The detection of proviral DNA by semi‐nested polymerase chain reaction and phylogenetic analysis of czech Maedi‐Visna Isolates Based on gag Gene Sequences

A semi‐nested polymerase chain reaction (snPCR) for detecting proviral DNA of ovine lentivirus (OvLV) in peripheral blood mononuclear cells was developed. Primers for snPCR were situated within the gag gene of the Maedi–Visna virus (MVV) genome. A comparison between the snPCR and serological tests (agar gel immunodiffusion test, immunoblot) were performed using 98 ovine blood samples. Thirty (30.6 %) of the 98 sheep examined had antibodies specific for the MVV. PCR showed 21 of them to be positive and nine seropositive animals to be PCR negative. Six of the 68 serologically negative sheep were found to be PCR positive, probably due to delayed seroconversion. The PCR amplification products of these six sheep were sequenced and subjected to phylogenetic analysis. The resulting phylogenetic tree of partial gag gene sequences confirmed that the ovine lentivirus genotype in the Czech Republic is more closely related to the prototype MVV isolates than to the caprine arthritis encephalitis viruses.     

Bovine leukaemia proviral DNA detection in cattle using the polymerase chain reaction

Bovine leukaemia virus (BLV) is the causative agent in enzootic bovine leukosis a disease occurring worldwide. This virus is normally detected by the agar gel immunodiffusion or ELISA assays which rely on the appearance of antibodies to a major surface protein of the virus, gp51, present in the serum of infected cattle. We have used the polymerase chain reaction, which depends on the amplification of specific DNA sequences as a sensitive assay for the detection of BLV. It was possible to detect proviral DNA in 100 pg of tumour DNA from an infected host using agarose gel electrophoresis followed by ethidium bromide staining. The sensitivity of the assay was increased by two log orders when hybridization analysis, using a BLV proviral DNA probe, was used in combination with amplification of the DNA. Proviral DNA was detected in both lymphocytic and tumour DNA and at all stages of infection in cattle.     

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.     

应用PCR检测布鲁氏菌病的可行性试验

在实验室条件下对布鲁氏菌的检测通常由细菌学和免疫学试验作出诊断。传统的细菌学诊断技术虽然可靠 ,但是很费时而且处理培养物必须相当谨慎且极易传染给人。其他试验如补体结合试验 ( CF)和 ELISA虽然已经开始普及 ,但其特异性和敏感性不高。聚合酶链式反应 ( PCR)不仅方便、高效 ,而且特异性很强 ,甚至可以区分感染动物和免疫接种动物。我单位在配备了 PCR试验相关仪器设备后 ,进行了 PCR检测布鲁氏菌可行性试验。1　材料与方法1 .1　引物设计　首先从 NCBI基因数据库网站查找 Brucella abortus 1 6S ribosomal基因序列 ,约1 43…     

Real-time polymerase chain reaction based on msa2c gene for detection of Babesia bovis

This paper reports a quantitative real-time polymerase chain reaction (q-PCR) based on the msa2c gene and standardized with Platinum SYBR Green/ROX for the detection of Babesia bovis in cattle. The msa2c q-PCR amplified a DNA fragment with average dissociation temperature of 77.41°C (± 0.25°C). No amplification was detected when DNA from B. bigemina, A. marginale or Bos taurus was used as the template. The detection limit of the msa2c q-PCR was 1000 copies per ml of blood sample, with a linear correlation between the number of msa2c copies and threshold cycle. The comparison between msa2c q-PCR and conventional PCR for cytochrome b revealed 88.8% agreement, with a Kappa index of 0.75. In the comparison between msa2c q-PCR and an enzyme-linked immunosorbent assay (ELISA) with semi-purified B. bovis antigen, agreement was 96.3% and the Kappa index was 0.91. The agreement between three tests was 85.8%. The msa2c q-PCR detected a higher number of positive cattle than conventional PCR in an enzootically stable area, but did not differ significantly from ELISA. No significant differences were detected between the three diagnostic tests with cattle from an enzootically unstable area. All animals raised on a tick-free facility were negative for B. bovis in the three tests. These results suggest that msa2c q-PCR is a useful test for the detection of B. bovis infection.     

Equine herpesvirus type 1: detection of viral DNA sequences in aborted fetuses with the polymerase chain reaction

Primers and probes were selected from the gene encoding glycoprotein 13 (gp 13) of equine herpesvirus 1 (EHV-1). The polymerase chain reaction (PCR) was run on infected and noninfected cultured cells and on 63 specimens from 29 aborted equine fetuses. The results were evaluated by electrophoresis and dot-blot hybridization using an oligonucleotide probe labeled with biotin. In the infected samples electrophoresis showed a PCR product of about 280 base pairs. The dot-blot hybridization confirmed that this product contained EHV-1 DNA sequences. PCR took 4 h and hybridization another 14 h; the results were thus achieved within 24 h and were highly specific for EHV-1. Close concordance was found between the results of PCR and virus isolation.     

Rapid detection of pseudorabies virus genomic sequences in biological samples from infected pigs using polymerase chain reaction DNA amplification

The presence of the pseudorabies virus (PRV) genome in infected hosts has previously been studied by standard hybridization techniques, which showed the viral genome to be present at very low levels in infected tissues. The recently introduced polymerase chain reaction (PCR) procedure provides an alternative and rapid means of amplifying small quantities of specific DNA sequences. We applied this technique to a study of pigs infected by PRV. The sequence selected for amplification consisted of 222 base pairs lying in the gene coding for the glycoprotein gp50. We used a pair of 20-mer oligonucleotides flanking this sequence as primer and a cloned Stu-Nde fragment containing the sequence as target DNA. To avoid the tedious DNA extraction procedure we performed PCR directly on disrupted cells and detected specific amplification after 25 cycles of PCR with the thermostable Taq DNA polymerase. Amplified products were detected by gel electrophoresis directly. Nasal samples from experimentally and naturally infected pigs were tested by this PCR technique. When compared with tissue culture and serological tests, detection by gel electrophoresis of PCR amplified fragments provided excellent specificity and sensitivity. We concluded that PCR amplification will be a valuable tool for rapid diagnosis of PRV infection in pigs, taking less than 1 h to complete.     

Direct and sensitive detection of Trypanosoma evansi by polymerase chain reaction.

The mechanically transmitted haemoflagellate, Trypanosoma evansi causes 'surra', a wasting disease of domestic animals and is highly endemic in distribution in Southeast Asia. The detection of T. evansi is important for improving the epizootiological and animal health status of the region. The specificity and sensitivity of polymerase chain reaction (PCR) using oligonucleotide primers constructed from T. evansi repetitive DNA sequences were studied in the present investigation. Using the assay, it was possible to amplify template DNA of T. evansi derived from buffaloes, camels and horses to a threshold sensitivity level of 0.5 pg and to detect DNA from as few as five organisms in 10 microliters crude blood samples. Following experimental infection of calves with 5 x 10(5) T. evansi, positive signals could be observed as early as 12 h post-infection. DNAs from two common haemoflagellates of cattle, Babesia bigemina and Theileria annulata were not amplified with the primers.     

A msp1alpha polymerase chain reaction assay for specific detection and differentiation of Anaplasma marginale isolates

Anaplasma marginale is the causative agent of bovine anaplasmosis, a disease which can be protected by vaccination with the less pathogenic Anaplasma species, A. centrale. Currently, there is no polymerase chain reaction (PCR) assay available which differentiates between different species of Anaplasma or which can differentiate isolates of A. marginale within outbreaks and between different countries. A molecular test specific for A. marginale would be ideal for the identification of Anaplasma species in wild ruminants, as possible reservoirs of anaplasmosis, and to differentiate between A. marginale from A. centrale. A PCR assay was designed to amplify the major surface protein 1alpha gene of the rickettsial bovine pathogen, A. marginale both as an inter- and intra-specific test. The test did not amplify A. centrale or A. ovis, and discriminated A. marginale by amplifying repeat regions within the msp1alpha gene which vary in number between many isolates. The nested A. marginale amplicons varied in size from 630 to 1190bp representing one to eight internal repeats. All 22 Australian isolates tested amplified a 630bp product (one repeat) in contrast to all 19 non-Australian isolates tested. Eight sequences from Australian isolates from different geographical regions confirmed the conserved nature of the Australian A. marginale msp1alpha genes. The Australian 'repeat unit' MSP1a deduced amino acid sequence has been designated as Australian type 1. The msp1alpha PCR method developed here enabled the amplification and comparison of A. marginale isolates originating from North and South America, Africa, Israel and Australia. The method is sensitive and specific for A. marginale. Although additional msp1alpha products were amplified from at least two Australian isolates, the results suggest limited introduction of A. marginale into Australia.     

金黄地鼠Dpl基因克隆及其组织特异性表达的研究

本文成功克隆了金黄地鼠Dpl(PrP-like protein doppel,Doppel)基因,并采用实时荧光定量PCR法对其组织特异性表达进行了研究.根据已发表家鼠Dpl基因序列设计引物,采用PCR直接扩增金黄地鼠(Ham.ster)的Dpl基因CDs区,序列测定和分析表明金黄地鼠的Dpl基因CDs区全长537 bp,编码178个氨基酸的前体蛋白,不存在基因多态性.经基因序列对比分析,金黄地鼠与其他10种属哺乳动物Dpl基因有较高同源性,均为70.8%以上,且与鼠科Dpl序列的同源性最高(86%).实时定量PCR结果表明,在所检测组织中,Dpl m.RNA在睾丸表达量最高,其次为脾脏、心脏、骨髓和脑,其在肝脏、肺脏、肾脏和肌肉中的表达低于检测水平;成年动物与未成年动物相比,在睾丸组织的表达量,成年鼠显著高于未成年鼠;在成年鼠脑组织检测不到Dpl m.RNA 的表达.本研究对金黄地鼠Dpl基因序列进行分析测定,并对其在不同组织的生理表达进行了定量,以期为其功能的进一步研究提供基础数据.     

Effects of temperature and time in transit on polymerase chain reaction detection of feline herpesvirus DNA.

The purpose of this study was to report methods currently recommended by commercial laboratories for collecting, shipping, and processing of samples for feline herpesvirus type 1 (FHV-1) testing using polymerase chain reaction (PCR) and to determine the effect of temperature and time on the ability of 1 PCR method to detect FHV-1 DNA in experimental and clinical samples. Eleven laboratories offering FHV-1 PCR were surveyed. There was notable variation in sample types and shipping conditions recommended and PCR protocols used by these laboratories. Subsequently, using a single PCR method, FHV-1 DNA was detected in samples exposed to various temperatures within the laboratory. Finally, FHV-1 PCR was performed on paired clinical samples collected from 25 cats and shipped at ambient temperatures via US Postal Service (USPS) or with an ice pack via a courier. Samples sent by USPS were exposed to significantly longer transit time and arrived at significantly higher temperature than did samples sent by courier. Despite this, all sample pairs yielded concordant results when tested for FHV-1 DNA using this PCR method. Although it may not be necessary for samples collected for detection of FHV-1 DNA using this PCR method to be shipped under the most expedient or temperature-controlled conditions, this should be verified for a variety of PCR assays and sample types.     

Genotypic characterization by polymerase chain reaction of Staphylococcus aureus isolates associated with bovine mastitis

Staphylococcus aureus is recognized worldwide as a pathogen causing many serious diseases in humans and animals, and is the most common aetiological agent of clinical and subclinical bovine mastitis. The importance of evaluating the combination of S. aureus virulence factors has been emphasized both in human and veterinary medicine, and knowledge about the genetic variability within different S. aureus populations would help in the design of efficient treatments. The aim of the present study was to determine the genetic profiles of S. aureus strains isolated from milk of cows suffering from clinical and subclinical mastitis in Belgium. The presence of about forty virulence-associated genes was investigated by specific polymerase chain reaction (PCR) amplification. A high number of genotypic subtypes were observed, demonstrating further the large variation in the presence of virulence genes in S. aureus isolates and the considerable diversity of strains populations that are able to cause mastitis in cows. In accordance with other studies, we showed that some genes are associated with mastitis-causing S. aureus isolates, whereas others are absent or rarely present. We also further highlighted the presence of conserved gene combinations, namely the enterotoxigenic egc-cluster and the bovine pathogenicity island SaPIbov. Importantly, the presence of isolates carrying genes coding for toxins involved in important human infections makes the milk of cows with mastitis a potential reservoir for these toxins, and therefore a potential danger in human health, which strengthens the importance to consider raw milk consumption and its processing very carefully.     

RT-PCR检测猪传染性胃肠炎病毒方法的建立

猪传染性胃肠炎 ( TGE)是一种以严重腹泻、呕吐和脱水为临床特征的高度接触性传染病 ,属于国际兽疫局 ( OIE)法典中 B类疫病必检的猪传染病。TGEV是一种有囊膜的正链 RNA冠状病毒。目前检测 TGEV的方法主要有中和试验、免疫荧光抗体试验、酶联免疫吸附试验和电镜技术 ,这些方法对于检测 TGEV和防制 TGE起到了重要作用 ,但也存在特异性不强、灵敏性差、耗时长等缺点。c DNA探针、RT- PCR近年来已成为国际上用于检测病原微生物的主要方法。由于 c DNA探针与临床样品中非相关核酸有一定的非特异性结合 ,尤其是检测粪便、尿液等…     

Evaluation of a quantitative enzyme-linked immunosorbent assay for feline leukemia virus p27 antigen and comparison to proviral DNA loads by real-time polymerase chain reaction

Feline leukemia virus (FeLV) is an oncogenic retrovirus of cats. While higher viral RNA and proviral DNA loads have been correlated with progressive infections and disease, a similar correlation has been suggested for p27 antigen concentrations. This analytical study compared the results of a quantitative ELISA for p27 antigen with quantitative real-time PCR results for FeLV proviral DNA in patient samples. A significant positive correlation between copies of proviral DNA and the concentration of p27 antigen was identified (r = 0.761, P < 0.0001). Samples with high proviral DNA loads, at least 1 × 10⁶ copies/mL of whole blood, typically had p27 antigen concentrations greater than 30 ng/mL in plasma. Samples with proviral DNA loads below this level all had concentrations of p27 antigen in plasma that were less than 10 ng/mL. Given this correlation, it is hypothesized that the concentration of p27 antigen at a given point in time may help to indicate the likelihood of a progressive or regressive infection similar to what has been demonstrated for proviral DNA loads.     

The frequent detection of a treponeme in bovine digital dermatitis by immunocytochemistry and polymerase chain reaction

A study was carried out to determine whether spirochaetes are frequently associated with digital dermatitis in United Kingdom (UK) dairy cattle. Histopathological examination of lesions using a silver stain showed a large number of unidentified spirochaete-like organisms present in digital dermatitis hoof skin tissue in all examined biopsies. Immunocytochemical staining demonstrated that spirochaetes in skin lesions were identified by polyclonal antisera to Borrelia burgdorferi, Treponema denticola and Treponema vincentii (again all biopsies were positively stained), whereas monoclonal antibodies to B. burgdorferi and any Treponema pallidum did not stain any organisms in all biopsies. A PCR of 16S rRNA, previously shown to be specific for a new treponeme, was employed and produced positive results from 82.4% of digital dermatitis tissues. It is concluded that this spirochaete (or related spirochaetes), which is similar to human oral treponemes, is frequently associated with, and may be responsible for, pathological changes in digital dermatitis.     

The polymerase chain reaction (PCR) for the detection of DNA of equine herpesviruses 1 and 4]

Formalin-fixed and Paraplast-embedded tissue samples of 42 aborted equine fetuses were examined by polymerase chain reaction for the presence of equine herpesvirus DNA. The used set of primers was located in the glycoprotein 13 open reading frame and allowed the amplification of both EHV 1 und EHV 4. By cleaving pattern analysis after Hinf I digestion EHV 1 could be distinguished from EHV 4. In 9 of the cases investigated EHV 1-DNA was detected. This finding is in absolute context with the results of the virological investigations.     

Detection and quantification of Renibacterium salmoninarum DNA in salmonid tissues by real-time quantitative polymerase chain reaction analysis.

Renibacterium salmoninarum is an important salmonid pathogen that is difficult to culture. We developed and assessed a real-time, quantitative, polymerase chain reaction (qPCR) assay for the detection and enumeration of R. salmoninarum. The qPCR is based on TaqMan technology and amplifies a 69-base pair (bp) region of the gene encoding the major soluble antigen (MSA) of R. salmoninarum. The qPCR assay consistently detected as few as 5 R. salmoninarum cells per reaction in kidney tissue. The specificity of the qPCR was confirmed by testing the DNA extracts from a panel of microorganisms that were either common fish pathogens or reported to cause false-positive reactions in the enzyme-linked immunosorbent assay (ELISA). Kidney samples from 38 juvenile Chinook salmon (Oncorhynchus tshawytscha) in a naturally infected population were examined by real-time qPCR, a nested PCR, and ELISA, and prevalences of R. salmoninarum detected were 71, 66, and 71%, respectively. The qPCR should be a valuable tool for evaluating the R. salmoninarum infection status of salmonids.