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At least 397 records · Page 22Linked to original sources
PubMed2005

Multiplex PCR for rapid differentiation of three species in the "Clostridium clostridioforme group".

Clostridium clostridioforme is a relatively antimicrobial resistant, phenotypically heterogeneous anaerobe that has been involved in a variety of infections. 16S rDNA sequencing analysis revealed three principal species in what has been called Clostridium clostridioforme - Clostridium bolteae, C. clostridioforme, and Clostridium hathewayi. Based on the 16S rDNA sequence information we obtained, we developed a cost-effective, timesaving one-step multiplex PCR assay for rapid and accurate differentiation of these three species. The established multiplex PCR identification scheme was applied to the identification of 88 clinical isolates that had previously been identified phenotypically as C. clostridioforme. The identification obtained from multiplex PCR assays showed 100% agreement with 16S rDNA sequencing identification. This scheme will permit more accurate assessment of the role of these three Clostridium species in infection and of the degree of antimicrobial resistance in each of the species.

Bacteriological Techniques↗
PubMed2005

Development and amplification of multiple co-dominant genetic markers from single spores of arbuscular mycorrhizal fungi by nested multiplex PCR.

Multiple co-dominant genetic markers from single spores of the arbuscular mycorrhizal (AM) fungi Glomus mosseae, Glomus caledonium, and Glomus geosporum were amplified by nested multiplex PCR using a combination of primers for simultaneous amplification of five loci in one PCR. Subsequently, each marker was amplified separately in nested PCR using specific primers. Polymorphic loci within the three putative single copy genes GmFOX2, GmTOR2, and GmGIN1 were characterized by sequencing and single strand conformation polymorphisms (SSCP). Primers specific for the LSU rDNA D2 region were included in the multiplex PCR to ensure correct identification of the Glomus spp. spores. Single AM fungal spores were characterized as multilocus genotypes by combining alleles of each amplified locus. Only one copy of each putative single copy gene could be amplified from each spore, indicating that spores are homokaryotic. All isolates of G. mosseae had unique genotypes. The amplification of multiple co-dominant genetic markers from single spores by the nested multiplex PCR approach provides an important tool for future studies of AM fungi population genetics and evolution.

Alleles↗
PubMed2005

Validation of a male-specific, 12-locus fluorescent short tandem repeat (STR) multiplex.

Y chromosome-specific short tandem repeat (Y-STR) analysis has become another widely accepted tool for human identification. The PowerPlex Y System is a fluorescent multiplex that includes the 12 loci: DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438 and DYS439. This panel of markers incorporates the 9-locus European minimal haplotype (EMH) loci recommended by the International Y-STR User Group and the 11-locus set recommended by the Scientific Working Group on DNA Analysis Methods (SWGDAM). Described here are inter-laboratory results from 17 developmental validation studies of the PowerPlex Y System and include the following results: (a) samples distributed between laboratories and commercial standards produced expected and reproducible haplotypes; (b) use of common amplification and detection instruments were successfully demonstrated; (c) full profiles were obtained with standard 30 and 32 cycle amplification protocols and cycle number (24-28 cycles) could be modified to match different substrates (such as direct amplification of FTA paper); (d) complete profiles were observed with reaction volumes from 6.25 to 50 microL; (e) minimal impact was observed with variation of enzyme concentration; (f) full haplotypes were observed with 0.5-2x primer concentrations; however, relative yield between loci varied with concentration; (g) reduction of magnesium to 1mM (1.5 mM standard) resulted in minimal amplification, while only partial loss of yield was observed with 1.25 mM magnesium; (h) decreasing the annealing temperature by 2-4 degrees C did not generate artifacts or locus dropout and most laboratories observed full amplification with the annealing temperature increased by 2 degrees C and significant locus dropout with a 4 degrees C increase in annealing temperature; (i) amplification of individual loci with primers used in the multiplex produced the same alleles as observed with the multiplex amplification; (j) all laboratories observed full amplification with >or = 125 pg of male template with partial and/or complete profiles observed using 30-62.5 pg of DNA; (k) analysis of < or = 500 ng of female DNA did not yield amplification products; (l) the minor male component of a male/female mixture was observed with < or =1200-fold excess female DNA with the majority of alleles still observed with 10,000-fold excess female; (m) male/male mixtures produced full profiles from the minor contributor with 10-20-fold excess of the major contributor; (n) average stutter for each locus; (o) precision of sizing were determined; (p) human-specificity studies displayed amplification products only with some primate samples; and (q) reanalysis of 102 non-probative casework samples from 65 cases produced results consistent with original findings and in some instances additional identification of a minor male contributor to a male/female mixture was obtained. In general, the PowerPlex Y System was shown to have the sensitivity, specificity and reliability required for forensic DNA analysis.

Animals↗
PubMed2005

Validation of male-specific, 12-locus fluorescent short tandem repeat (STR) multiplex.

Y chromosome-specific short tandem repeat (Y-STR) analysis has become another widely accepted tool for human identification. The PowerPlex Y System is a fluorescent multiplex that includes the 12 loci: DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438 and DYS439. This panel of markers incorporates the 9-locus European minimal haplotype (EMH) loci recommended by the International Y-STR User Group and the 11-locus set recommended by the Scientific Working Group on DNA Analysis Methods (SWGDAM). Described here are inter-laboratory results from 17 developmental validation studies of the PowerPlex Y System and include the following results: (a) samples distributed between laboratories and commercial standards produced expected and reproducible haplotypes; (b) use of common amplification and detection instruments were successfully demonstrated; (c) full profiles were obtained with standard 30 and 32 cycle amplification protocols and cycle number (24-28 cycles) could be modified to match different substrates (such as direct amplification of FTA paper); (d) complete profiles were observed with reaction volumes from 6.25 to 50 microL; (e) minimal impact was observed with variation of enzyme concentration; (f) full haplotypes were observed with 0.5-2x primer concentrations; however, relative yield between loci varied with concentration; (g) reduction of magnesium to 1mM (1.5 mM standard) resulted in minimal amplification, while only partial loss of yield was observed with 1.25 mM magnesium; (h) decreasing the annealing temperature by 2-4 degrees C did not generate artifacts or locus dropout and most laboratories observed full amplification with the annealing temperature increased by 2 degrees C and significant locus dropout with a 4 degrees C increase in annealing temperature; (i) amplification of individual loci with primers used in the multiplex produced the same alleles as observed with the multiplex amplification; (j) all laboratories observed full amplification with >or = 125 pg of male template with partial and/or complete profiles observed using 30-62.5 pg of DNA; (k) analysis of < or = 500 ng of female DNA did not yield amplification products; (l) the minor male component of a male/female mixture was observed with < or =1200-fold excess female DNA with the majority of alleles still observed with 10,000-fold excess female; (m) male/male mixtures produced full profiles from the minor contributor with 10-20-fold excess of the major contributor; (n) average stutter for each locus; (o) precision of sizing were determined; (p) human-specificity studies displayed amplification products only with some primate samples; and (q) reanalysis of 102 non-probative casework samples from 65 cases produced results consistent with original findings and in some instances additional identification of a minor male contributor to a male/female mixture was obtained. In general, the PowerPlex Y System was shown to have the sensitivity, specificity and reliability required for forensic DNA analysis.

Animals↗
PubMed2005

Multiplex mRNA profiling for the identification of body fluids.

We report the development of a multiplex reverse transcription-polymerase chain reaction (RT-PCR) method for the definitive identification of the body fluids that are commonly encountered in forensic casework analysis, namely blood, saliva, semen, and vaginal secretions. Using selected genes that we have identified as being expressed in a tissue-specific manner we have developed a multiplex RT-PCR assay which is composed of eight body fluid-specific genes and that is optimized for the detection of blood, saliva, semen, and vaginal secretions as single or mixed stains. The genes include beta-spectrin (SPTB) and porphobilinogen deaminase (PBGD) for blood, statherin (STATH) and histatin 3 (HTN3) for saliva, protamine 1 (PRM1) and protamine 2 (PRM2) for semen, and human beta-defensin 1 (HBD-1) and mucin 4 (MUC4) for vaginal secretions. The known or presumed functions of these genes suggest an extremely restricted pattern of gene expression, which is a basic requirement for incorporation into a tissue-specific assay. The methodology is based upon gene expression profiling analysis in which the body fluid-specific genes are identified by detecting the presence of appropriate mRNA species using capillary electrophoresis/laser induced fluorescence. An mRNA-based approach, such as the multiplex RT-PCR method described in the present work, allows for the facile identification of the tissue components present in a body fluid stain and could supplant the battery of serological and biochemical tests currently employed in the forensic serology laboratory.

Blood↗
PubMed2005

Multiplex genotyping of human minor histocompatibility antigens.

Minor histocompatibility antigens (mHAg) induce major histocompatibility complex-restricted, T cell-mediated immune responses that may contribute to increased risk of graft-versus-host disease and graft-versus-leukemia effects. Unlike human leukocyte antigen genes, mHAg are encoded by genetically and functionally unrelated genes located throughout the chromosome. The role of mHAg in stem cell transplantation and the population frequencies of mHAg alleles remain unknown due in part to the lack of suitable high throughput methods for genotyping these diverse genes. Here we describe the development and utility of a multiplexed Luminex assay for genotyping human mHAg, including HA-1, HA-2, HA-3, HA-8, HB-1, CD31(125), and CD31(563). The assay uses a multiplexed, allele-independent, gated amplification of mHAg genes followed by differential detection of allele-specific primer extension products using the MultiCode PLx system (EraGen Biosciences, Madison, WI). The alleles are interrogated using a multiplex allele-specific primer extension reaction using primers tagged with EraCodes. The products are hybridized to Luminex beads and the hybridization duplexes are detected using streptavidin-phycoerythrin. The assay resolved the mHAg genotypes of 259 Caucasian donors and provided population estimates of mHAg gene and phenotypic frequencies. All mHAg alleles evaluated in this study exhibited Hardy-Weinberg equilibrium, although some mHAg phenotypes were present in large majority of individuals tested (HA-2, HB-1). This assay will provide a valuable tool for determining mHAg frequencies in other ethnic populations, as well as for establishing the clinical importance of mHAg disparities in stem cell transplantation.

Alleles↗
PubMed2005

Development and evaluation of a Mycobacterium avium subspecies paratuberculosis (MAP) specific multiplex PCR assay.

There are specificity questions inherent in most of the current PCR systems that amplify the MAP IS900 sequence as an indicator for Mycobacterium avium subspecies paratuberculosis (MAP) presence due to false positives associated with IS900-like sequences that exists in other Mycobacterium species besides MAP. We developed a multiplex PCR system designed to enhance specificity for MAP detection in a single PCR reaction. This PCR assay co-amplifies the mycobacterium species 16S rRNA gene, MAP IS900 and f57 sequences. The assay incorporates an internal PCR amplification control (IC) template system enabling PCR amplification conditions to be assessed in each reaction. The assay's specificity was confirmed by testing 10 isolates of MAP and 59 isolates of other bacterial species. In parallel we also applied on the same samples, one of the established nested PCR systems that targets only the IS900 sequence. The multiplex PCR assay was highly specific for MAP, the nested PCR system was also positive with several other mycobacterium species. In this context, we report for the first time false positive IS900 PCR signals in Mycobacterium chelonae, Mycobacterium terrae and Mycobacterium xenopi strains associated with one of the established PCR systems widely used for MAP detection. Finally, the potential application of this multiplex PCR system in milk analysis is demonstrated through analysis of raw milk samples artificially contaminated with MAP.

Animals↗
PubMed2006

Rapid identification of nontypeable and serotype b Haemophilus influenzae from nasopharyngeal secretions by the multiplex PCR.

OBJECTIVE: Heamophilus influenzae (H. influenzae) is an important pathogen responsible for both invasive and non-invasive infectious diseases. While encapsulated type b strain recognized as a major cause of severe invasive diseases, nontypeable strains are the major causes of non-invasive infectious diseases. Detection of this pathogen from nasopharyngeal secretions (NPS) is important. METHODS: We developed a multiplex polymerase chain reaction (PCR) for rapid identification of nontypeable and serotype b H. influenzae from nasopharyngeal secretions. RESULTS: A total 25 nasopharyngeal secretions were evaluated in this study. The multiplex PCR provided rapid and unequivocal results for determining either nontypeable or encapsulated typeable especially type b strains including a determination of beta-lactamase productions. CONCLUSION: The multiplex PCR based serotyping provided more reliable results than slide agglutination test (SAT) and is a valuable and expeditious method for identification of H. influenzae with determining capsular serotypes.

Haemophilus influenzae↗
PubMed2007

Improved multiplex-PCR to identify hepatitis B virus genotypes A-F and subgenotypes B1, B2, C1 and C2.

BACKGROUND: There are eight genotypes (A-H) and numerous subgenotypes of hepatitis B virus (HBV). The genotype has been shown to affect the course of HBV infection. OBJECTIVES: To develop an efficient genotyping and subgenotyping method for large-scale epidemiological surveys of HBV infection in countries with high prevalence of HBV B and C such as China. STUDY DESIGN: We designed genotype and subgenotype-specific primer pairs, and adjusted PCR conditions for a multiplex-PCR using common Taq polymerase to identify HBV genotypes A-F in one reaction and for the main subgenotypes B1/B2 and C1/C2 in another reaction. RESULTS: We have developed a multiplex-PCR system, which specifically amplifies DNA of HBV genotypes and the corresponding main subgenotypes B and C from the sera of HBV patients. Our patients were infected with HBV of subgenotypes B2 (n=18), C1 (n=2) and C2 (n=48). Eleven patients were doubly infected and three showed a triple infection with HBV A, B and C. CONCLUSIONS: The low-cost multiplex-PCR for identification of HBV genotypes A-F and main subgenotypes of HBV B and C, is rapid, reliable and sufficient for large-scale epidemiological surveys and clinical studies.

Base Sequence↗
PubMed2006

Comparison of three multiplex cytokine analysis systems: Luminex, SearchLight and FAST Quant.

Multiplex cytokine analysis technologies have become readily available in the last five years. Two main formats exist: multiplex sandwich ELISA and bead based assays. While these have each been compared to individual ELISAs, there has been no direct comparison between the two formats. We report here the comparison of two multiplex sandwich ELISA procedures (FAST Quant and SearchLight) and a bead based assay (UpState Luminex). All three kits differed from each other for different analytes and there was no clear pattern of one system giving systematically different results than another for any analyte studied. We suggest that each system has merits and several factors including range of analytes available, prospect of development of new analytes, dynamic range of the assay, sensitivity of the assay, cost of equipment, cost of consumables, ease of use and ease of data analysis need to be considered when choosing a system for use. We also suggest that results obtained from different systems cannot be combined.

CD8-Positive T-Lymphocytes↗
PubMed2006

Comparison of a multiplex, bead-based fluorescent assay and immunofluorescence methods for the detection of ANA and ANCA autoantibodies in human serum.

Detection of antinuclear (ANA) and antineutrophil cytoplasmic (ANCA) antibodies is extensively used for establishing a diagnosis in patients with clinical features suggestive of autoimmune disorders. The most common methods for the identification of positive patients' sera for ANA or ANCA are indirect immunofluorescence (IIF) and ELISA-based procedures. Considerable effort has been made in developing simpler automated assays for routine laboratory use. Recently a commercially available microsphere-based fluorescent assay has been introduced for the detection of ANA and ANCA. The aim of this study was to compare this technology with routinely used IIF and ELISA procedures, in patients with a suggested autoimmune disorder. A highly significant correlation between ELISA procedures for specific antibodies and the microsphere-based assays were obtained for both ANA and ANCA as well as for extractable nuclear antigens ELISA screening, indicating that multiplex technology could replace individual ELISA tests for the measurement of specific autoantibodies. However, a low sensitivity for identifying IIF-positive cases was obtained for both ANA (58.0%) and ANCA (59.1%), although there was a significant correlation between the assays. In conclusion, our data show that a microsphere-based fluorescent assay may be a valid platform for the simultaneous determination of circulating individual ANA and ANCA autoantibodies. Furthermore, multiplexing technology offers several advantages that will probably make it an attractive tool in the future. Nevertheless, until further studies are conducted that determine the clinical performance of the multiplex technology, the initial screening of patients for autoantibodies with IIF is still considered necessary.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed2004

Simultaneous detection of enteric viruses by multiplex real-time RT-PCR.

A multiplex real-time RT-PCR protocol for the simultaneous detection of noroviruses ("Norwalk-like viruses") of genogroups I and II, human astroviruses and enteroviruses is described. The protocol was developed and evaluated using the LightCycler and corresponding SYBR Green reagents. New primers were designed within conserved genome regions to optimize the detection range of virus subtypes of each genus. To enable the development of a multiplex PCR assay within one tube (capillary), similar mastermix- and cycling-conditions were respected for each individual primer system. Subsequent melting curve analysis allowed the determination of possible dual-contaminations of entero- and noro- or astroviruses by the formation of dual peaks. Special care was taken to minimize the loss of sensitivity, since the detection of small viral contaminations is a crucial parameter especially for food analysis. The multiplex assay was compared successfully to the single SYBR Green assay, and revealed to be at least 10 times more sensitive than the one obtained with an endpoint PCR thermocycler protocol published previously.

Benzothiazoles↗
PubMed2004

Multiplex RT-nested PCR differentiation of gill-associated virus (Australia) from yellow head virus (Thailand) of Penaeus monodon.

A multiplex RT-nested PCR has been developed to detect and differentiate the closely related prawn viruses, gill-associated virus (GAV) from Australia and yellow head virus (YHV) from Thailand. RT-PCR using primers to conserved sequences in the ORF1b gene amplified a 794 bp region of either GAV or YHV. Nested PCR using a conserved sense primer and either a GAV- or YHV-specific antisense primer to a divergent sequence differentially amplified a 277 bp region of the primary PCR amplicon. Multiplexing the YHV antisense primer with a GAV antisense primer to another divergent sequence allowed the viruses to be distinguished in a single nested PCR. Nested PCR enhanced detection sensitivity between 100- and 1000-fold and GAV or YHV RNA was detectable in approximately 10 fg lymphoid organ total RNA. The multiplex RT-nested PCR was also able to co-detect GAV and YHV RNA mixed over a wide range of concentrations to simulate potential dual-infection states. The robustness of the test was examined using RNA samples from Penaeus monodon prawns infected either chronically or acutely with GAV or YHV and collected at different locations in Eastern Australia and Thailand between 1994 and 1998. GAV- (406 bp) or YHV-specific (277 bp) amplicons were differentially generated in all cases, including five YHV RNA samples in which no primary RT-PCR amplicon was detected. Sequence analysis of GAV and YHV PCR amplicons identified minor variations in the regions targeted by the virus-specific antisense primers. However, none occurred at positions that critically affected the PCR.

Amino Acid Sequence↗
PubMed2004

Multiplex real-time quantitative RT-PCR assay for hepatitis B virus, hepatitis C virus, and human immunodeficiency virus type 1.

A multiplex real-time quantitative reverse transcription (RT)-PCR assay was developed for simultaneous detection, identification and quantification of hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) in plasma or serum samples. Genomic amplification of one virus was unaffected by the simultaneous amplification of the other two. Competition between HCV and HIV-1 amplifications slightly affected the yield of HIV-1 amplification. However, quantitation was possible when a single virus was present. The 95% detection limits were 30, 167 and 680IU/ml for HBV DNA, HCV RNA and HIV-1 RNA, respectively. The multiplex assay detected with similar efficiency strains of HBV genotypes A-F, HCV genotypes 1-6, and HIV-1 subtypes A-G. Applied to 267 pools of 10 plasmas from blood donors, multiplex screening indicated that the assay was reproducible, sensitive, and specific. This assay has the potential to be used for large-scale nucleic acid testing (NAT) of blood donations.

Base Sequence↗
PubMed2004

Multiplexed RT-PCR for streamlined detection and separation of barley and cereal yellow dwarf viruses.

Two novel multiplexed RT-PCR assays that can efficiently detect and distinguish among different barley and cereal yellow dwarf viruses (B/CYDVs) are described. The basic multiplex can produce two fragments simultaneously, a approximately 830-bp fragment indicating the presence of the BYDV-PAV, BYDV-MAV, or BYDV-SGV viruses and a approximately 372-bp fragment indicating the presence of the CYDV-RPV, BYDV-RMV, or BYDV-GPV viruses. The enhanced multiplex produces two additional fragments, which further differentiate between BYDV-PAV, BYDV-MAV, and BYDV-SGV. These assays fulfill the critical need for a streamlined diagnostic procedure for B/CYDVs that can be cost-effectively applied to large numbers of small samples. The assays are useful not only in the basic diagnosis of B/CYDVs, but also for studies examining the ecological roles of B/CYDVs in natural systems and for longer-term epidemiological studies of grasses and cereals.

DNA, Complementary↗
PubMed2005

Development of a ssRNA internal control template reagent for a multiplex RT-PCR to detect turkey astroviruses.

Turkey astrovirus (TAstV), a positive sense single-stranded RNA (ssRNA) virus, is an important causative agent of enteritis of poults. The detection and diagnosis of astroviruses have been mainly dependent on electron microscopy (EM). However, EM is not very sensitive. Reverse transcription-polymerase chain reaction (RT-PCR) has high specificity and sensitivity. Thus, monoplex RT-PCR and multiplex RT-PCR for detection of TAstVs were developed in our laboratory. RT-PCR could be adversely affected by many factors, which would result in lowering the sensitivity of the reaction. To minimize this pitfall of RT-PCR, a ssRNA internal control (IC) template reagent containing two sets of primer sequences (SRV and AFCP) specific to the capsid region of TAstV genomes was developed, and applied to the multiplex RT-PCR. Sixty-four fecal specimens from 2-week-old poults with enteritis were tested using the multiplex RT-PCR with the IC serving as a co-amplification template. An overall test inhibition rate of 12.5% was found for the RT-PCR, which can be used to decrease the false-negative rate. The approach to the generation of the IC developed in this study is simple, convenient and productive, and can be used as a universal protocol to generate a ssRNA IC template reagent for RT-PCR.

Animals↗
PubMed2005

Nested multiplex PCR assay for detection of human enteric viruses in shellfish and sewage.

Environmental samples and contaminated shellfish present frequently low concentrations of more than one viral species. For this reason, a nested multiplex RT-PCR was developed for the detection of adenoviruses, enteroviruses and hepatitis A viruses in different environmental samples such as urban sewage and shellfish. This assay will save time and cost for detection of these enteric viruses with a smaller sample volume, which otherwise can be a limiting factor in routine analysis. The limit of detection was approximately 1 copy for adenovirus and 10 copies for enterovirus and hepatitis A virus per PCR reaction using titrated cell-cultured viruses as template material. In shellfish and environmental samples, this multiplex PCR was optimized to detect all three viruses simultaneously when the concentration of each virus was equal or lower than 1000 copies per PCR reaction. This is the level found predominantly in the environment and in shellfish when the numbers of fecal bacterial and phage indicators are low. The detection of human adenoviruses by PCR has been suggested as a molecular index of fecal contamination of human origin in the environment and food and the multiplex assay developed may be a tool for evaluating the presence of viral contamination in shellfish and water and to expand microbiological control to include viral markers.

Adenoviruses, Human↗
PubMed2005

Rapid detection and differentiation of bovine herpesvirus 1 and 5 glycoprotein C gene in clinical specimens by multiplex-PCR.

A multiplex polymerase chain reaction (multiplex-PCR) to detect and differentiate bovine herpesvirus 1 (BoHV-1) and 5 (BoHV-5) was developed using primers for the gene sequence that encodes the glycoprotein C. The technique was assessed against the BoHV-1 and BoHV-5 cell culture adapted strains, and clinical samples collected from animals with clinical signs of BoHV-1 (n = 10) or BoHV-5 (n = 7) infection and with diagnosis confirmed by virus isolation in cell culture and semi-nested PCR. Fifteen clinical samples from asymptomatic animals were included as control group. For the evaluation of the amplifiability of the extracted nucleic acid from clinical specimens was included a bovine internal control that amplified a 626 bp fragment of the ND5 gene present in the bovine mitochondrial DNA. For DNA extraction, a combination of the phenol/chloroform/isoamyl alcohol and silica/guanidine isothiocyanate methods was used. The specificity of the BoHV-1 and BoHV-5 amplicons from standard strains were confirmed by sequence analysis. All the positive clinical samples for BoHV included in this study were characterized as BoHV-1 or BoHV-5 by the difference in length of the amplified product visualized in a agarose gel (354 bp size for BoHV-1, and 159 bp for BoHV-5). The internal control was amplified in all clinical specimens. Non-specific reactions were not observed when the multiplex-PCR was assessed with other viruses (bovine viral diarrhea virus and rabies virus) and BoHV-negative clinical samples from fetuses and adult cattle obtained from a slaughterhouse.

Animals↗