80 blood samples collected from Oklahoma cattle were positive, the RPA-nfo assays detected all A. marginale cattle blood samples with varying prevalence rates of infection, 83% of the 24 cELISA A. marginale-positive serum samples, and all A. phagocytophilum cell culture samples. Overall, although early detection of three Anaplasma species was not specifically addressed, the described RPA technique represents an improvement for detection of three Anaplasma in regions where access to laboratory equipment is limited.

TASPERT: Target-Specific Reverse Transcript Pools to Improve HTS Plant Virus Diagnostics.

High-throughput sequencing (HTS) is becoming the new norm of diagnostics in plant quarantine settings. HTS can be used to detect, in theory, all pathogens present in any given sample. The technique's success depends on various factors, including methods for sample management/preparation and suitable bioinformatic analysis. The Limit of Detection (LoD) of HTS for plant diagnostic tests can be higher than that of PCR, increasing the risk of false negatives in the case of low titer of the target pathogen. Several solutions have been suggested, particularly for RNA viruses, including rRNA depletion of the host, dsRNA, and siRNA extractions, which increase the relative pathogen titer in a metagenomic sample. However, these solutions are costly and time-consuming. Here we present a faster and cost-effective alternative method with lower HTS-LoD similar to or lower than PCR. The technique is called TArget-SPecific Reverse Transcript (TASPERT) pool. It relies on pathogen-specific reverse primers, targeting all RNA viruses of interest, pooled and used in double-stranded cDNA synthesis. These reverse primers enrich the sample for only pathogens of interest. Evidence on how TASPERT is significantly superior to oligodT, random 6-mer, and 20-mer in generating metagenomic libraries containing the pathogen of interest is presented in this proof of concept.

Exploring the Use of High-Resolution Melting Analysis and Helicase-Dependent Amplification for Discrimination of Bemisia tabaci (Hemiptera: Aleyrodidae) Cryptic Species and Trialeurodes vaporariorum.

The sweetpotato whitefly, Bemisia tabaci (Gennadius) (Hemiptera; Aleyrodidae), and greenhouse whitefly, Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae), are highly problematic plant pests and virus vectors with worldwide distributions. Identification of whitefly species is typically accomplished by observation of distinct morphological characters; however, because of morphological inconsistency and indistinguishability, the discrimination of B. tabaci species variants is dependent on molecular techniques based on genetic differences. New assays were designed for the detection of B. tabaci A, B, and Q mitotype groups, and T. vaporariorum. Specific primer sets were designed for amplification of the mitochondrial cytochrome c oxidase I gene of the four targets to perform in end-point PCR, real-time PCR coupled to high-resolution melting analysis (HRM), and the isothermal helicase-dependent amplification (HDA). Primer specificities were validated using end-point PCR, then tested in HRM and HDA. Bemisia tabaci A, B, and Q mitotypes, and T. vaporariorum-targeted primer sets discriminately amplified specimens of different populations within their target whitefly group. These tests provide three novel discrimination assays for the high-consequence, exotic B. tabaci B and Q groups, along with the native B. tabaci A group and T. vaporariorum.

Combining multiplex PCR and high-resolution melting for the detection and discrimination of arthropod transmitted viruses of cereals.

The Great Plains of the United States is a region comprised of approximately 45 million hectares of grasslands where several economically important cereal crops are grown. Arthropod-transmitted, cereal-infecting viruses vary in incidence from year-to-year and are often difficult to detect in large acreages. To facilitate the detection of economically important viruses of cereals that often exist in co-infections, a multiplex reverse transcriptase PCR (RT-PCR) platform assay was developed. This method can be used in combination with high resolution melting (HRM) to detect and allow for discrimination between three arthropod-transmitted plant viruses; Wheat streak mosaic virus (WSMV), Maize mosaic virus (MMV) and Barley yellow dwarf virus (BYDV). Multiplex PCR in combination with HRM allowed for successful detection of WSMV, MMV, and BYDV, as well as discrimination between three BYDV species, BYDV-PAS, BYDV-PAV and BYDV-MAV. All primer pairs amplified products of the predicted size. The BYDV-RT-PCR primers amplified products of identical length for all three species of BYDV. HRM was then used to discriminate between these products by determining significant differences between the melting rates for each (p < 0.05). This study demonstrates the flexibility of combining multiplex PCR with HRM to increase the specificity of plant virus diagnostics based on the needs of the diagnostician performing the assay.

Recombinase polymerase amplification applied to plant virus detection and potential implications.

Several isothermal techniques for the detection of plant pathogens have been developed with the advent of molecular techniques. Among them, Recombinase Polymerase Amplification (RPA) is becoming an important technique for the rapid, sensitive and cost-effective detection of plant viruses. The RPA technology has the advantage to be implemented in field-based scenarios because the method requires a minimal sample preparation, and is performed at constant low temperature (37-42 °C). The RPA technique is rapidly becoming a promising tool for use in rapid detection and further diagnostics in plant clinics and monitoring quarantine services. This paper presents a review of studies conducted using RPA for detection/diagnosis of plant viruses with either DNA genomes (Banana bunchy top virus, Bean golden yellow mosaic virus, Tomato mottle virus, Tomato yellow leaf curl virus) or RNA genomes (Little Cherry virus 2, Plum pox virus and Rose rosette virus).

Development of a loop-mediated isothermal amplification (LAMP) assay for rapid screening of ticks and fleas for spotted fever group rickettsia.

BACKGROUND: The importance of tick and flea-borne rickettsia infections is increasingly recognized worldwide. While increased focus has shifted in recent years to the development of point-of-care diagnostics for various vector-borne diseases in humans and animals, little research effort has been devoted to their integration into vector surveillance and control programs, particularly in resource-challenged countries. One technology which may be helpful for large scale vector surveillance initiatives is loop-mediated isothermal amplification (LAMP). The aim of this study was to develop a LAMP assay to detect spotted fever group (SFG) rickettsia DNA from field-collected ticks and fleas and compare with published end-point PCR results. METHODOLOGY/PRINCIPAL FINDINGS: A Spotted Fever Group rickettsia-specific loop-mediated isothermal amplification (SFGR-LAMP) assay was developed using primers based on a region of the R. rickettsii 17kDa protein gene. The sensitivity, specificity, and reproducibility of the assay were evaluated. The assay was then compared with the results of end-point PCR assays for pooled tick and flea samples obtained from field-based surveillance studies. The sensitivity of the SFGR-LAMP assay was 0.00001 ng/µl (25µl volume) which was 10 times more sensitive than the 17kDa protein gene end-point PCR used as the reference method. The assay only recognized gDNA from SFG and transitional group (TRG) rickettsia species tested but did not detect gDNA from typhus group (TG) rickettsia species or closely or distantly related bacterial species. The SFGR-LAMP assay detected the same positives from a set of pooled tick and flea samples detected by end-point PCR in addition to two pooled flea samples not detected by end-point PCR. CONCLUSIONS/SIGNIFICANCE: To our knowledge, this is the first study to develop a functional LAMP assay to initially screen for SFG and TRG rickettsia pathogens in field-collected ticks and fleas. With a high sensitivity and specificity, the results indicate the potential use as a field-based surveillance tool for tick and flea-borne rickettsial pathogens in resource-challenged countries.

A field based detection method for Rose rosette virus using isothermal probe-based Reverse transcription-recombinase polymerase amplification assay.

Rose rosette disease, caused by Rose rosette virus (RRV; genus Emaravirus) is a major threat to the rose industry in the U.S. The only strategy currently available for disease management is early detection and eradication of the infected plants, thereby limiting its potential spread. Current RT-PCR based diagnostic methods for RRV are time consuming and are inconsistent in detecting the virus from symptomatic plants. Real-time RT-qPCR assay is highly sensitive for detection of RRV, but it is expensive and requires well-equipped laboratories. Both the RT-PCR and RT-qPCR cannot be used in a field-based testing for RRV. Hence a novel probe based, isothermal reverse transcription-recombinase polymerase amplification (RT-exoRPA) assay, using primer/probe designed based on the nucleocapsid gene of the RRV has been developed. The assay is highly specific and did not give a positive reaction to other viruses infecting roses belonging to both inclusive and exclusive genus. Dilution assays using the in vitro transcript showed that the primer/probe set is highly sensitive, with a detection limit of 1 fg/µl. In addition, a rapid technique for the extraction of viral RNA (<5min) has been standardized from RRV infected tissue sources, using PBS-T buffer (pH 7.4), which facilitates the virus adsorption onto the PCR tubes at 4°C for 2min, followed by denaturation to release the RNA. RT-exoRPA analysis of the infected plants using the primer/probe indicated that the virus could be detected from leaves, stems, petals, pollen, primary roots and secondary roots. In addition, the assay was efficiently used in the diagnosis of RRV from different rose varieties, collected from different states in the U.S. The entire process, including the extraction can be completed in 25min, with less sophisticated equipments. The developed assay can be used with high efficiency in large scale field testing for rapid detection of RRV in commercial nurseries and landscapes.

Evaluación de diferentes métodos de extracción de ARN a partir del hongo nativo Xylaria sp / Evaluation of different RNA extraction methods from the native fungus Xylaria sp

RESUMEN La extracción de ARN de calidad constituye el primer paso para el análisis de la expresión génica. Sin embargo, su obtención no es sencilla debido a la susceptibilidad de esta molécula a la presencia de contaminantes como ARNasas, proteínas y polisacáridos. Adicionalmente, debido a la diversa composición de la pared celular de los hongos se requiere optimizar los procesos de extracción de ARN para organismos específicos. Este estudio evalúo el uso de diferentes metodologías de homogeneización de tejido (nitrógeno líquido y liofilización) y extracción de ARN (Trizol, CTAB y RNeasy mini kit) a partir del hongo nativo ascomiceto Xylaria sp. Se determinó la pureza, concentración e integridad del ARN obtenido por medio de espectrofotometría y electroforesis. Adicionalmente, se diseñaron cebadores de referencia para el gen β-Tubulina a partir del alineamiento de secuencias de este gen obtenidas de diferentes ascomicetes. Estos cebadores fueron utilizados para evaluar si el ARN extraído es amplificable mediante RT-PCR. Se determinó que la homogeneización de tejido por medio de liofilización generó mayores rendimientos de extracción independientemente del protocolo de extracción utilizado; sin embargo, éstos alteraron la integridad del ARN. Se obtuvo un ARN con mayor pureza con el protocolo CTAB y un mayor rendimiento con el RNeasy mini kit. Los resultados indican que el ARN extraído, independientemente de la metodología de homogeneización y extracción utilizada, es amplificable mediante RT-PCR. No obstante, se recomienda homogeneizar el tejido con nitrógeno líquido y extraer con RNeasy mini kit por la brevedad del protocolo de extracción y calidad obtenida.

ABSTRACT Obtaining high quality RNA is the first step for gene expression analysis. However, the low stability of this molecule and high presence of contaminants such as RNases, proteins and polysaccharides may trouble extractions. Fungi cell wall composition is highly diverse; therefore optimizing RNA extraction procedures is necessary when studying specific organisms. In this study, different methods of tissue homogenization (liquid nitrogen and lyophilization) and RNA extraction (Trizol, CTAB and RNeasy mini kit) were assessed with a native ascomycete, Xylaria sp. RNA purity, concentration and integrity were determined by spectrophotometry and electrophoresis. In addition, a set of housekeeping gene primers was designed targeting the β-tubulin gene. The primers were used to determine if the RNA extracted allowed RT-PCR amplification. It was demonstrated that homogenization of tissue by lyophilization allowed higher yields of RNA regardless of the extraction protocol used, however, the RNA integrity was affected. The higher RNA purity was obtained using CTAB and the higher yields using the RNeasy mini kit. The extracted RNA is amplifiable by RT-PCR regardless of the homogenization and extraction methodology used. However, it is recommended to homogenize the tissue with liquid nitrogen and to extract RNA with the RNeasy mini kit due the shortness and efficiency of these protocols.

Single-Target and Multiplex Discrimination of Whiteflies (Hemiptera: Aleyrodidae) Bemisia tabaci and Trialeurodes vaporariorum With Modified Priming Oligonucleotide Thermodynamics.

The whitefly species Bemisia tabaci (Gennadius) and Trialeurodes vaporariorum (Westwood) are worldwide agricultural pests and virus vectors. Bemisia tabaci, in particular, is often transported internationally via trade routes leading to potential introductions of exotic whiteflies or plant viruses. Quick identification of agriculturally important whiteflies can facilitate interventions that prevent these cross-border introductions. Polymerase chain reaction (PCR) primers were designed to amplify the mitochondrial cytochrome oxidase I gene (mtCOI) sequence of members of the B. tabaci complex, MEAM1, MED, and NW, and T. vaporariorum. Primers incorporated an A/T-rich overhang sequence at the 5' terminus (5' flap) to test for increased primer sensitivity and assay efficiency. Single-target and multiplex endpoint PCR assays with the eight primer sets were performed using genomic DNA template extracted from individual adult whiteflies. Resultant PCR amplicons obtained for B. tabaci MEAM1, MED, and NW, and T. vaporariorum primers with the 5' flap were 559-, 717-, 353-, and 258-bp, respectively, and without the 5' flap were 550-, 712-, 329-, and 252-bp in length, respectively. In single-target and multiplex reactions, specific amplification was achieved using both the unmodified and 5' flap-modified primers. Sequencing and phylogenetic analysis confirmed primer-target amplification specificity. Using these primer sets in single-target or multiplex PCR allows for quick discrimination and specific identification of B. tabaci complex members and T. vaporariorum, and the addition of 5'A/T-rich overhang sequences increases the sensitivity and amplification of some primer sets.

A rapid assay for detection of Rose rosette virus using reverse transcription-recombinase polymerase amplification using multiple gene targets.

Rose rosette disease caused by Rose rosette virus (RRV; genus Emaravirus) is the most economically relevant disease of Knock Out® series roses in the U.S. As there are no effective chemical control options for the disease, the most critical disease management strategies include the use of virus free clean plants for propagation and early detection and destruction of infected plants. The current diagnostic techniques for RRV including end-point reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR (RT-qPCR) are highly sensitive, but limited to diagnostic labs with the equipment and expertise; and is time consuming. To address this limitation, an isothermal reverse transcription-recombinase polymerase amplification (RT-RPA) assay based on multiple gene targets for specific detection of RRV was developed. The assay is highly specific and did not cross react with other viruses belonging to the inclusive and exclusive genus. Dilution assays using the in vitro transcripts showed that the primer sets designed (RPA-267, RPA-131, and RPA-321) are highly sensitive, consistently detecting RRV with a detection limit of 1fg/µL. Testing of the infected plants using the primer sets indicated that the virus could be detected from leaves, stems and petals of roses. The primer pair RPA-267 produced 100% positive detection of the virus from infected leaf tissues, while primer set RPA-131 produced 100% detection from stems and petals. The primer set RPA-321 produced 83%, 87.5% and 75% positive detection from leaves, petals and stem tissues, respectively. In addition, the assay has been efficiently used in the detection of RRV infecting Knock Out® roses, collected from different states in the U.S. The assay can be completed in 20min as compared to the end-point RT-PCR assay (3-4h) and RT-qPCR (1.5h). The RT-RPA assay is reliable, rapid, highly sensitive, and can be easily used in diagnostic laboratories for detection of RRV with no need for any special equipment.