Evaluation of FTA cards as a laboratory and field sampling device for the detection of foot-and-mouth disease virus and serotyping by RT-PCR and real-time RT-PCR.
ABSTRACT Foot-and-mouth disease virus (FMDV) samples transported to the laboratory from far and inaccessible areas for serodiagnosis pose a major problem in a tropical country like India, where there is maximum temperature fluctuation. 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. The present study evaluated the usefulness of FTA Classic Cards for the collection, shipment, storage and identification of the FMDV genome by RT-PCR and real-time RT-PCR. The stability of the viral RNA, the absence of infectivity and ease of processing the sample for molecular methods make the FTA cards a useful option for transport of FMDV genome for identification and serotyping. The method can be used routinely for FMDV research as it is economical and the cards can be transported easily in envelopes by regular document transport methods. Live virus cannot be isolated from samples collected in FTA cards, which is a limitation. This property can be viewed as an advantage as it limits the risk of transmission of live virus.
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ABSTRACT: Current avian influenza (AI) virus surveillance programs involving wild birds rely on sample collection methods that require refrigeration or low temperature freezing to maintain sample integrity for virus isolation and/or reverse-transcriptase (RT) PCR. Maintaining the cold chain is critical for the success of these diagnostic assays but is not always possible under field conditions. The aim of this study was to test the utility of Finders Technology Associates (FTA) cards for reliable detection of AI virus from cloacal and oropharyngeal swabs of wild birds. The minimum detectable titer was determined, and the effect of room temperature storage was evaluated experimentally using multiple egg-propagated stock viruses (n = 6). Using real time RT-PCR, we compared results from paired cloacal swab and samples collected on FTA cards from both experimentally infected mallards (Anasplatyrhynchos) and hunter-harvested waterfowl sampled along the Texas Gulf Coast. Based on the laboratory trials, the average minimal detectable viral titer was determined to be 1 x 10(4.7) median embryo infectious dose (EID50)/ml (range: 1 x 10(4.3) to 1 x 10(5.4) EID50/ml), and viral RNA was consistently detectable on the FTA cards for a minimum of 20 days and up to 30 days for most subtypes at room temperature (23 C) storage. Real-time RT-PCR of samples collected using the FTA cards showed fair to good agreement in live birds when compared with both real-time RT-PCR and virus isolation of swabs. AI virus detection rates in samples from several wild bird species were higher when samples were collected using the FTA cards compared with cloacal swabs. These results suggest that FTA cards can be used as an alternative sample collection method when traditional surveillance methods are not possible, especially in avian populations that have historically received limited testing or situations in which field conditions limit the ability to properly store or ship swab samples.Avian Diseases 03/2012; 56(1):200-7. · 1.73 Impact Factor
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ABSTRACT: A one-step, real-time reverse transcription-loop-mediated isothermal amplification assay (RT-LAMP) for rapid detection and serotyping of Indian foot-and-mouth disease virus (FMDV) is described. The RT-LAMP assay was found to be 10(3)-10(5) fold more sensitive in comparison with RT-PCR, with a detection limit ranging from 10(-3) to 10(-5) TCID(50) of virus samples of all three serotypes. The RT-LAMP assay and qRT-PCR could detect 100 percent of clinical samples of three serotypes, whereas the RT-PCR detected 69.7% of type O, 58.1% of type A and 60.0% of Asia 1 samples. The qRT-PCR has the same sensitivity as the RT-LAMP. The assay conditions with absence of cross reactivity within the three serotypes of FMDV and FMDV negative samples were established. The RT-LAMP assay could detect 100% of samples stored in FTA(®) cards. In comparison with the performance of the RT-PCR; the RT-LAMP appears to be more sensitive, rapid and specific, with the potential for use as a point-of-care (POC) test, especially in developing countries. The use of FTA(®) cards for the preservation of RNA samples coupled with the RT-LAMP assay for the identification of serotypes may help in achieving improved FMDV serotype identification both in the field and in the laboratory.Journal of virological methods 08/2012; · 2.13 Impact Factor
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ABSTRACT: The genetic characterization of wild-type measles viruses plays an important role in the description of viral transmission pathways and the verification of measles elimination. The 450 nucleotides that encode the carboxyl-terminus of the nucleoprotein (N-450) are routinely sequenced for genotype analysis. The objectives of this study were to develop improved primers and controls for RT-PCR reactions used for genotyping of measles samples and to develop a method to provide a convenient, safe, and inexpensive means to distribute measles RNA for RT-PCR assays and practice panels. A newly designed, genetically defined synthetic RNA and RNA isolated from cells infected with currently circulating genotypes were used to compare the sensitivity of primer pairs in RT-PCR and nested PCR. FTA(®) cards loaded with lysates of measles infected cells were tested for their ability to preserve viral RNA and destroy virus infectivity. A new primer pair, MeV216/MeV214, was able to amplify N-450 from viruses representing 10 currently circulating genotypes and a genotype A vaccine strain and demonstrated 100-fold increased sensitivity compared to the previously used primer set. A nested PCR assay further increased the sensitivity of detection from patient samples. A synthetic positive control RNA was developed that produced PCR products that are distinguishable by size from PCR products amplified from clinical samples. FTA(®) cards completely inactivated measles virus and stabilized RNA for at least six months. These improved molecular tools will advance molecular characterization of circulating measles viruses globally and provide enhanced quality control measures.Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology 06/2013; · 3.12 Impact Factor