No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Detection of foot-and-mouth disease virus (FMDV) in biological material
Abstract
Zakład Pryszczycy Państwowego Instytutu Weterynaryjnego, ul. Wodna 7, 98-220 Zduńska Wola Foot-and-mouth disease (FMD) still exists in Europe. Rapid diagnosis is essential for a precise and effective eradication of the disease. The aim of this work was to compare two methods used in diagnosing FMD: by viral isolation (VI) - ELISA and RT-PCR. Samples of epithelium from FMDV infected animals and supernatant from inoculated susceptible cell culture from the authors own collection of FMD infected materials were used. The results obtained were: 81.3% positive results using VI and 53.1% using ELISA. The effectiveness in detecting FMDV increased to 87.5% after applying both methods. RT-PCR appeared to be the most effective method of viral RNA (vRNA) detection in all tested samples. The reaction was performed with primers from the 3D coding region of viral genomes. The specificity of PCR was confirmed by restriction analysis of amplicans. The specificity and sensitivity of RT-PCR justifies its inclusion into routine FMDV diagnosis. Thanks to this technique it was possible to detect vRNA in material which had been thawed several times as well as in poor quality (contaminated) materials.
... In the scenario of overall distribution pattern of FMD virus types, serotype 'O' (60.00%) was the predominant type, followed by serotype 'A' (40.00%), (Table 3). specimens negative in sandwich ELISA test were found to be positive in RT-PCR.It indicates that the RT-PCR is more sensitive than and sandwich ELISA test (Donn et al., 1996;Reid et al., 1999;Alexandersen et al., 2000;Paprocka and Kesy, 2001;Paprocka et al., 2002;Clavijo et al., 2003;King et al., 2006;Verma et al., 2010Verma et al., , 2012). The higher sensitivity of RT-PCR may be because of its ability to detect very small number of virus as well as detection of RNA of non-viable FMD virus. ...
The aim of this study was to compare the effectiveness of ELISA and RT-PCR cell culture isolation methods for the detection and differentiation of FMDV isolates. Samples of epithelium taken from FMDV infected animals and supernatant from inoculated susceptible cell culture from the laboratory collection of FMD infected biological materials were used. RT-PCR was performed with the 3 sets of primers specific for FMDV serotypes A, O and C. This method allowed to serotype FMDV isolates on the basis of different length of the amplification products. Similar results were obtained using isolation/ELISA and RT-PCR methods, the only difference occurring in 100% positive results with RT-PCR and in 87.5% of positive results by means of standard methods. In conclusion, it can be stated that RT-PCR is a very sensitive and specific method for FMDV detection and differentiation of FMDV serotypes A, O and C.
The aim of these studies was to prepare the digoxigenin (DIG)-labeled molecular probes for the identification of RT-PCR products. FMD infectious biological material from the laboratory collection was used. The PCR reaction of cDNA of all FMDV samples was performed using 30 cycles. The specificity of amplicons was confirmed by Southern Blot hybridization using DIG-labeled probes. By the use of DIG for the labeling of FMD cDNA it was possible to obtain very specific and sensitive molecular probes. The hybridization method allowed us to verify RT-PCR results and can be applied as an additional diagnostic method for FMD.
A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
A PCR assay for the detection and characterization of foot-and-mouth disease virus was developed. The procedure allows RT-PCR amplification following direct adsorption of viral suspensions to microtiter plates, avoiding previous steps of phenol-extraction or heating. Using this procedure, FMDV-specific (based on 3D gene sequences), as well as serotype-specific (based on VP1 gene sequences) amplification were achieved for viral samples of serotypes A, O and C, either from cell culture supernatants or from lesions of infected animals. The assay allowed detection of around 15 PFU, being 500-fold more sensitive than a conventional indirect ELISA. This new method constitutes a simple, rapid and efficient alternative for the diagnosis and characterization of FMDV by PCR.
Molecular detection of foot-and-mouth disease virus (FMDV) using the polymerase chain reaction (PCR) is a rapid and accurate method. In this study we present PCR for the detection of FMDV RNA in infected BHK cells. Using PCR and two primers selected from the RNA polymerase gene, a conserved sequence in all types and subtypes of FMDV, we were able to detect FMDV RNA present in RNA extracted from the FMDV-infected cells. RNA from uninfected BHK cells gave negative results. Another set of primers selected from the nucleotide sequence of the variable VP1 gene permitted the demonstration of variations among different FMDV Israeli isolates by PCR. Two 01 type FMDV isolates out of a total of 6 FMDV field isolates (including 01 Geshur) gave a positive PCR while two other 01 isolates and two ASIA isolates were detected with the RNA polymerase gene primers but not with the VP1 primers. Serial dilutions of the RNA used in each reaction showed that a very small amount of RNA may be detected by PCR. The PCR products from the RNA polymerase and the VP1 genes were sequenced and the nucleotide sequences obtained were compared with a known nucleotide sequence of the FMDV 01 genome.
A method for extracting RNA from animal-derived materials that provides foot-and-mouth disease viral template suitable for Tth polymerase-dependent synthesis of cDNA and subsequent PCR is described. Viral genomes were detected in less than 24 h. Nasal swabs that can be easily and repeatedly collected, proved suitable for virus detection by PCR, even during the asymptomatic stages of infection.
Reverse transcription coupled with PCR was used for the detection of foot-and-mouth disease virus serotypes A, C, and O in organ extracts from experimentally infected cattle. Primers were selected from conserved sequences flanking the genome region coding for the major antigenic site of the capsid located in the C-terminal part of viral protein 1 (VP1). Because this region of the capsid is highly variable its coding sequence is considered to be the most appropriate for the characterization of virus isolates and, therefore, for the determination of the epidemiological relationships between viruses of the same serotype. For differentiation between serotypes and for detailed characterization of individual virus isolates restriction enzyme cleavage and nucleotide sequence analysis of the respective PCR products were carried out. In order to minimize the time required for sample preparation from clinical material, viral RNA was released from particles by heating the sample for 5 min at 90 degrees C. Finally, an air thermocycler was used, which allows performance of a PCR of 30 cycles in approximately 20 min. The results show that reverse transcription PCR followed by restriction enzyme analysis and/or nucleotide sequence analysis of the PCR products is useful for the rapid detection and differentiation of foot-and-mouth disease virus.
The polymerase chain reaction method (PCR) has been applied to the diagnosis of foot-and-mouth disease viral RNA in tissues and, particularly, oesophageal-pharyngeal fluid (probang) samples from cattle. Using primer sets which corresponded to conserved regions of the VP1 sequence of the viral genome, it was possible to amplify sequences regardless of the serotype/strain of the virus. In comparison with infectivity assays, the PCR was generally more sensitive although there were a number of examples where only infectivity was detectable. In experiments with uninfected probang samples deliberately seeded with a dilution series of virus, the PCR proved to be approximately 10(4) times more sensitive than infectivity assays. This greater sensitivity was attributed, in part, to the ability of the PCR to amplify specifically from non-infectious RNA preparations. This enabled the identification, by sequencing, of viral RNA from chemically inactivated virus concentrates typical of those used for commercial vaccine production. Amplification of specific PCR products was also achieved with virus eluted from commercial vaccine, including preparations which had been stored for more than 10 years at 4 degrees C. The PCR technique is of considerable value, therefore, both as a complement to infectivity assays and as a powerful tool in vaccine-associated studies.
Foot-and-mouth disease is one of the most economically important virus diseases of livestock. Two important requirements for the control of this disease are rapid laboratory diagnosis and epidemiological investigation. The use of the polymerase chain reaction method (PCR) to amplify specific nucleic acid regions offers the unique possibility of combining swift viral detection with the production of genetic material suitable for sequencing and other methods of molecular epidemiological analysis. The sequencing of the region of foot-and-mouth disease virus (FMDV) genome encoding the capsid proteins of the virus (approximately 2260 bps), provides valuable information that adds to the molecular characterisation of an isolate. This paper describes the use of the PCR for the amplification of this region of the FMDV genome from bovine clinical samples and cell culture isolates. Suitable pairs of oligonucleotide primers were selected from the published sequence of FMDV type O1, Kaufbeuren. One primer set amplified 2091 bps of the capsid coding region of all seven serotypes of FMDV. The other primer set amplified 216 bp from this region of FMDV type O1, BFS 1860, in nucleic acid extracts from several clinical samples. Nucleic acid extracts from the picornaviruses, bovine enterovirus and swine vesicular disease virus, which affect the same animals, were not amplified. Direct sequencing was carried out on the amplified fragments and showed that the PCR products were > 98% homologous to published FMDV sequences.
A polymerase chain reaction (PCR) technique was used to detect the presence of foot-and-mouth disease virus (FMDV) in oesophageal-pharyngeal(OP) samples from experimentally infected steers. Ten-fold dilutions of OP samples were also diluted, inoculated onto lamb kidney cell cultures, and incubated overnight. The cultures that did not show overt cytopathogenic effects (CPE) of FMDV infection were frozen and thawed; both the fluid and the cell pellet were tested by the PCR technique. The PCR detected FMDV in the fluids of 57% of the cell cultures inoculated with 2-20 tissue culture infective doses-50% (TCID50) and of 33% of cell cultures inoculated with the 0.4-2 TCID50. The PCR detected FMDV in cell pellets of all cell cultures inoculated with 20 TCID50, of 71% of cell cultures inoculated with 2-20 TCID50 and of 50% of cell cultures inoculated with 0.2-2 TCID50. A diagnostic scheme using PCR and cell culture that would provide rapid and sensitive detection of FMDV in OP samples is proposed.
Reverse transcription followed by the polymerase chain reaction method (PCR) allowed the detection of foot-and-mouth disease virus (FMDV), regardless of the serotype. A primer set corresponding to highly conserved regions of the 2B sequence was selected. By combining in a single reaction tube specific primer pairs for FMDV, swine vesicular disease virus, (SVDV), encephalomyocarditis virus (EMCV) and bovine viral diarrhea virus (BVDV), all four viruses could be identified and differentiated in one amplification reaction, based on the different lengths of the respective amplified segments. In a similar way, FMDV types O, A, C, SAT 2 and Asia 1 could be identified and differentiated, using primers selected from the ID (VP1) genome region. All results were confirmed by direct sequencing of the PCR product. The very fast RNA extraction, reverse transcription and PCR permitted us to read the agarose gels within three hours after samples (cell culture isolates as well as clinical material) arrived, which is of great importance in case of an FMDV suspicion. Furthermore, a very high sensitivity was achieved (less than one PFU). Therefore, our powerful detection assay by means of PCR for FMDV as well as for SVDV, EMCV and BVDV, has advantages compared to the presently used procedures.
A reverse transcription polymerase chain reaction (RT-PCR) method was compared with virus isolation in cell culture and the antigen detection ELISA for the primary diagnosis of foot-and-mouth disease (FMD) on 166 clinical samples from the field. Eighty samples were positive by virus isolation/ELISA and 78 by RT-PCR. The RT-PCR detected FMD viral RNA in 11 of the 86 samples assessed as negative by virus isolation/ELISA but conversely failed to diagnose 13 samples identified as positive by the latter procedures. This RT-PCR is not serotype-specific so a cDNA product is indicative of the presence of FMD viral RNA only. Confirmation of the specificity of the cDNA product and the identification of the serotype requires nucleotide sequence analysis. The value of the RT-PCR is that it can rapidly facilitate the molecular analysis of field isolates and thus provide important epidemiological information regarding the source of outbreaks. However, it is a sophisticated technique requiring specialised equipment, expertise and refined reagents and has to be used in conjunction with current procedures for FMD diagnosis.