Flinders Technology Associates Cards Research Articles

A Comparison Study of Cryopreservation of Seminal Fluid between Flinders Technology Associates (FTA-card) and Swab for Multiplex Short Tandem Repeats (STR) Analysis

Background Whatman™ FTA (Flinders Technology Associates) is a form of card soaked in chemicals that denatures proteins while protecting DNA and ensuring the safe handling of dried body fluid spots (blood, semen, and saliva) and buccal cells. To this day, these cards are still infrequently used in forensic science. Therefore, reference samples including biological material may be collected on FTA cards for genetic analysis that have been widely used for DNA preservation and analysis, particularly in forensic sciences and genetic studies. The goal of the study was to evaluate the effects of cryopreservation on DNA quantitative and short tandem repeats (STR) profiling by comparing the yield and quality of DNA extracted before and after cryopreservation using FTA card and swabs in the forensic analysis. The mean total DNA concentration and the purity of seminal fluid from FTA-cards and swabs before and after sperm cryopreservation were measured by using nano-drop spectrophotometer (93.688 ± 12.5) (90.94 ± 13.9) ng/ µl; ( 221.07 ± 20.97)( 135.47 ± 19.6) ng/ µl respectively. Also a highly significant difference (p<0.001) was detected when the results of concentration DNA on FTA card were compared with dilution using wash media before and after sperm cryopreservation. Whereas significant difference (p<0.05) was observed between DNA concentration on FTA card with and without dilution after sperm cryopreservation. STR loci were present in the majority of data, particularly in FTA-card and swab samples where results explained that 100% of STR profiles were present in all 15 loci, both before and after sperm cryopreservation. In contrast to the conventional method of storing and analyzing STR profiling, it can be concluded that seminal fluid samples taken by FTA card and swabs can be taken and stored for a time by cryopreservation, leads to maintaining DNA integration in the field of criminal research.

Stability and Detection Limit of Avian Influenza, Newcastle Disease Virus, and African Horse Sickness Virus on Flinders Technology Associates Card by Conventional Polymerase Chain Reaction.

The Flinders Technology Associates (FTA) card, a cotton-based cellulose membrane impregnated with a chaotropic agent, effectively inactivates infectious microorganisms, lyses cellular material, and fixes nucleic acid. The aim of this study is to assess the stability and detection limit of various RNA viruses, especially the avian influenza virus (AIV), Newcastle disease virus (NDV), and African horse sickness virus (AHSV), on the FTA card, which could significantly impact virus storage and transport practices. To achieve this, each virus dilution was inoculated onto an FTA card and stored at room temperature in plastic bags for durations ranging from 1 week to 6 months. Following storage, the target genome was detected using conventional reverse transcription polymerase chain reaction. The present study demonstrated that the detection limit of AIV ranged from 1.17 to 6.17 EID50 values over durations ranging from 1 week to 5 months, while for NDV, it ranged from 2.83 to 5.83 ELD50 over the same duration. Additionally, the detection limit of AHSV was determined as 4.01 PFU for both 1 and 2 weeks, respectively. Based on the demonstrated effectiveness, stability, and safety implications observed in the study, FTA cards are recommended for virus storage and transport, thus facilitating the molecular detection and identification of RNA viral pathogens.

A size and taxonomic assessment of non-lethal DNA sampling of gastropods using Flinders Technology Associates (FTA) cards

Sampling the DNA of rare animal species should have minimal impacts on individual health. This can be accomplished through non-lethal/non-invasive sampling. Few of these methods have been developed for invertebrates, including the Mollusca, which are in global decline. Tissue clipping the foot is a common non-lethal method for gastropods. However, it causes permanent damage and is inappropriate for smaller snails. This study used Flinders Technology Associates (FTA) cards to sample DNA from snail mucus for species of different sizes and habitat types, and across evolutionarily distant lineages. In a survival assay, the death rate of individuals sampled with FTA cards (12.1%) was greater than in the controls (3.7%), but the difference was not significant. Of 224 individuals representing 27 snail species (17 Hawaiian native, ten non-native) sampled using both FTA cards and tissue clipping, 80.4% of FTA samples and 91.6% of tissue samples amplified for COI, a significant difference. COI sequencing success did not differ significantly between the two methods. For individuals that failed to produce a COI sequence, an attempt was made to sequence 16S. For 16S, amplification and sequencing rates did not differ significantly between FTA and tissue samples. Habitat type and shell size did not affect FTA sampling success. Phylogenetically basal taxa exhibited lower success rates, but this may have been because of difficulty in sampling operculate taxa, and not because of identity. These results indicate that the FTA sampling is a viable non-lethal alternative to tissue clipping and can be used for diverse gastropods.

Extracting DNA from Preserved Mosquito Blood Meals.

Mosquito species vary in their host associations. Although some species are relative generalists, most specialize, to varying extents, on particular types of host animals. Mosquito host associations are among the most important factors that influence the transmission dynamics of mosquito-vectored pathogens, and understanding these associations can provide insight on how such pathogens move within ecosystems. Characterization of the host associations of mosquito species requires applying blood meal analysis to the largest possible sample size of mosquito blood meals. Processing large samples of mosquito blood meals can be time-consuming, especially when chain-termination sequencing is used, necessitating individual processing of each specimen. Various methods and commercially available kits and products are available for extracting DNA from mosquito blood meals. The hot sodium hydroxide and Tris (HotSHOT) method is a rapid and inexpensive method of DNA extraction that is compatible with the recovery of DNA from mosquito blood meals preserved on QIAcard Flinders Technology Associates (FTA) Classic Cards (FTA cards). FTA cards allow nucleic acids found in blood meals to be preserved easily, even in field conditions. DNA prepared using this method is suitable for polymerase chain reaction (PCR)-based blood meal analysis.

Sensitivity of RNA viral nucleic acid-based detection of avian influenza virus, Newcastle disease virus, and African horse sickness virus on flinders technology associates card using conventional reverse-transcription polymerase chain reaction.

The flinders technology associates (FTA) card is a cotton-based cellulose membrane impregnated with a chaotropic agent that inactivates infectious microorganisms, lyses cellular material, and fixes DNA and/or RNA within the fiber matrix. However, little is known about the effectiveness of these cards for detecting RNA viruses in animals. This study aimed to evaluate the sensitivity of RNA virus detection using conventional reverse-transcription polymerase chain reaction (RT-PCR) on FTA cards. A highly virulent Newcastle disease virus (NDV) and an avian influenza virus (AIV) with low pathogenicity were propagated using chicken embryonic eggs. Three days after inoculation, the allantoic fluid was harvested, stored at -80°C, and the stock virus was tested for virus titration. African horse sickness virus (AHSV) was obtained from a live attenuated vaccine that was dissolved and stored at -80°C. For sample preparation, each stock virus was 10-fold serially diluted and each dilution was inoculated onto an FTA card, followed by drying in a Class II safety cabinet. Both the stock virus and infected FTA card were genomically isolated using an extraction kit, FTA purification kit, and extraction kit with Tris-EDTA (TE) buffer. The target genome was then detected by one-step RT-PCR for NDV and AIV, and two-step RT-PCR for African horse sickness, including gel electrophoresis for the detection of specific nucleic acids. The detection limit of stock AIV was compared on FTA cards, using the FTA purification kit, and with TE buffer with an extraction kit. The corresponding results were 1.47, 1.17, and 2.18 log10 EID50, respectively, while for NDV the results were 4.13, 4.83, and 4.84 log10 ELD50. Finally, detection limit of stock AHSV and AHSV on the FTA card extracted using TE buffer with an extraction kit were 4.30 and 4.01 log10 plaque-forming units, respectively. This study demonstrated that the detection limit or sensitivity of all tested RNA viruses on FTA cards did not differ when compared with those of the stock virus and in both methods for RNA isolation on FTA cards. These cards are suitable for collecting and transporting samples infected with RNA viruses, particularly AIV, NDV, and AHSV. Flinders technology associates cards also provide hazard-free samples, a reliable source of RNA for molecular characterization, and sufficient quantity for diagnostic applications based on nucleic acid-based detection.

FTA Cards as a Rapid Tool for Collection and Transport of Infective Samples: Experience with Foot-and-Mouth Disease Virus in Libya.

Foot-and-mouth disease (FMD) is a viral disease, widespread and highly contagious, that mainly affects cloven-hoofed domestic and wild animals. FMD can lead to high economic losses due to the reduction in animal production such as a drop in milk production, loss of body weight, and a high mortality rate in young ruminants. Sixteen samples were collected from animals showing typical clinical signs of FMD during the last FMD outbreak in Libya in 2018-2019. Flinders Technology Associates (FTA) cards impressed with blood, swabs, or vesicular epithelium samples were shipped to the WOAH FMD reference laboratory in Brescia, Italy, and tested for the detection of FMD viruses. Nucleic acids were extracted from the FTA cards, and molecular testing based on real-time RT-PCR assays was carried out, of which one was specifically designed for the detection of the FMD virus of serotype O, topotype O/East Africa-3 (O/EA-3), that was further confirmed by a sequence analysis of the VP1 gene. The phylogenetic analysis of the VP1 gene showed a nucleotide identity of more than 99% between the virus circulating in Libya and the FMD virus strains isolated in Algeria in 2019.

Comparison of Biosafety and Diagnostic Utility of Biosample Collection Cards.

Six different biosample collection cards, often collectively referred to as FTA (Flinders Technology Associates) cards, were compared for their ability to inactivate viruses and stabilize viral nucleic acid for molecular testing. The cards were tested with bluetongue virus, foot-and-mouth disease virus (FMDV), small ruminant morbillivirus (peste des petits ruminants virus), and lumpy skin disease virus (LSDV), encompassing non-enveloped and enveloped representatives of viruses with double-stranded and single-stranded RNA genomes, as well as an enveloped DNA virus. The cards were loaded with virus-containing cell culture supernatant and tested after one day, one week, and one month. The inactivation of the RNA viruses was successful for the majority of the cards and filters. Most of them completely inactivated the viruses within one day or one week at the latest, but the inactivation of LSDV presented a greater challenge. Three of the six cards inactivated LSDV within one day, but the others did not achieve this even after an incubation period of 30 days. Differences between the cards were also evident in the stabilization of nucleic acid. The amount of detectable viral genome on the cards remained approximately constant for all viruses and cards over an incubation period of one month. With some cards, however, a bigger loss of detectable nucleic acid compared with a directly extracted sample was observed. Using FMDV, it was confirmed that the material applied to the cards was sufficiently conserved to allow detailed molecular characterization by sequencing. Furthermore, it was possible to successfully recover infectious FMDV by chemical transfection from some cards, confirming the preservation of full-length RNAs.

Arbovirus detection in synanthropic mosquitoes from the Brazilian Amazon and in mosquito saliva using Flinders Technology Associates cards

Although arbovirus transmission and identifying target vectors may provide a baseline for planning disease control strategies, there are many gaps in knowledge regarding these mosquitoes and viral species in urban, rural, or sylvatic habitats in the Brazilian Amazon. Our goal was to screen for dengue, chikungunya, and Zika viruses in synanthropic mosquitoes and with Flinders Technology Associates (FTA) cards using insect saliva. Mosquitoes were caught using ovitraps and aspirators in the city of Porto Velho, Rondônia, Brazil. Honey-baited FTA cards were placed in mosquito cages for 15 days; whole mosquitoes and FTA cards were analysed for viral RNA using RT-qPCR assays. One pool of Aedes aegypti females was found to be infected with the Zika virus and one male mosquito was infected with dengue-4, suggesting natural vertical/venereal transmission. Our study also reported evidence of vertical/venereal transmission of ZIKV in Culex quinquefasciatus males for the first time in the Brazilian Amazon, and the feasibility of using FTA cards to detect arboviruses in the saliva of field-collected mosquitoes. Vertical/venereal transmission of viruses by atypical mosquito species reinforces the need for combined viral and entomological screening in arbovirus surveillance programs.

Comparison between Whatman FTA Elute Cards and Conventional Swab for the Detection of Pathogenic Enteric Bacteria Using an RT-qPCR Assay.

The emergence of outbreaks of foodborne illness is closely associated with food contamination caused by various enteric pathogens, such as Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica serovar Enteritidis, and Staphylococcus aureus. The control of enteric pathogens poses a challenge due to the fact that these pathogens can persist for a long period of time in the environment. The rapid detection of pathogenic organisms plays a crucial role in the prevention and identification of crises related to health, safety, and well-being. Improper sample handling and processing may influence the diagnostic efficacy and accuracy. The aim of the present study was to compare the preservation capacity for enteric bacteria between Whatman Flinders Technology Associates (FTA) cards and swabs for reverse transcription-quantitative PCR (RT-qPCR) detection. It was found that Whatman FTA cards exhibited an improved preservation capacity for five types (both laboratory and environmental strains) of enteric bacteria, including Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica serovar Enteritidis, and Staphylococcus aureus for RT-qPCR detection. Hence, Whatman FTA cards may be a suitable tool for the routine isolation of foodborne bacteria for molecular diagnosis. Therefore, the use of Whatman FTA cards for sample collection and preservation may increase sensitivity and accuracy for bacteria isolation and diagnosis.

A comparison between two different dried blood substrates in determination of psychoactive substances in postmortem samples

PurposeWhatman™ 903 cards represent a valid type of support for collection, storage, and analysis of dried blood spots (DBS). Whatman™ FTA (Flinders Technology Associates) are a type of cards soaked in chemicals that cause denaturation of proteins, while preserving DNA and ensuring the safe handling of DBS; to date, these cards are still rarely employed in forensic toxicology. The aim of this study was to analyze several psychoactive substances on not-dried blood on the two different cards and to compare the qualitative and quantitative results.MethodsTwenty cardiac postmortem blood samples were collected and deposed on Whatman™ 903 and Whatman™ FTA cards. Spots and not-dried blood were analyzed following our validated and previously published liquid chromatography–mass spectrometry methods.ResultsWe were able to identify: eight drugs of abuse and their metabolites (15 cases), five benzodiazepines and their metabolites (3 cases), six antidepressants (6 cases) and two antipsychotics (3 cases). We observed a perfect qualitative correspondence and a general good quantitative correlation between data obtained from not-dried blood and the two different DBS cards, except for alprazolam, diazepam, desmethyldiazepam, fluoxetine and sertraline, that showed a lower concentration on FTA. Additional experiments suggest that the chemicals, adsorbed on FTA, are not the cause of the loss of signal observed for the substances previously mentioned and that methanol should be preferred as extraction solvent.ConclusionsThis study proved that FTA cards are a good and a hazard-free alternative sample storage method for analysis of several psychoactive substances in postmortem blood.

Viromics on Honey-Baited FTA Cards as a New Tool for the Detection of Circulating Viruses in Mosquitoes.

Worldwide, emerging and re-emerging infectious diseases (EIDs) are a major burden on public and animal health. Arthropod vectors, with mosquitoes being the main contributors of global disease, transmit more than 70% of the recognized EIDs. To assess new alternatives for arthropod-borne viral diseases surveillance, and for the detection of new viruses, honey-baited Flinders Technology Associates (FTA) cards were used as sugar bait in mosquito traps during entomological surveys at the Llobregat River Delta (Catalonia, Spain). Next generation sequencing (NGS) metagenomics analysis was applied on honey-baited FTA cards, which had been exposed to field-captured mosquitoes to characterize their associated virome. Arthropod- and plant-infecting viruses governed the virome profile on FTA cards. Twelve near-complete viral genomes were successfully obtained, suggesting good quality preservation of viral RNAs. Mosquito pools linked to the FTA cards were screened for the detection of mosquito-associated viruses by specific RT-PCRs to confirm the presence of these viruses. The circulation of viruses related to Alphamesonivirus, Quaranjavirus and unclassified Bunyavirales was detected in mosquitoes, and phylogenetic analyses revealed their similarities to viruses previously reported in other continents. To the best our knowledge, our findings constitute the first distribution record of these viruses in European mosquitoes and the first hint of insect-specific viruses in mosquitoes’ saliva in field conditions, demonstrating the feasibility of this approach to monitor the transmissible fraction of the mosquitoes’ virome. In conclusion, this pilot viromics study on honey-baited FTA cards was shown to be a valid approach for the detection of viruses circulating in mosquitoes, thereby setting up an alternative tool for arbovirus surveillance and control programs.

Evaluating Flinders Technology Associates card for transporting bacterial isolates and retrieval of bacterial DNA after various storage conditions.

Background and Aim:Flinders Technology Associates (FTA) cards simplify sample storage, transport, and extraction by reducing cost and time for diagnosis. This study evaluated the FTA suitability for safe transport and storage of Gram-positive and Gram-negative bacterial cells of animal origin on its liquid culture form and from organ impression smears (tissues) under the same routine condition of microbiological laboratory along with detecting their nucleic acid over different storage conditions.Materials and Methods:Increase in bacterial count from 104 to 107 (colony-forming units/mL) of 78 isolates representing seven bacterial species was applied onto cards. FTA cards were grouped and inoculated by these bacteria and then stored at different conditions of 24-27°C, 4°C, and −20°C for 24 h, for 2 weeks, for 1 and 3 month storage, respectively. Bacteriological examination was done, after which bacterial DNA was identified using specific primers for each bacterial type and detected by polymerase chain reaction (PCR).Results:The total percentage of recovered bacteria from FTA cards was 66.7% at 24-27°C for 24 h, the detection limit was 100% in Gram-positive species, while it was 57.4% in Gram-negative ones. Regarding viable cell detection from organ impression smears, it was successful under the previous conditions. No live bacterial cells were observed by bacteriological isolation rather than only at 24-27°C for 24 h storage. All bacterial DNA were sufficiently confirmed by the PCR technique at different conditions.Conclusion:Overall, the FTA card method was observed to be a valid tool for nucleic acid purification for bacteria of animal origin in the form of culture or organ smears regardless of its Gram type and is used for a short time only 24 h for storage and transport of live bacteria specifically Gram-positive type. Moreover, the bacterial nucleic acid was intact after storage in −20°C for 3 months and was PCR amplifiable.

Further insight into the geographic distribution of Leishmania species in Peru by cytochrome b and mannose phosphate isomerase gene analyses.

To obtain further insight into geographic distribution of Leishmania species in Peru, a countrywide survey, including central to southern rainforest areas where information on causative parasite species is limited, was performed based on cytochrome b (cyt b) and mannose phosphate isomerase (mpi) gene analyses. A total of 262 clinical samples were collected from patients suspected of cutaneous leishmaniasis (CL) in 28 provinces of 13 departments, of which 99 samples were impregnated on FTA (Flinders Technology Associates) cards and 163 samples were Giemsa-stained smears. Leishmania species were successfully identified in 83 (83.8%) of FTA-spotted samples and 59 (36.2%) of Giemsa-stained smear samples. Among the 142 samples identified, the most dominant species was Leishmania (Viannia) braziliensis (47.2%), followed by L. (V.) peruviana (26.1%), and others were L. (V.) guyanensis, L. (V.) lainsoni, L. (V.) shawi, a hybrid of L. (V.) braziliensis and L. (V.) peruviana, and Leishmania (Leishmania) amazonensis. Besides the present epidemiological observations, the current study provided the following findings: 1) A hybrid of L. (V.) braziliensis and L. (V.) peruviana is present outside the Department of Huanuco, the only place reported, 2) Many cases of CL due to L. (V.) lainsoni, an uncommon causative species in Peru, were observed, and 3) L. (V.) shawi is widely circulating in southern Amazonian areas in Peru.

The use of FTA cards to acquire DNA profiles from postmortem cases.

Filter papers have been used for many years in different applications of molecular biology and have been proven to be a stable way to store DNA waiting to be analyzed. Sampling of DNA on FTA (Flinders Technology Associates) cards is convenient and cost effective compared to alternative approaches involving DNA extractions and storage of DNA extracts. FTA cards are analyzed at many forensic laboratories, and the way to perform direct genetic profiling on buccal swab cards has developed into an almost industrial process. The possibility to include postmortem (PM) samples into an FTA-based workflow would facilitate and speed up the genetic identification process compared to conventional methods, both on a regular basis and in a mass casualty event. In this study, we investigated if FTA cards may be used to carry tissue DNA from deceased and present a high-quality DNA profile from the individual in order to be useful for the identification process. The study also aimed to investigate if a specific body tissue would be preferable, and if decomposed tissue is suitable at all to put on an FTA card in order to obtain a DNA profile. We have compared the quality of the DNA profiles acquired from postmortem tissue on FTA cards, with the results acquired with conventional methods from reference bone/muscle samples from the same individual. Several types of tissues have been tested from different identification cases and scenarios. We concluded that tissue cells from inner organs are suitable to put on FTA cards, and that the obtained DNA profiles have the potential to serve as PM data for identification purposes. In cases including compromised samples, however, it is recommended to keep the tissue sample as a backup if further DNA has to be extracted.

Cytological preparations for molecular analysis: A review of technical procedures, advantages and limitations for referring samples for testing.

Minimally invasive procedures such as endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) must yield not only good quality and quantity of material for morphological assessment, but also an adequate sample for analysis of molecular markers to guide patients to appropriate targeted therapies. In this context, cytopathologists worldwide should be familiar with minimum requirements for refereeing cytological samples for testing. The present manuscript is a review with comprehensive description of the content of the workshop entitled Cytological preparations for molecular analysis: pre-analytical issues for EBUS TBNA, presented at the 40th European Congress of Cytopathology in Liverpool, UK. The present review emphasises the advantages and limitations of different types of cytology substrates used for molecular analysis such as archival smears, liquid-based preparations, archival cytospin preparations and FTA (Flinders Technology Associates) cards, as well as their technical requirements/features. These various types of cytological specimens can be successfully used for an extensive array of molecular studies, but the quality and quantity of extracted nucleic acids rely directly on adequate pre-analytical assessment of those samples. In this setting, cytopathologists must not only be familiar with the different types of specimens and associated technical procedures, but also correctly handle the material provided by minimally invasive procedures, ensuring that there is sufficient amount of material for a precise diagnosis and correct management of the patient through personalised care.

BioSpotlights/Citations

BioSpotlights/Citations

Telomere length measurement by qPCR in birds is affected by storage method of blood samples

Given the potential role of telomeres as biomarkers of individual health and ageing, there is an increasing interest in studying telomere dynamics in a wider range of taxa in the fields of ecology and evolutionary biology. Measuring telomere length across the lifespan in wild animal systems is essential for testing these hypotheses, and may be aided by archived blood samples collected as part of longitudinal field studies. However, sample collection, storage, and DNA extraction methods may influence telomere length measurement, and it may, therefore, be difficult to balance consistency in sampling protocol with making the most of available samples. We used two complementary approaches to examine the impacts of sample storage method on measurements of relative telomere length (RTL) by qPCR, particularly focusing on FTA (Flinders Technology Associates) cards as a long-term storage solution. We used blood samples from wandering albatrosses collected over 14 years and stored in three different ways (n = 179), and also blood samples from captive zebra finches (n = 30) that were each stored using three different methods. Sample storage method influenced RTL in both studies, and samples on FTA cards had significantly shorter RTL measurements. There was no significant correlation between RTL measured in zebra finch blood on FTA cards and the same samples stored either as frozen whole blood or as extracted DNA. These results highlight the importance of consistency of sampling protocol, particularly in the context of long-term field studies, and suggest that FTA cards should not be used as a long-term storage solution to measure RTL without validation.

Long-term storage and safe retrieval of human papillomavirus DNA using FTA elute cards

Biobanking or collection and storage of specimens for future research purposes have become an essential tool in many fields of biomedical research and aims to provide a better understanding of disease mechanisms as well as the identification of disease-specific biomarkers that can navigate in complex diseases. In this study, we assessed the use of Flinders Technology Associates (FTA) cards as a long-term storage device for cervical specimens with suspected human papillomavirus (HPV) infections. HPV detection and genotyping results in liquid-based transport media were compared to HPV results from FTA cards. The overall agreement for the presence of any HPV infection between liquid-based medium and FTA cards stored for 1 year at ambient temperature was 100%. Reproducibility analysis of HPV detection and genotyping from FTA cards demonstrated that FTA cards are a reliable medium to store and preserve viral nucleic acids. Biobanking of cervical cells on FTA cards may provide a key resource for epidemiological and retrospective HPV studies.

Evaluation of the Flinders Technology Associates Cards for Storage and Temperature Challenges in Field Conditions for Foot-and-Mouth Disease Virus Surveillance.

Foot-and-mouth disease virus (FMDV) samples transported to the laboratory from far and inaccessible areas for diagnosis and identification of FMDV pose a major problem in a tropical country like India, where wide fluctuation of temperature over a large geographical area is common. Inadequate storage methods lead to spoilage of FMDV samples collected from clinically positive animals in the field. Such samples are declared as non-typeable by the typing laboratories with the consequent loss of valuable epidemiological data. In this study, an attempt was made to evaluate the robustness of Flinders Technology Associates (FTA) cards for storage and transportation of FMDV samples in different climatic conditions which will be useful for FMDV surveillance. Simulation transport studies were conducted using FTA impregnated FMDV samples during post-monsoon (September-October 2010) and summer season (May-June 2012). FMDV genome or serotype could be identified from the FTA cards after the simulation transport studies with varying temperature (22-45°C) and relative humidity (20-100%). The stability of the viral RNA, the absence of infectivity and ease of processing the sample for molecular methods make the FTA cards an useful option for transport of FMDV genome for identification and type determination. The method can be used routinely for FMDV research as it is economical and the cards can be transported easily in envelopes by regular courier/postal systems. The absence of live virus in FTA card can be viewed as an advantage as it restricts the risk of transmission of live virus.

Use of FTA Technology to ExtractWheat streak mosaic virusandCandidatusLiberibacter Solanacearum from Single Vectors

Detection and quantification of plant pathogens is a fundamental component of plant disease epidemiology. For vector-borne diseases, pathogen detection and quantification within the vector is often necessary to understand development and spread of disease. However, extraction and testing of pathogens from a single vector can be expensive and tedious, especially with large numbers of samples. Therefore, to simplify sample collection and reduce cost of analysis, we used a new nucleic acid elution approach using existing Flinders Technology Associates (FTA) technology from Whatman GE Healthcare. We applied this method in two very distinct vector-borne pathogen systems for Wheat streak mosaic virus (Tritimovirus) (McKinney 1937) and Candidatus Liberibacter solanacearum (Liefting et al. 2009). Total DNA and RNA were extracted from adult and nymphal psyllids (family Psyllidae) and wheat curl mite, Aceria tosichella Keifer, respectively using FTA cards (Whatman GE Health care, Pittsburgh, PA), and results were compared to extraction with DNeasy/RNeasy® kits (Qiagen Inc., Valencia, CA). Additionally, composite samples of multiple mite and psyllid vectors were used to compare the efficiencies of both methods. FTA elution and kit extractions were significantly different (P < 0.001). Slightly lower cycle threshold (Ct) values of 27.8 ± 0.19 and 25.5 ± 0.17 and 29.8 ± 0.09 and 27.45 ± 0.05 were determined for the composite adult and nymphal psyllids for the FTA and kit extractions, respectively. Wheat curl mite Ct values averaged 32.5 ± 0.75 and 27.6 ± 1.5 for the FTA elution and kit extractions, respectively. Coefficient of variation values were found to be lower for the FTA elution method when compared to the kit extractions for both the adult and nymphal psyllid (0.7 and 0.9%) and wheat curl mite (2.3 and 5.5%). Despite lower Ct values the FTA elution method is more cost efficient, protects nucleic acid integrity, and proved to be a valuable and reliable tool for detection and comparative studies of both pathogens within a single-vector sample.