Detection and quantification of equine herpesvirus-1 viremia and nasal shedding by real-time polymerase chain reaction.

Equine herpesvirus-1 (EHV-1) infection is common in young horses throughout the world, resulting in respiratory disease, epidemic abortion, sporadic myelitis, or latent infections. To improve on conventional diagnostic tests for EHV-1, a real-time polymerase chain reaction (PCR) technique was developed, using primers and probes specific for the EHV-1 gB gene. Amplification efficiencies of 100% +/- 5% were obtained for DNA isolated from a plasmid, infected peripheral blood mononuclear cells (PBMCs), and nasal secretions from infected ponies. The dynamic range of the assay was 8 log10 dilutions, and the lower limit of detection was 6 DNA copies. Fifteen ponies, seronegative for EHV-1, were experimentally infected with EHV-1, and nasal samples were used to quantify shedding of virus by both virus isolation and real-time PCR analysis. Virus isolation identified nasal shedding of EHV-1 in 12/15 ponies on a total of 25 days; real-time PCR detected viral shedding in 15/15 ponies on 75 days. Viremia was quantified using PBMC DNA, subsequent to challenge infection in 3 additional ponies. Viremia was identified in 1/3 ponies on a single day by virus isolation; real-time PCR detected viremia in 3/3 ponies on 17 days. When real-time PCR was used to analyze PBMC DNA from 11 latently infected ponies (documented by nested PCR), EHV-1 was not detected. We conclude that real-time PCR is a sensitive and quantitative test for EHV-1 nasal shedding and viremia and provides a valuable tool for EHV-1 surveillance, diagnosis of clinical disease, and investigation of vaccine efficacy.     

Development of quantitative real-time polymerase chain reaction for duck enteritis virus DNA

Duck enteritis virus (DEV) is a herpesvirus that causes an acute, contagious, and fatal disease. In the present article, we introduce a quantitative real-time polymerase chain reaction (PCR) assay for DEV DNA using TaqMan technology and a two-step protocol. It was confirmed to be rapid, sensitive, and specific for DEV detection. The primers and probe were designed and directed to the DNA polymerase gene of DEV. The method will provide a valuable tool for rapid laboratory diagnosis of DEV infection. By virtue of its high-throughput format and its ability to accurately quantify the viral DNA, the method may be useful for large epidemiological surveys and clarification of pathogenesis, such as latency and reactivation of the virus.     

Detection of chicken anaemia virus DNA from formalin-fixed tissues by polymerase chain reaction

Chicken anaemia virus was detectable from various samples such as cell-free virus, infected cells, unfixed liver homogenates, formalin-fixed liver homogenate or formalin-fixed paraffin-embedded ( ) tissues from experimental or field infected chicks using assay. The detection limit of the first PCR assay was 1 infected cell or 10^−1·5, ₅₀ of cell-free virus (strain A2). The nested assay increased the sensitivity 10- or 100-fold. was detectable in the other 14 Japanese strains isolated from 1976 to 1994 by the assay. All the amplified products were digested with BglII, HindIII, PstI and SacI. These results suggest that the region amplified was highly conserved among the strains. The nested assay was very sensitive. However, was detectable in most field samples using the first assay. Therefore, the nested assay may not always be necessary. In contrast, the nested PCR assay was necessary to detect in tissues or formalin-fixed material. Use of the assay in detection from tissues may be most valuable in diagnosis of diseases caused by or associated with , because it allows detection of both microscopic lesions and .     

Quantification of MDR-1 gene expression in canine tissues by real-time reverse transcription quantitative polymerase chain reaction

MDR-1 gene product mediated multidrug resistance is thought to play a major role in the outcome of chemotherapy in some canine tumors, especially malignant lymphoma. In the present study, MDR-1 RNA expression in normal lymph node and liver tissue as well as in tumor biopsies from 23 dogs with lymphomas and two dogs with liver tumors was measured by real-time RT-quantitative PCR. MDR-1 gene expression was detected in all samples analyzed. Comparably high MDR-1 RNA levels were measured in all normal liver tissues, one of the lymphomas and a cholangiocarcinoma. MDR-1 expression levels in canine lymphomas were found to vary over a wide range with most tumors expressing relative low levels. Interestingly, gastrointestinal lymphomas expressed higher MDR-1 RNA levels than multicentric lymphomas (p = 0.03). In conclusion, real-time RT-quantitative PCR appears to be a suitable method for sensitive and quantitative determination of MDR-1 gene expression in canine normal and neoplastic tissues.     

Relative quantification of canine CD56 mRNA expression by real-time polymerase chain reaction method in normal tissues and activated lymphocytes

Real-time PCR was optimized for the quantification of canine CD56 mRNA expression. This study was conducted to easily quantify canine CD56 expression and to identify its expression in normal tissues, peripheral blood mononuclear cells and activated lymphocytes in dogs. This assay revealed the highest level of CD56 mRNA expression in the normal canine brain, followed by the lung, kidney and liver. CD56 mRNA expression level in peripheral blood mononuclear cells was considerably lower; among activated lymphocytes in vitro, CD56 mRNA expression was increased.     

Detection of Mycobacterium paratuberculosis in formalin-fixed paraffin-embedded tissues by the polymerase chain reaction.

The polymerase chain reaction (PCR) utilizing primers specific for the IS900 sequence of Mycobacterium paratuberculosis was applied to tissue sections of formalin-fixed, paraffin-embedded ileum from cattle with Johne's disease and the results compared to those obtained with acid-fast (Ziehl-Neelsen) and immunohistochemical staining. The PCR was positive in 19/21 tissues (90%) while Ziehl-Neelsen staining was positive in 18/21 (86%) and immunohistochemical staining in 21/21 (100%). The Ziehl-Neelsen and immunohistochemical stains are not, however, specific for M. paratuberculosis. The PCR for detection of M. paratuberculosis using the IS900 sequence is a specific and relatively sensitive method for confirmation of Johne's disease and its application to formalin-fixed, paraffin-embedded tissues may be useful for confirmation of dubious cases, for retrospective studies and for epidemiological analyses.     

实时荧光定量PCR对犬乳腺肿瘤组织TNFR1基因的定量检测

乳腺肿瘤是母犬中最为常见的肿瘤,运用实时荧光定量PCR检测了37例犬乳腺肿瘤病例和37份正常乳腺组织.结果表明:犬乳腺肿瘤组织中肿瘤坏死因子受体(TNFR1)的表达量显著低于正常对照组织(P<0.05).该试验结果说明,犬乳腺肿瘤的发生很可能与TNFR1表达降低有关,为深人研究肿瘤形成的分子机理提供了重要参考.     

Detection of Salmonella spp in fecal specimens by use of real-time polymerase chain reaction assay

OBJECTIVE: To use real-time polymerase chain reaction (PCR) technology to develop a highly sensitive and specific diagnostic assay for the detection of Salmonella spp in fecal specimens. SAMPLE POPULATION: 299 fecal specimens from cattle, horses, and dogs. PROCEDURE: Enrichment of fecal specimens was followed by genomic DNA extraction by use of commercially available isolation kits. Real-time PCR assay was performed to target a Salmonella spp-specific DNA segment. Results of real-time PCR assay were compared with bacterial culture results to determine relative sensitivity and specificity. RESULTS: Use of the spaQ primer-probe set resulted in a relative sensitivity of 100% and a specificity of 98.2%, compared with bacterial culture results when tested on 299 clinical fecal specimens. CONCLUSIONS AND CLINICAL RELEVANCE: A rapid, sensitive, and specific assay for the detection of Salmonella spp from enriched clinical fecal specimens was developed. This technique would be highly valuable in clinical settings to help avoid or mitigate the complications arising from an outbreak of salmonellosis in a herd or among patients of a veterinary hospital.     

Detection of Chlamydia psittaci DNA in avian clinical samples by polymerase chain reaction

A polymerase chain reaction (PCR) assay was developed to detect Chlamydia psittaci DNA in faeces and tissue samples from avian species. Primers were designed to amplify a 264 bp product derived from part of the 5′ non-translated region and part of the coding region of the ompA gene which encodes the major outer membrane protein. Amplified sequences were confirmed by Southern hybridization using an internal probe. The sensitivity of the combined assay was found to be between 60 to 600 fg of chlamydial DNA (approximately 6 to 60 genome copies). The specificity of the assay was confirmed since PCR product was not obtained from samples containing several serotypes of C. trachomatis, strains of C. pneumoniae, the type strain of C. pecorum, nor from samples containing microorganisms commonly found in the avian gut flora. In this study, 404 avian faeces and 141 avian tissue samples received by the Central Veterinary Laboratory over a 6 month period were analysed by PCR, antigen detection ELISA and where possible, cell culture isolation. PCR performed favourably compared with ELISA and cell culture, or with ELISA alone. The PCR assay was especially suited to the detection of C. psittaci DNA in avian faeces samples. The test was also useful when applied to tissue samples from small contact birds associated with a case of human psittacosis where ELISA results were negative and chlamydial isolation was a less favourable method due to the need for rapid diagnosis.     

Detection of avian polyomavirus infection by polymerase chain reaction using formalin-fixed, paraffin-embedded tissues

Avian polyomavirus infection in psittacines was diagnosed in tissues by the use of polymerase chain reaction (PCR) test. The tissues used in the procedure were either formalin-fixed tissues embedded in paraffin blocks or fresh tissues (heart, liver, and spleen) collected from the psittacines during necropsy. DNA was extracted from these tissues and was tested with the published primers for avian polyomavirus VP1 gene in the PCR that yielded an amplicon of 550 base pair size, which was then visualized by electrophoresis. The amplicon size was consistent with avian polyomavirus. The PCR test was found to be an effective method for identifying avian polyomavirus infection in both formalin-fixed, paraffin-embedded and fresh tissues from psittacine birds of different age groups.     

Detection of infectious laryngotracheitis virus in formalin-fixed, paraffin-embedded tissues by nested polymerase chain reaction

Infectious laryngotracheitis virus (ILTV) is routinely diagnosed by histopathologic examination of trachea, eyelid, and lung tissues. Lesions consistent with infectious laryngotracheitis (ILT) infection include syncytial cell formation with intranuclear inclusion bodies. These changes are present during the acute phase of infection. To increase the sensitivity of detecting ILT, a nested polymerase chain reaction (PCR) was developed for detection of ILTV DNA. Nested PCR assay was specific for the amplification of ILTV DNA and did not amplify a variety of other avian pathogens. To further validate the ability of this assay to detect ILT, nested PCR was performed in formalin-fixed, paraffin-embedded tissues from 35 cases of respiratory disease. Of the 35 cases, 12 were considered ILT suspects on the basis of initial clinical observation. Eleven of the 12 ILT-suspect cases were diagnosed as ILT, and the remaining 24 were diagnosed as nonspecific tracheitis (NST) by histopathologic examination. Histopathologically positive samples were confirmed by direct fluorescent antibody test and virus isolation. Of the 11 ILT-positive cases, 10 were positive by nested PCR. In addition, ILTV DNA was detected in 7 of the 24 samples diagnosed as NST upon histopathologic examination. Therefore, by nested PCR, ILTV DNA was detected in tissues independently of the presence of syncytial cells, intranuclear inclusions, or both. ILT nested PCR is a specific and sensitive assay capable of detecting ILT at different stages of infection and can be utilized in combination with histopathological examination to accelerate the diagnosis of ILT infection.     

Detection of feline immunodeficiency proviral DNA in peripheral blood lymphocytes by the polymerase chain reaction.

Feline immunodeficiency virus (FIV) proviral DNA was detected by the polymerase chain reaction method (PCR). PCR products were detected by gel electrophoresis and ethidium bromide staining. The P-10, P-15 and P-24 regions of the gag gene of FIV were chosen as the target sequences for amplification, and three primer pairs were prepared. The PCR products subjected to amplification with each primer pair were found to possess sites of digestion by a restriction enzyme, as hypothesized. They did not react with feline leukemia virus (FeLV)-infected or feline syncytium-forming virus (FeSFV)-infected cell-derived DNA, and specifically amplified FIV-infected cell-derived DNA. FIV proviral DNA was detected by the PCR method with either primer pair (one-step amplification: single PCR) in DNA derived from peripheral blood lymphocytes (PBL) from 7 of 12 FIV antibody-positive cats. When PCR products in each of the 12 cats were subjected to a second amplification using the same primer pair (two-step amplification: double PCR), FIV proviral DNA was detected in all of the cats. When PBL samples collected from three cats that were negative and three that were positive in the single PCR were cultured for a few weeks in the presence of interleukin 2, FIV proviral DNA was detected in all six cats by the single PCR method. The results suggest that either the use of cultured PBL as the sample or the performance of the double PCR method enables simple and specific detection of FIV proviral DNA in PBL.     

Detection of pigeon circovirus by polymerase chain reaction

Pigeon circovirus was identified by polymerase chain reaction (PCR) in young pigeons belonging to 12 different lofts. Viral DNA was extracted from formalin-fed, paraffin-imbedded tissues containing primarily bursa and occasionally liver and spleen with a commercial kit. PCR primers were selected from a published sequence for columbid circovirus and evaluated in a PCR assay. The histopathologic examination of various tissues revealed basophilic globular intracytoplasmic inclusions in the mononuclear cells of the bursa of Fabricius and occasionally in the spleen characteristic for a circovirus. Transmission electron microscopy of a few bursas of Fabricius revealed virus particles measuring 18-21 nm. All the samples were negative by PCR for psittacine beak and feather disease (PBFD) virus and chicken infectious anemia virus. The primers for both pigeon circovirus and PBFD virus did not react in PCR with the chicken anemia virus DNA. Most of the circovirus-infected pigeons had concurrent infections of Escherichia coli, Salmonella, Pasteurella, Aspergillus, candidiasis, nematodiasis, or capillariasis.     

Detection of avian adenovirus by polymerase chain reaction

An avian adenovirus-specific polymerase chain reaction was developed. The origin of primers was from the DNA sequence data of the chicken embryo lethal orphan avian adenovirus virus genome. An avian adenovirus-specific 421-bp DNA product was amplified by these primers from group I of adenovirus containing 12 serotypes and serotypes of adenovirus from group II and group III. The adenovirus-specific DNA product was also amplified from the 19 field isolates of avian adenoviruses but not from the mammalian adenovirus and other avian pathogenic viruses and bacteria. As little as 1 fg of avian adenovirus DNA was detected by gel electrophoresis and Southern blot analysis.     

Duplex real-time polymerase chain reaction for simultaneous detection and quantification of Anaplasma marginale and Anaplasma centrale

Anaplasma marginale and Anaplasma centrale are rickettsial pathogens responsible for acute disease and mild infections, respectively, in cattle herds. A duplex real-time polymerase chain reaction (PCR) assay with probes labeled with different fluorophores was developed for simultaneous detection and quantification of A. marginale and A. centrale DNA in bovine blood samples. The assay was able to detect as few as 10(1) and 10(2) DNA copies for A. marginale and A. centrale, respectively, with optimal specificity and reproducibility. Analysis by real-time and nested PCR carried out on 54 samples previously tested by reverse line blot hybridization showed that the established duplex real-time PCR assay can detect and quantify the 2 Anaplasma spp., even if present simultaneously in the same blood samples. Such an assay could be used in pathogenesis studies on bovine acute anaplasmosis.