Ethidium Bromide-stained Agarose Gels Research Articles

Cloning of porcine cytokine-specific cDNAs and detection of porcine tumor necrosis factor alpha, interleukin 6 (IL-6), and IL-1 beta gene expression by reverse transcription PCR and chemiluminescence hybridization.

A reverse transcription PCR assay with porcine cytokine-specific primers was developed to clone cDNA fragments and generate cDNA probes that were specific for porcine tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), and IL-1 beta. The specificities of the cDNA PCR products were confirmed by sequence analysis on the basis of known porcine cytokine gene sequences. The reverse transcription PCR assay was also used to study cytokine mRNA expression in lipopolysaccharide (LPS)-stimulated and control unstimulated porcine alveolar macrophages. The cDNA products were analyzed in ethidium bromide-stained agarose gels, and the transcription level of each cytokine was determined relative to the endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA level of each cytokine by measuring the intensity of the chemiluminescence hybridization signals by densitometric scanning. Various levels of cytokine mRNAs were detected in both LPS-stimulated and control unstimulated cells. Thus, TNF-alpha mRNA levels were enhanced in the cell cultures stimulated for 6 h with LPS compared with those in control cell cultures. No differences in TNF-alpha transcription levels between LPS-stimulated and control cells were observed after incubation for 24 or 55 h. Enhancements of IL-6 and IL-1 beta mRNA levels were also observed in the cultures stimulated with LPS for 6 and 24 h compared with the cytokine mRNA levels in control cell cultures. The presence of cytokine mRNA transcripts in the LPS-stimulated macrophage cultures correlated with the detection of these soluble cytokines by the bioassays. In contrast, no soluble cytokine was detected in control macrophage culture supernatants in the presence of cytokine mRNA transcripts.

HLA-DQA1 and Amelogenin Coamplification: A Handy Tool for Identification

A protocol for HLA-DQA1 and gender identification by single amplification is described. The use of the commercial HLA-DQA1 amplification kit (Perkin Elmer) permits a positive response for sex determination by adding primers for a short sequence on the first intron of Amelogenin gene. The suggested amplification protocol results in PCR products easily and clearly detectable on ethidium bromide stained agarose gel or silver stained polyacrylamide gel. In both gels the HLA-DQA1 observations at 242-239 bp are accomplished with a single band at 106 bp in females and a doublet 112-106 bp in males. HLA-DQA1 reverse dot-blot hybridization is unaffected by the presence of X and Y amplified fragments.

A rapid method for mRNA detection in single-cell biopsies from preimplantation-stage bovine embryos

Major questions concerning the control of development and gene expression at the cellular level are still unanswered. Nowhere is this more evident than during the earliest stages of development and embryogenesis. This study describes the detection of specific gene transcripts in single cells derived from bovine embryos. Following in vitro fertilization (IVF) and in vitro culture (IVC) of bovine embryos, small groups of cells and even single blastomeres from 32 to 64-cell embryos were micromanipulated into individual tubes for analysis of cytoplasmic RNAs. Reverse transcriptase-PCR was applied to cell lysates for the amplification of β-actin mRNA transcripts. Primers were designed to flank an intron expected to be present within genomic DNA sequences, thus allowing for simple differentiation between DNA- and RNA-derived amplification products. Using a 50-cycle amplification profile, a 260 bp band could be seen as a PCR product derived from a single blastomere following electrophoresis in an ethidium bromide-stained agarose gel. The identity of the band was verified by DNA sequence determination and diagnostic restriction digestion. Lysates derived from single blastomeres in this way have been used for simultaneously phenotyping multiple RNA products. This capability allows the spatial analysis of gene expression and development within embryos from the earliest stages of cellular differentiation.

Competitor internal standards for quantitative detection of mycoplasma DNA

Homologous internal controls were used as competitor DNA in the polymerase chain reaction for the quantitative detection of mycoplasma DNA. PCR primer sets were designed on the basis of the most conserved nucleotide sequences of the 16S rRNA gene of mycoplasma species. Amplification of this gene was examined in five different mycoplasma species: Mycoplasma orale, M. hyorhinus, M. synoviae, M. gallisepticum and M. pneumoniae. To evaluate the primers, a number of different cell lines were assayed for the detection of mycoplasma infections. All positive cell lines showed a distinct product on agarose gels while uninfected cells showed no DNA amplification. Neither bacterial nor eukaryotic DNA produced any cross-reaction with the primers used, thus confirming their specificity. Internal control DNA to be used for quantitation was constructed by modifying the sizes of the wild-type amplified products and cloning them in plasmid vectors. These controls used the same primer binding sites as the wild-type and the amplified products were differentiated by a size difference. The detection limits for all the mycoplasma species by competitive quantitative PCR were estimated to range from 4 to 60 genome copies per assay as determined by ethidium bromide-stained agarose gels. These internal standards also serve as positive controls in PCR-based detection of mycoplasma DNA, and therefore accidental contamination of test samples with wild-type positive controls can be eliminated. The quantitative PCR method developed will be useful in monitoring the progression and significance of mycoplasma in the disease process.

Detection of California serogroup viruses using universal primers and reverse transcription-polymerase chain reaction.

Universal primers have been identified and a protocol developed that are suitable for rapid detection of California encephalitis (CE) complex viruses in a reverse transcription-polymerase chain reaction (RT-PCR) assay. These primers correspond to sequences in the coding regions of the G2 glycoprotein of the middle-size RNA segment. The identities of the amplified products were confirmed by sequencing on the clones or PCR products. The technique is capable of detecting 40 plaque-forming units (PFU) directly on an ethidium bromide-stained agarose gel and the sensitivity increases to 0.4-1 PFU when a radiolabeled probe is used as the detector.

Competitor internal standards for quantitative detection of mycoplasma DNA

Homologous internal controls were used as competitor DNA in the polymerase chain reaction for the quantitative detection of mycoplasma DNA. PCR primer sets were designed on the basis of the most conserved nucleotide sequences of the 16S rRNA gene of mycoplasma species. Amplification of this gene was examined in five different mycoplasma species: Mycoplasma orale, M. hyorhinus, M. synoviae, M. gallisepticum and M. pneumonias. To evaluate the primers, a number of different cell lines were assayed for the detection of mycoplasma infections. All positive cell lines showed a distinct product on agarose gels while uninfected cells showed no DNA amplification. Neither bacterial nor eukaryotic DNA produced any cross-reaction with the primers used, thus confirming their specificity. Internal control DNA to be used for quantitation was constructed by modifying the sizes of the wild-type amplified products and cloning them in plasmid vectors. These controls used the same primer binding sites as the wild-type and the amplified products were differentiated by a size difference. The detection limits for all the mycoplasma species by competitive quantitative PCR were estimated to range from 4 to 60 genome copies per assay as determined by ethidium bromide-stained agarose gels. These internal standards also serve as positive controls in PCR-based detection of mycoplasma DNA, and therefore accidental contamination of test samples with wild-type positive controls can be eliminated. The quantitative PCR method developed will be useful in monitoring the progression and significance of mycoplasma in the disease process.

Rapid deoxyribonucleic acid analysis by allele-specific polymerase chain reaction for detection of mutations in the steroid 21-hydroxylase gene.

Rapid DNA analysis based on allele-specific polymerase chain reaction (PCR) using mutation site-specific primers was developed to detect mutations in the CYP21 gene known to cause steroid 21-hydroxylase deficiency. In contrast to the previous method, in which PCR of genomic DNA was followed by dot blot analysis with radioactive probes and multiple rounds of stripping and reprobing for each of the 8 most common mutation sites, the results using this new method were immediately visualized after the PCR run by ethidium bromide-stained agarose gel electrophoresis. Using allele-specific PCR, mutation(s) were identified on 148 affected chromosomes out of 160 tested. Although mutation(s) were identified on only one chromosome of 11 of these patients, their parents showed a consistent pattern on DNA analysis. The only exception was that in one family, in which the parents each had a detectable mutation, a mutation was detected on only one allele of the patient. Most likely there is a mutation in the patient's other allele that could have arisen de novo or was inherited from the parent and was not evident in the transmitting parent's phenotype. When compared with the dot blot procedure, allele-specific PCR is more rapid, less labor-intensive, and avoids the use of radioactivity.

Tandem repeat sequence variation and length heteroplasmy in the mitochondrial DNA D-loop of the threatened Gulf of Mexico sturgeon, Acipenser oxyrhynchus desotoi.

Genetic variability within the Suwannee River, Florida, population of Gulf of Mexico sturgeon, Acipenser oxyrhynchus desotoi, was assessed by examining sequence and length variation within the control region, or D-loop, of the mitochondrial genome. Although once abundant throughout the Gulf of Mexico, Gulf sturgeon are now listed as a threatened species by the U.S. Fish and Wildlife Service. Mitochondrial DNA was analyzed for length variation from 168 individual Gulf sturgeon by PCR amplification and visualization of PCR products using ethidium bromide-stained agarose gels. Of the 168 individual Gulf sturgeon, 31 (18.5%) were heteroplasmic for one to four copies of an 81-base pair, tandemly repeated sequence in the D-loop region. However, no individuals homoplasmic for multiple copies of the repeat sequence were observed. The existence and nature of these tandem repeats in heteroplasmic individuals was confirmed by direct sequencing of the PCR products for a subset of 22 individuals. The results are consistent with the apparent nature and mechanism of heteroplasmy observed in a congeneric species, A. transmontanus. In addition, sequences for 187 base pairs outside of the tandem repeats were identical among all 16 individuals assayed for this region. Lack of variable sequences is concordant with earlier studies involving mtDNA restriction fragment length profiles of Gulf sturgeon found in the Suwannee River. The absence of sequence variation exclusive of the tandem repeats is consistent with the hypothesis that the subspecies has undergone a population or evolutionary bottleneck.

An improved, sensitive, non‐radioactive in situ hybridization method for the detection of cytokine mRNAs

We established an improved non-radioactive in situ hybridization (ISH) method to detect mRNA of cytokines in cell preparations and tissues. Via this method we could demonstrate various cytokines in stimulated peripheral blood mononuclear cells (PBMC), lymphoid cell lines and human lymphoid tissues. The probes for the in situ hybridization were made by labelling cytokine-specific PCR products with digoxigenin (Dig) in a repeated PCR. This resulted in an intrinsic labelling of the probe with several Dig-UTP molecules. Incorporation of Dig-11-dUTPs was shown on ethidium bromide-stained agarose gels by a higher molecular weight of the PCR products with incorporated Dig-dUTPs when compared to control PCR products without digoxigenin. Cytospin-centrifuged cells of PHA-stimulated PBMC or lymphoid cell lines and frozen sections of various human lymphoid tissues were hybridized with the Dig-labelled cytokine probes and the hybridized probes were detected immuno-histochemically. In this way, we detected and localized cytokine mRNAs (IL-2, IL-4, IL-6, IL-8, IL-10) in PBMC, in the human T-cell line Jurkat, in the follicular lymphoma cell line DoHH2, and in human lymph nodes and tonsils. The in situ hybridization had a high sensitivity as the results correlated closely with the detection of cytokine mRNA by reverse transcriptase-PCR (RT-PCR) data from the same samples. We showed that Jurkat and DoHH2 cells produce several cytokines constitutively and that, after activation with the phorbol ester PMA, expression of several cytokine mRNAS was enhanced.

Allele-specific competitive blocker PCR: a one-step method with applicability to pool screening.

We have developed a novel one-step pool screening PCR procedure which is based on the principles of amplification refractory mutation system (ARMS) and competitive oligonuleotide priming (COP) PCR. In addition to the usual primers, this approach uses two allele-specific competitive oligonucleotides, one of which is 3'-end labeled with a dideoxynucleotide and blocks amplification of the wild-type allele. An allele-specific product is generated only in the presence of the mutation. The introduction of an allele-specific competitive blocker oligonucleotide improves the specificity and robustness of ARMS-PCR. Further its sensitivity is dramatically increased, which allows detection of one mutant allele in a large excess of wild-type-bearing genomic DNA by electrophoresis in an ethidium bromide-stained agarose gel (up to 1 in 10(4) alleles). This makes the method ideal for nonradioactive pool screening. The successful application of the method has been demonstrated for four different point mutations, two in the apolipoprotein B gene (R3500Q, R3531C) which result in familial defective apolipoprotein B-100, one in the CFTR gene (R1162X), and one in the gene for lipoprotein lipase (G188E).

Detection ofErwinia amylovoraby Nested PCR and PCR-Dot-Blot and Reverse-Blot Hybridizations

The sensitivity and specificity of four methods based on the polymerase chain reaction (PCR) for detection of Erwinia amylovora were compared. A previously developed single-round PCR assay was used to amplify a specific 1-kb DNA fragment of plasmid pEA29, known to be unique to and conserved in E. amylovora. The ends of the 1-kb single-round PCR product were sequenced, and two new oligonucleotide primers were designed from sequences internal to the original primers and used in nested PCR. These primers directed amplification of a 844-bp fragment from the 1-kb first-round PCR product directly from E. amylovora cells but not from other bacteria associated with apple. Less than one cell of E. amylovora from pure culture was detected with nested PCR-an increase in sensitivity of 1,000-fold compared to single-round PCR. The lower limit of detection of PCR-dot-blot and reverse-blot hybridization methods was approximately 20 cells. Weak positive signals were sometimes produced by bacteria other than E. amylovora in hybridization experiments but only if nonspecific PCR products were visible on ethidium bromide-stained agarose gels. When asymptomatic apple tissue was screened, E. amylovora was detected in 61, 84, and 100 % of leaf samples; 0, 66, and 80 % of axillary bud samples; and 4, 27, and 75 % of mature fruit calyx samples by first-round PCR, PCR-dot-blot hybridization, and nested PCR, respectively. The potential applications of each method are discussed

Quantitation of bovine TNF-α mRNA by reverse transcription and competitive polymerase chain reaction amplification

A reverse transcription (RT) and competitive polymerase chain reaction (PCR) amplification technique was developed to quantify bovine tumor necrosis factor-alpha (TNF-α) mRNA. A bovine TNF-α cDNA insert containing plasmid was used to produce a 237-bp cDNA competitive template which, when PCR-amplified with primers designed to amplify bovine TNF-α cDNA, resulted in a 147-bp product distinguishable by agarose gel electrophoresis from the 378-bp TNF-α RT-PCR-amplified cDNA product. Dilutions of competitive template were co-amplified with constant amounts of total cellular RNA harvested from bovine alveolar macrophages (BAM). Products were separated in ethidiumbromide-stained agarose gels and relative amounts of TNF-α mRNA were determined by densitometric scanning of TNF-α cDNA and competitive template product bands on photographic negatives. Digestions with Pvu II and Sty I restriction endonucleases verified that RT-PCR-amplified TNF-α cDNA products were authentic. A time study indicated that TNF-α mRNA concentrations peaked 3 h after endotoxin and virus induction of BAMs.

PCR Detection ofErwinia carotovorasubsp.atrosepticaAssociated with Potato Tissue

The nucleotide sequence for a DNA hybridization probe specific for Erwinia carotovora subsp. atroseptica was determined, and primers were selected for detection of the blackleg pathogen using the polymerase chain reaction (PCR). Primers ECA1f (5'-CGGCATCATAAAAACACG-3') and ECA2r (5'-GCACACTTCATCCAGCGA-3') specifically amplified a 690-bp DNA fragment of all E. carotovora subsp. atroseptica strains tested but not strains of other E. carotovora subspecies isolated from various hosts and geographic regions or other plant- and soil-associated bacteria. Visualization of the E. carotovora subsp. atroseptica-specific PCR product on ethidium bromide-stained agarose gels required a minimum of 250 to 500 CFU per ml. A total of 170 samples of potato stem and tuber tissue was tested by PCR and compared with reactions in enzyme-linked immunosorbent assay (ELISA) with a monoclonal antibody specific for the lipopolysaccharide of E. carotovora subsp. atroseptica. Although 50.6% of these samples were positive in PCR compared to 46.5% in ELISA, some of the ELISA-positive samples were negative in PCR. When E. carotovora subsp. atroseptica DNA was added to these samples, amplified products were obtained in all but two samples after repeating the PCR, indicating that in most cases the failure to obtain PCR amplification for some of the ELISA-positive samples was not due to the presence of inhibitors.

Double-nested polymerase chain reaction for detection of caprine arthritis-encephalitis virus proviral DNA in blood, milk, and tissues of infected goats

A nested polymerase chain reaction (PCR) for detecting proviral DNA of caprine arthritis-encephalitis virus (CAEV) in biological samples was developed. Primers for both gag and pol sequences of the CAEV genome were included in a single tube for simultaneous amplification (‘double’ PCR), and the resulting bands were resolved visually in ethidium bromide-stained agarose gels. Internal gag and pol probes were used to verify the identity of the amplified products by non-radioactive Southern hybridization. Final confirmation of the identity of representative PCR bands was provided by DNA sequence analysis. A comparison between the PCR and an antibody ELISA (with recombinant CAEV p28 as target) using 141 caprine blood samples indicated very strong agreement between the two assays ( κ = 0.912). Four of 7 goats with indeterminate ELISA results were PCR-positive as were 5 of 40 (12.5%) seronegative goats, most probably indicating delayed seroconversion. Eleven of 27 goats (41%) PCR-positive on blood had detectable CAEV proviral DNA in milk. Proviral DNA was also detected in lung, mesenteric lymph node, bone marrow, synovial membrane, and mammary gland of a seropositive, clinically affected goat, but not in equivalent tissues of a healthy seronegative goat.

Unique double-stranded RNAs associated with the Trichomonas vaginalis virus are synthesized by viral RNA-dependent RNA polymerase.

Most Trichomonas vaginalis isolates are carriers of the multisegmented double-stranded RNA (dsRNA) virus. In vitro polymerase assays were performed to demonstrate the RNA-dependent RNA polymerase (RDRP) activity of purified particles. Transcripts which comigrated with the dsRNAs of the virus were readily detected as synthesized products, indicating viral RDRP activity. In addition, smaller-sized dsRNA species, possibly two of approximately 700 bp (s1) and one of 500 bp (s2), were synthesized by purified virus particles of the CsCl gradient surrounding the virus peak. No cross-hybridization with either s1 or s2 and the dsRNA segments occurred, suggesting that s1 and s2 were synthesized from different templates. An RNase A protection assay revealed that the synthesized s1 and s2 polymerase products were double stranded. Furthermore, hybridization of products with strand-specific RNA of s1 generated from cDNA indicated that only one strand was synthesized in vitro. s1 and s2 were not visualized in ethidium bromide-stained agarose gels of dsRNA of infected trichomonads grown in batch cultures. However, dsRNA profiles of the same infected organisms cultivated under defined continuous-flow conditions contained readily detectable levels of s1 and s2, indicating that amplification of s1 and s2 occurred under specific environmental conditions. These newly discovered dsRNAs were not detected in all of the virus-carrying isolates. Finally, it is noteworthy that the s1 and s2 dsRNAs and the RDRP activity were not detected in trichomonal isolates without virus or in virus-negative progeny derived from virus-positive parental isolates. These data indicate the possibility of variations in the number of dsRNAs and/or of the presence of satellites in trichomonads infected with the multisegmented virus.

A Highly Sensitive, Rapid, and Simple Polymerase Chain Reaction-Based Method to Detect Human Malaria (Plasmodium Falciparum and Plasmodium Vivax) in Blood Samples

A highly sensitive, rapid and simple method to detect human malaria in blood samples was developed. Malaria parasite DNA in blood from a fingerprick was directly amplified by the polymerase chain reaction (PCR) using two sets of primers to yield a 206-basepair (bp) product for Plasmodium falciparum and a 183-bp product for P. vivax. Both were easily visualized in an ethidium bromide-stained agarose gel, allowing identification of the two human malaria species in a single amplification reaction. As little as a one P. falciparum and/or P. vivax parasite per microliter of blood was detectable by this method, a sensitivity superior to that of thick blood film microscopy. The total time required for diagnosis of 48 blood samples, starting from fingerprick blood collection, was approximately 4 hr. When compared with microscopic examination by an expert microscopist, results showed a sensitivity of 89% for P. falciparum and 91% for P. vivax and an overall specificity of 94%. Six infected blood samples classified by microscopy as single species were diagnosed by the PCR method as being mixed P. falciparum and P. vivax infections. The high sensitivity, rapidity, and simplicity of the method should make it attractive for a large-scale epidemiology study, follow-up of drug treatment, and immunization trials.

Rare detection of hepatitis B and hepatitis C virus genomes by polymerase chain reaction in seronegative donors with elevated alanine aminotransferase.

Since screening for antibody to hepatitis C virus (HCV) was introduced in 1990, posttransfusion hepatitis has been reduced to nearly background levels. This has led to reconsideration of the value of testing donated blood for elevated alanine aminotransferase (ALT). The contribution of ALT testing in detecting seronegative infection was evaluated by the performance of polymerase chain reaction (PCR) for hepatitis B virus (HBV) or HCV in plasma from ALT-elevated blood units. Testing was performed on 375 units of plasma, derived from an equivalent of 47,500 blood donations, with a highly sensitive hemi-nested PCR procedure. Using a triplet of primers directed at the conserved regions of HBV DNA and 5'-noncoding regions of HCV RNA, the hemi-nested PCR assay can reliably amplify 10 viral molecules to levels detectable in ethidium bromide-stained agarose gels. Pools of plasma from groups of four donors were screened with hemi-nested PCR. For any reactive pools, the plasma from individual donors was retested twice on different aliquots. Two of 375 units, both with midrange ALT elevation, were repeatedly reactive in hemi-nested PCR (one each for HBV DNA and HCV RNA). However, samples from the two suspect donors tested 9 and 5 months later revealed no seroconversion, elevated ALT, or viral genomes in hemi-nested PCR. The lack of confirmed HBV or HCV infection in this study representing an estimated 47,500 voluntary blood donations suggests that routine ALT testing for further prevention of posttransfusion hepatitis after exclusion of HBV- and/or HCV-seropositive blood may be superfluous.

Characterization of Random Amplified Polymorphic DNA (RAPD) Products from Xanthomonas campestris and Some Comments on the Use of RAPD Products in Phylogenetic Analysis

As part of our research to determine phylogenetic relationships of organisms within the phytobacterial species Xanthomonas campestris, we have examined the use of the random amplified polymorphic DNA (RAPD) technique. The objective of this aspect of our research was to determine if a valid cladistic character analysis could be carried out by direct comparison of RAPD products separated on ethidium bromide-stained agarose gels. RAPD products were amplified from 47 Xanthomonas campestris DNA templates using a single oligonucleotide primer. These RAPD products were compared and variation was characterized by Southern analysis of both RAPD products and genomic DNA of the 47 bacterial strains using two cloned RAPD products as probes. Analysis of the data set revealed that the RAPD products were not necessarily homologous or independent, crucial prerequisites for characters to be analyzed in a cladistic phylogenetic analysis. It has been commonly assumed that RAPD variation occurs due to insertion/deletion events or alterations in the primer binding site. Within our data set, we demonstrate absence phenotypes arising from the apparent absence of corresponding loci and also due to the preferred synthesis of alternative RAPD products from unrelated loci. These different types of variation are a reflection of different types of genotypic variation, and direct examination of RAPD products did not allow us to distinguish by which mechanism a particular absence phenotype arose. Although this may not be important for phenetic analyses, for analyses of homologous characters using a cladistic approach it is critical. We also detected unrelated, co-migrating RAPD products and multiple related RAPD products within reaction mixtures. These could both contribute to errors in estimates of similarity, important in any phylogenetic analysis. All of these characteristics of RAPD products should be taken into consideration when RAPD products are used for phylogenetic comparisons.

Rapid quantitation of mRNA species in ethidium bromide-stained gels of competitive RT-PCR products.

A rapid method for quantifying the low-abundant mRNAs of the low density lipoprotein receptor and the 3-hydroxy-3-methylglutaryl coenzyme A reductase by competitive polymerase chain reaction is presented. This approach requires neither special labeling nor blotting procedures. For each analysis, a defined amount of total cellular RNA is co-reverse transcribed and co-amplified with a titration series of in vitro synthesized competitor RNA that carries an internal deletion. The equivalence point, which defines the amount of specific RNA in the sample, can be scored in ethidium bromide-stained agarose or polyacrylamide gels of the reaction products. As an example, responses to pravastatin, a competitive inhibitor of the HMG-CoA reductase, in a human tumor cell line were analyzed with this new technique. As a control, the expression of the unregulated gene, glyceraldehyde-3-phosphate dehydrogenase was measured in parallel using the same methodology. The results obtained were compared with those obtained by conventional Northern blotting.

Detection of Epizootic Hemorrhagic Disease Virus Serotypes 1 and 2 in Cell Culture and Clinical Samples Using Polymerase Chain Reaction

The potential for the use of the polymerase chain reaction (PCR) in detecting epizootic hemorrhagic disease virus (EHDV) ribonucleic acid in cell cultures and clinical samples was studied. Using oligoribonucleotide primers selected from genome segment 6 of EHDV-2 (Alberta strain), the PCR-based assay resulted in a 387-base pair (bp) PCR product. EHDV RNA from US prototype serotypes 1 and 2 and a number of EHDV field isolates, propagated in cell cultures, were detected by this EHDV PCR-based assay. Amplification products were visualized on ethidium bromide-stained agarose gels or detected by chemiluminescent hybridization. The sensitivity of the PCR assay was 100 fg of virus RNA (equivalent to 6 x 10(3) virus particles) with ethidium bromide-stained agarose gels. Chemiluminescent hybridization increased the sensitivity of the PCR assay 1,000 times, and specific signals were detected from 0.1 fg of virus RNA (equivalent to 6 virus particles). Amplification product was not detected when the PCR-based assay was applied to RNA from the US bluetongue (BT) virus prototype serotypes 2, 10, 11, 13, and 17 or total nucleic acid extracts from uninfected baby hamster kidney-21 cells, Vero cells, and blood cells from deer that were EHDV seronegative and virus isolation negative. Application of this EHDV PCR-based assay to clinical samples resulted in detection of EHDV RNA from blood and spleen samples from a deer in California with clinical hemorrhagic disease. This EHDV PCR-based assay could provide a rapid, sensitive, and specific assay for detection of EHDV infection in susceptible ruminants.