BACKGROUND
foot-and-mouth disease  is a highly contagious viral disease that affects cloven-hoofed domestic and wild animals.
foot-and-mouth disease is considered the most economically important animal disease worldwide, and outbreaks of fmd require immediate notification to the world organization for animal health .
outbreaks of fmd result in sanitary barriers that prevent export of bovine and swine products  <cit> .
furthermore, fmd causes enormous losses to the animal industry due to costs associated with control or eradication measures, including massive vaccination and/or destruction of infected herds, as well as decreases in milk and beef production as a result of clinical disease.
clinically affected cattle have vesicular lesions in the oral cavity, foot, and udder, which are associated with fever, lameness, salivation, and anorexia.
foot-and-mouth disease usually has a high morbidity and low mortality, with mortality occurring mostly in young animals  <cit> .
in brazil, the number of cases of fmd has been steadily decreasing since the 1980s  <cit> .
the national program for control and eradication of fmd , implemented in  <dig>  has resulted in recognition by the oie of vast areas of the brazilian territory as free of fmd with vaccination  <cit> .
however, the recognition of most of these areas was suspended due to the outbreak of fmd in the state of mato grosso do sul in  <dig>  from which part of the biological samples used in the current study was obtained.
the foot-and-mouth disease virus  has a single-stranded, positive-sense rna genome of approximately  <dig>  kb.
there are  <dig> serotypes with a large number of variants spread over several regions in the world  <cit> .
serotypes a, o, and c have been detected in brazil  <cit> .
considering that fmd is highly contagious and has clinical signs similar to other vesicular diseases, a quick definitive diagnosis is extremely important  <cit> .
a presumptive clinical diagnosis associated with laboratory tests such as serology, virus isolation, and antigen detection are the basis for the diagnosis at the herd level.
serological tests  are not time-consuming, but they are indirect tests that do not always allow for differentiation between infected and vaccinated cattle due to the use of poor vaccine or previous infection in endemic areas.
serological techniques are not the technique of first choice to detect an acute infection  <cit> .
a definitive diagnosis is based on detection of virus in fluids or epithelium from vesicular lesions or esophageal-pharyngeal fluid collected with a probang  <cit> .
virus isolation is the most reliable diagnostic method, but it is labor-intensive, time-consuming, and requires properly equipped facilities.
sandwich elisa is a much faster approach to detect viral antigens, but it has low sensitivity, so its primary indication is to confirm and type the fmdv after isolation in cell culture  <cit> .
therefore, several groups have been developing faster diagnostic methods for fmd based on amplification of specific sequences of the viral genome by reverse transcription polymerase chain reaction   <cit> , which can be applied to different kinds of biological samples such as fluids and tissues, and in some cases, this approach allows identification of infected animals even before development of clinical signs or positive virus isolation as well as identification of positive cattle at the end of the course of infection when virus isolation may be negative  <cit> .
in addition, in a study in which results were compared between reference laboratories, rt-pcr results were more consistent among laboratories as compared to virus isolation and elisa results, which varied from low to high sensitivity when aliquots of the same samples were processed by  <dig> different laboratories  <cit> .
reverse transcription polymerase chain reaction is also useful for typing fmdv isolates  <cit> .
however, conventional rt-pcr does not have optimal results in terms of specificity and sensitivity  <cit> .
thus, recently, a method based on real-time rt-pcr amplification and a fluorescent probe resulted in significantly improved sensitivity and specificity under experimental conditions  <cit> .
a previously described real-time rt-pcr method was employed in the current study, which demonstrated high specificity and sensitivity under laboratory and experimental conditions  <cit> , for the diagnosis of fmd under field conditions in brazil.
methods
source of samples
a total of  <dig> oral swabs from adult cattle of both sexes were used in this study.
these samples originated from different regions according to the following groups.
group  <dig> consisted of  <dig> healthy cattle from an area free of fmd with vaccination .
group  <dig> consisted of  <dig> cattle from carero da várzea, state of amazonas , which were sampled during an outbreak of fmd  in  <dig>  <cit> .
these cattle did not have any clinical signs and were sampled more than  <dig> days after the identification of the index case.
group  <dig> consisted of  <dig> cattle suspected of fmd .
these swabs were obtained during an outbreak of fmd  in  <dig>  <cit> .
sampling
dacron-tipped swabs were obtained from the oral cavity by swabbing the oral mucosa and tongue, saturating the swab with saliva while avoiding contamination with ingested material.
immediately after sampling, the swabs were placed into a 2-ml sterile cryogenic tube containing  <dig>  ml of dulbecco's minimal essential medium   with antibiotic and antimycotic .
the tubes containing the swabs from cattle from an area free of fmd  were immediately frozen on dry ice and stored at -70°c until further processing.
however, due to the unavailability of dry ice in the areas of the outbreaks  some samples were kept at 4°c for up to  <dig> days prior to freezing and storage at -70°c.
rna extraction
rna samples were extracted using the mini rneasy kit  following the manufacturer's instructions.
briefly, the tubes containing the swabs were thawed and thoroughly homogenized by vortexing, and  <dig> μl of the medium were added to  <dig> μl of the rlt buffer that is included in the kit, followed by  <dig> μl of 70% ethanol, and then transferred to a mini rneasy column previously inserted into a 2-ml collecting tube.
rna was immobilized in the column by centrifugation, sequentially washed, and eluted in  <dig> μl of rnase-free water.
in addition to the rna samples obtained from oral swabs as described above, rna samples were purified using trizol  from  <dig> reference strains  representing the  <dig> serotypes that occur in brazil , and  <dig> previously characterized field isolates .
these rna samples were used as positive controls.
real-time reverse transcription polymerase chain reaction
the rrt-pcr amplification protocol used in the current study was previously described  <cit> .
this method amplifies a conserved segment of the fmdv rna polymerase gene .
the following primers were used 5'-actgggttttacaaacctgtga-3' and 5'-gcgagtcctgccacgga-3' along with the probe 5'-tcctttgcacgccgtgggac-3' labeled with 6-carboxyfluorescein at the 5' end, and the quencher tetramethyl rhodamine at the 3' end.
this method amplifies the rna from all serotypes of fmdv, but does not amplify nucleic acids from other viruses that cause vesicular diseases  <cit> .
reactions were performed using  <dig> μl cepheid tubes  containing all the dehydrated reagents required for amplification of the fmdv rna  that were rehydrated with  <dig>  μl of rehydration buffer .
as controls,  <dig>  μl of rna samples or an oligonucleotide used as positive control  or te buffer  instead of rna as a negative control, were added to the respective reaction tubes.
the real-time rt-pcr reactions were carried out in a smart cycler ii thermocycler .
the one-step real-time rt-pcr amplification started with reverse transcription for  <dig> hr at 60°c, followed by pcr with the following parameters:  <dig> cycles of  <dig> sec at 95°c and  <dig> sec at 60°c.
one positive and  <dig> negative control were included in each reaction.
virus isolation
the samples  were filtered using a  <dig> -μm filter, and  <dig> μl was then inoculated onto a monolayer of ibrs - <dig> cells grown in 25-cm <dig> flasks and kept without washing for the duration of the assay .
after inoculation, the cultures were checked for cytopathic effects  every  <dig> hr for  <dig> hr.
cultures that did not develop cpe were lysed by freezing at -70°c and reinoculated  onto a new monolayer of ibrs- <dig> cells, which was then evaluated for an additional  <dig> hr.
cultures with cpe were stored at -70°c until processing for indirect sandwich elisa.
indirect sandwich enzyme-linked immunosorbent assay
samples from cultures that had cpe were tested by indirect sandwich elisa, according to the protocol recommended by the pan american foot-and-mouth disease center .
briefly, anti-fmdv serotypes a, o, and c rabbit antiserum was incubated in elisa plates for  <dig> hr at 4°c.
antiserum was generated by using a pool of inactivated virus subtypes o <dig> campos, a <dig> cruzeiro, and c <dig> indaial as antigen.
the plate was washed and incubated with 1% ovalbumin for  <dig> hr at room temperature.
after washing, the reference antigens and testing samples were added to the wells and incubated for  <dig> hr at 37°c under agitation.
the plate was then washed again and incubated with guinea pig anti-fmdv  antiserum for  <dig> min at 37°c under agitation, followed by washing and incubation with an anti-guinea pig antibody conjugated with peroxidase for  <dig> min at 37°c under agitation.
the plate was washed and incubated with a solution of hydrogen peroxide  as a substrate for  <dig> min at room temperature, and read in an elisa reader at  <dig> nm.
RESULTS
representative results of real-time rt-pcr reactions with samples from the area free of fmd and from areas with outbreaks of fmd are illustrated in figure  <dig>  all  <dig> cattle from the area free of fmd with vaccination were negative by real-time rt-pcr and combined virus isolation/elisa.
the  <dig> samples from carero da várzea  were negative by real-time rt-pcr and combined virus isolation/elisa.
among the  <dig> samples from eldorado and japorã ,  <dig> were positive by real-time rt-pcr,  <dig> of which were also positive by combined virus isolation/elisa; whereas  <dig> samples were considered inconclusive with cycle threshold  values higher than  <dig>  samples considered inconclusive by real-time rt-pcr were all negative by combined virus isolation/elisa.
importantly, no false-negative results by rt-pcr were observed since none of the samples was positive by virus isolation/elisa and negative by real-time rt-pcr.
the results are summarized in table  <dig>  the samples from the  <dig> reference strains  and the  <dig> previously characterized field isolates  were all positive by real-time rt-pcr with low ct values indicating high concentration of viral rna .
* origin of samples was eldorado/japorã, state of minas gerais.
ct  corresponds to the number of cycles required for a given sample to reach the threshold above which it is considered positive.
a sample was considered positive when the ct value was lower than  <dig>  and inconclusive when the ct value was higher than  <dig>  cpe = cytopathic effect; nd = not determined.
only samples that resulted in cpe were processed for indirect sandwich elisa.
* ct  corresponds to the number of cycles required for a given sample to reach the threshold above which it is considered positive.
a sample was considered positive when the ct value was lower than  <dig>
discussion
the real-time rt-pcr method used in the current study has proven to be highly sensitive and specific under laboratory and experimental conditions , which is supported by our results as all previously characterized field isolates resulted in positive reactions with low ct values.
the  <dig> samples that were positive by combined virus isolation/elisa were also positive by real-time rt-pcr .
in addition, neither false-positives nor false-negatives were detected.
indeed, a recent study demonstrated that this method performed similarly to another real-time rt-pcr protocol for amplification of the 5' untranslated region  of the fmdv genome  <cit> .
the recommended protocol for storage of samples prior to rna extraction was strictly followed in the case of swabs collected from the area free of fmd with vaccination .
however, it was not possible to supply dry ice to the areas affected by fmd outbreaks, which resulted in storage of samples at 4°c up to  <dig> days.
presumably, this inadequate procedure for storage of samples likely resulted in a decrease in the sensitivity of the test.
however, samples from the area free of fmd, which were properly stored, were all negative by real-time rt-pcr as well as by virus isolation and elisa, indicating a high specificity of this test as previously demonstrated  <cit> .
a comprehensive recent study of specificity of this method confirmed a very high specificity, but even with the inconclusive reactions considered false-positives, the specificity was  <dig> %, which may provide enough accuracy for a diagnosis during an outbreak, but may not be specific enough for surveillance in fmd-free areas  <cit> .
in spite of inadequate storage of samples from cattle suspect of fmd,  <dig> positive animals were identified by real-time rt-pcr, whereas only  <dig> of those samples were positive by combined virus isolation/elisa, indicating comparable sensitivity between these two diagnostic methods, which is in good agreement with previous  <cit> .
in contrast, none of the  <dig> samples collected from cattle without lesions more than  <dig> month after identification of the index case were positive by real-time rt-pcr.
although these samples were also inadequately stored, negative results are likely to occur in cattle that recovered from clinical lesions since the likelihood of virus isolation is extremely reduced with more than 7– <dig> days after the appearance of gross lesions  <cit> .
CONCLUSIONS
in summary, the current study supports the notion that real-time rt-pcr is a powerful technique for reliable detection of fmdv in a fraction of the time required for combined virus isolation/elisa.
however, it is noteworthy that lack of infrastructure in certain areas with high risk of fmd may be a limiting factor for using real-time rt-pcr as a routine diagnostic tool.
authors' contributions
tap, avcn, and rls collected samples in fmd-free areas; nop collected samples in areas of outbreaks; jrr performed virus isolation procedures; msb performed elisas; tap, avcn, rrs, and rls did the real time rt-pcr reactions; trb, bmm, npc, lga, and rls designed the study, analyzed the data, and supervised all procedures; lga and rls coordinated the group; and all co-authors were involved in writing and revising the manuscript.

 foot-and-mouth disease is a highly contagious viral disease that affects cloven-hoofed domestic and wild animals. foot-and-mouth disease is considered the most economically important animal disease worldwide, and outbreaks of fmd require immediate notification to the world organization for animal health. outbreaks of fmd result in sanitary barriers that prevent export of bovine and swine products. furthermore, fmd causes enormous losses to the animal industry due to costs associated with control or eradication measures, including massive vaccination and/or destruction of infected herds, as well as decreases in milk and beef production as a result of clinical disease. clinically affected cattle have vesicular lesions in the oral cavity, foot, and udder, which are associated with fever, lameness, salivation, and anorexia. foot-and-mouth disease usually has a high morbidity and low mortality, with mortality occurring mostly in young animals. in brazil, the number of cases of fmd has been steadily decreasing since the 1980s. the national program for control and eradication of fmd, implemented in has resulted in recognition by the oie of vast areas of the brazilian territory as free of fmd with vaccination. however, the recognition of most of these areas was suspended due to the outbreak of fmd in the state of mato grosso do sul in from which part of the biological samples used in the current study was obtained. the foot-and-mouth disease virus has a single-stranded, positive-sense rna genome of approximately kb. there are serotypes with a large number of variants spread over several regions in the world. serotypes a, o, and c have been detected in brazil. considering that fmd is highly contagious and has clinical signs similar to other vesicular diseases, a quick definitive diagnosis is extremely important. a presumptive clinical diagnosis associated with laboratory tests such as serology, virus isolation, and antigen detection are the basis for the diagnosis at the herd level. serological tests are not time-consuming, but they are indirect tests that do not always allow for differentiation between infected and vaccinated cattle due to the use of poor vaccine or previous infection in endemic areas. serological techniques are not the technique of first choice to detect an acute infection. a definitive diagnosis is based on detection of virus in fluids or epithelium from vesicular lesions or esophageal-pharyngeal fluid collected with a probang. virus isolation is the most reliable diagnostic method, but it is labor-intensive, time-consuming, and requires properly equipped facilities. sandwich elisa is a much faster approach to detect viral antigens, but it has low sensitivity, so its primary indication is to confirm and type the fmdv after isolation in cell culture. therefore, several groups have been developing faster diagnostic methods for fmd based on amplification of specific sequences of the viral genome by reverse transcription polymerase chain reaction, which can be applied to different kinds of biological samples such as fluids and tissues, and in some cases, this approach allows identification of infected animals even before development of clinical signs or positive virus isolation as well as identification of positive cattle at the end of the course of infection when virus isolation may be negative. in addition, in a study in which results were compared between reference laboratories, rt-pcr results were more consistent among laboratories as compared to virus isolation and elisa results, which varied from low to high sensitivity when aliquots of the same samples were processed by different laboratories. reverse transcription polymerase chain reaction is also useful for typing fmdv isolates. however, conventional rt-pcr does not have optimal results in terms of specificity and sensitivity. thus, recently, a method based on real-time rt-pcr amplification and a fluorescent probe resulted in significantly improved sensitivity and specificity under experimental conditions. a previously described real-time rt-pcr method was employed in the current study, which demonstrated high specificity and sensitivity under laboratory and experimental conditions, for the diagnosis of fmd under field conditions in brazil. methods source of samples a total of oral swabs from adult cattle of both sexes were used in this study. these samples originated from different regions according to the following groups. group consisted of healthy cattle from an area free of fmd with vaccination. group consisted of cattle from carero da várzea, state of amazonas, which were sampled during an outbreak of fmd in. these cattle did not have any clinical signs and were sampled more than days after the identification of the index case. group consisted of cattle suspected of fmd. these swabs were obtained during an outbreak of fmd in. sampling dacron-tipped swabs were obtained from the oral cavity by swabbing the oral mucosa and tongue, saturating the swab with saliva while avoiding contamination with ingested material. immediately after sampling, the swabs were placed into a 2-ml sterile cryogenic tube containing ml of dulbecco's minimal essential medium with antibiotic and antimycotic. the tubes containing the swabs from cattle from an area free of fmd were immediately frozen on dry ice and stored at -70°c until further processing. however, due to the unavailability of dry ice in the areas of the outbreaks some samples were kept at 4°c for up to days prior to freezing and storage at -70°c. rna extraction rna samples were extracted using the mini rneasy kit following the manufacturer's instructions. briefly, the tubes containing the swabs were thawed and thoroughly homogenized by vortexing, and μl of the medium were added to μl of the rlt buffer that is included in the kit, followed by μl of 70% ethanol, and then transferred to a mini rneasy column previously inserted into a 2-ml collecting tube. rna was immobilized in the column by centrifugation, sequentially washed, and eluted in μl of rnase-free water. in addition to the rna samples obtained from oral swabs as described above, rna samples were purified using trizol from reference strains representing the serotypes that occur in brazil, and previously characterized field isolates. these rna samples were used as positive controls. real-time reverse transcription polymerase chain reaction the rrt-pcr amplification protocol used in the current study was previously described. this method amplifies a conserved segment of the fmdv rna polymerase gene. the following primers were used 5'-actgggttttacaaacctgtga-3' and 5'-gcgagtcctgccacgga-3' along with the probe 5'-tcctttgcacgccgtgggac-3' labeled with 6-carboxyfluorescein at the 5' end, and the quencher tetramethyl rhodamine at the 3' end. this method amplifies the rna from all serotypes of fmdv, but does not amplify nucleic acids from other viruses that cause vesicular diseases. reactions were performed using μl cepheid tubes containing all the dehydrated reagents required for amplification of the fmdv rna that were rehydrated with μl of rehydration buffer. as controls, μl of rna samples or an oligonucleotide used as positive control or te buffer instead of rna as a negative control, were added to the respective reaction tubes. the real-time rt-pcr reactions were carried out in a smart cycler ii thermocycler. the one-step real-time rt-pcr amplification started with reverse transcription for hr at 60°c, followed by pcr with the following parameters: cycles of sec at 95°c and sec at 60°c. one positive and negative control were included in each reaction. virus isolation the samples were filtered using a -μm filter, and μl was then inoculated onto a monolayer of ibrs - cells grown in 25-cm flasks and kept without washing for the duration of the assay. after inoculation, the cultures were checked for cytopathic effects every hr for hr. cultures that did not develop cpe were lysed by freezing at -70°c and reinoculated onto a new monolayer of ibrs- cells, which was then evaluated for an additional hr. cultures with cpe were stored at -70°c until processing for indirect sandwich elisa. indirect sandwich enzyme-linked immunosorbent assay samples from cultures that had cpe were tested by indirect sandwich elisa, according to the protocol recommended by the pan american foot-and-mouth disease center. briefly, anti-fmdv serotypes a, o, and c rabbit antiserum was incubated in elisa plates for hr at 4°c. antiserum was generated by using a pool of inactivated virus subtypes o campos, a cruzeiro, and c indaial as antigen. the plate was washed and incubated with 1% ovalbumin for hr at room temperature. after washing, the reference antigens and testing samples were added to the wells and incubated for hr at 37°c under agitation. the plate was then washed again and incubated with guinea pig anti-fmdv antiserum for min at 37°c under agitation, followed by washing and incubation with an anti-guinea pig antibody conjugated with peroxidase for min at 37°c under agitation. the plate was washed and incubated with a solution of hydrogen peroxide as a substrate for min at room temperature, and read in an elisa reader at nm.  representative results of real-time rt-pcr reactions with samples from the area free of fmd and from areas with outbreaks of fmd are illustrated in figure all cattle from the area free of fmd with vaccination were negative by real-time rt-pcr and combined virus isolation/elisa. the samples from carero da várzea were negative by real-time rt-pcr and combined virus isolation/elisa. among the samples from eldorado and japorã, were positive by real-time rt-pcr, of which were also positive by combined virus isolation/elisa; whereas samples were considered inconclusive with cycle threshold values higher than samples considered inconclusive by real-time rt-pcr were all negative by combined virus isolation/elisa. importantly, no false-negative results by rt-pcr were observed since none of the samples was positive by virus isolation/elisa and negative by real-time rt-pcr. the results are summarized in table the samples from the reference strains and the previously characterized field isolates were all positive by real-time rt-pcr with low ct values indicating high concentration of viral rna. * origin of samples was eldorado/japorã, state of minas gerais. ct corresponds to the number of cycles required for a given sample to reach the threshold above which it is considered positive. a sample was considered positive when the ct value was lower than and inconclusive when the ct value was higher than cpe = cytopathic effect; nd = not determined. only samples that resulted in cpe were processed for indirect sandwich elisa. * ct corresponds to the number of cycles required for a given sample to reach the threshold above which it is considered positive. a sample was considered positive when the ct value was lower than discussion the real-time rt-pcr method used in the current study has proven to be highly sensitive and specific under laboratory and experimental conditions, which is supported by our results as all previously characterized field isolates resulted in positive reactions with low ct values. the samples that were positive by combined virus isolation/elisa were also positive by real-time rt-pcr. in addition, neither false-positives nor false-negatives were detected. indeed, a recent study demonstrated that this method performed similarly to another real-time rt-pcr protocol for amplification of the 5' untranslated region of the fmdv genome. the recommended protocol for storage of samples prior to rna extraction was strictly followed in the case of swabs collected from the area free of fmd with vaccination. however, it was not possible to supply dry ice to the areas affected by fmd outbreaks, which resulted in storage of samples at 4°c up to days. presumably, this inadequate procedure for storage of samples likely resulted in a decrease in the sensitivity of the test. however, samples from the area free of fmd, which were properly stored, were all negative by real-time rt-pcr as well as by virus isolation and elisa, indicating a high specificity of this test as previously demonstrated. a comprehensive recent study of specificity of this method confirmed a very high specificity, but even with the inconclusive reactions considered false-positives, the specificity was %, which may provide enough accuracy for a diagnosis during an outbreak, but may not be specific enough for surveillance in fmd-free areas. in spite of inadequate storage of samples from cattle suspect of fmd, positive animals were identified by real-time rt-pcr, whereas only of those samples were positive by combined virus isolation/elisa, indicating comparable sensitivity between these two diagnostic methods, which is in good agreement with previous. in contrast, none of the samples collected from cattle without lesions more than month after identification of the index case were positive by real-time rt-pcr. although these samples were also inadequately stored, negative results are likely to occur in cattle that recovered from clinical lesions since the likelihood of virus isolation is extremely reduced with more than 7– days after the appearance of gross lesions.  in summary, the current study supports the notion that real-time rt-pcr is a powerful technique for reliable detection of fmdv in a fraction of the time required for combined virus isolation/elisa. however, it is noteworthy that lack of infrastructure in certain areas with high risk of fmd may be a limiting factor for using real-time rt-pcr as a routine diagnostic tool. authors' contributions tap, avcn, and rls collected samples in fmd-free areas; nop collected samples in areas of outbreaks; jrr performed virus isolation procedures; msb performed elisas; tap, avcn, rrs, and rls did the real time rt-pcr reactions; trb, bmm, npc, lga, and rls designed the study, analyzed the data, and supervised all procedures; lga and rls coordinated the group; and all co-authors were involved in writing and revising the manuscript. 
foot-and-mouth disease is an economically important and highly contagious viral disease that affects cloven-hoofed domestic and wild animals. however, it is noteworthy that lack of infrastructure in certain areas with high risk of fmd may be a limiting factor for using real-time rt-pcr as a routine diagnostic tool. a previously described real-time reverse transcriptase polymerase chain reaction assay with high sensitivity and specificity under laboratorial and experimental conditions was used in the current study. three samples from areas with outbreaks of fmd were positive by real-time rt-pcr, and of those samples were positive by virus isolation and elisa. four other samples were considered inconclusive by real-time rt-pcr; whereas all samples from an area free of fmd were real-time rt-pcr negative. real-time rt-pcr is a powerful technique for reliable detection of fmdv in a fraction of the time required for virus isolation and elisa. to verify the applicability of this assay under field conditions in brazil, oral swabs from cattle were collected in areas free of fmd and from areas with outbreaks of fmd. virus isolation and enzyme-linked immunosorbent assay are the gold standard tests for diagnosis of fmd.
 foot-and-mouth disease is an economically important and highly contagious viral disease that affects cloven-hoofed domestic and wild animals. virus isolation and enzyme-linked immunosorbent assay are the gold standard tests for diagnosis of fmd. as these methods are time consuming, assays based on viral nucleic acid amplification have been developed.  a previously described real-time reverse transcriptase polymerase chain reaction assay with high sensitivity and specificity under laboratorial and experimental conditions was used in the current study. to verify the applicability of this assay under field conditions in brazil, oral swabs from cattle were collected in areas free of fmd and from areas with outbreaks of fmd. three samples from areas with outbreaks of fmd were positive by real-time rt-pcr, and of those samples were positive by virus isolation and elisa. four other samples were considered inconclusive by real-time rt-pcr; whereas all samples from an area free of fmd were real-time rt-pcr negative.  real-time rt-pcr is a powerful technique for reliable detection of fmdv in a fraction of the time required for virus isolation and elisa. however, it is noteworthy that lack of infrastructure in certain areas with high risk of fmd may be a limiting factor for using real-time rt-pcr as a routine diagnostic tool. 