Detection of bovine viral diarrhea virus, using degenerate oligonucleotide primers and the polymerase chain reaction.

A technique for detection of bovine viral diarrhea virus (BVDV) from circulating blood leukocytes, using the polymerase chain reaction, is described. The published nucleotide sequences of 2 strains of BVDV and that of hog cholera virus were aligned and the information was used to design oligonucleotides coding for 2 regions of amino acid homology. The oligonucleotides were a mixed population including all possible codons for the conserved amino acids. These degenerate oligonucleotides were used in the polymerase chain reaction to detect viral RNA in cells infected in vitro, or in circulating blood leukocytes from infected animals. Virus was detected in over 60 samples from diverse isolates. The detection of BVDV by the polymerase chain reaction is a rapid, sensitive, and specific technique, which represents an improvement over existing technology.     

Detection of Babesia equi (Laveran, 1901) by nested polymerase chain reaction

We describe a nested polymerase chain reaction (PCR) for the detection of Babesia equi in equine infected erythrocytes using oligonucleotides designed on the published sequence of a B. equi merozoite antigen gene (ema-1). A 102bp DNA fragment is specifically amplified from B. equi but not from Babesia caballi, Babesia bovis or Babesia bigemina DNA. In a mock infection we were able to detect down to six infected cells in 10(8) equine erythrocytes or to detect the parasite in blood with an equivalent parasitemia of 0.000006%. Furthermore, gene polymorphism was found by performing a PCR-RFLP (PCR combined with restriction fragment length polymorphism) on both the 102bp and the entire ema-1 gene DNA amplified from two B. equi isolates, Florida (USA) and Pelotas (Southern Brazil) isolates. The polymorphism was confirmed by sequencing the entire ema-1 gene from the B. equi isolate Pelotas. Our results demonstrate that the ema-1 based nested PCR is a valuable technique for routine detection of B. equi in chronically infected horses. It may be used for epidemiological and phylogenetic studies of the parasite as well as monitoring B. equi infected horses in chemotherapeutic trials.     

Evaluation of a real-time quantitative polymerase chain reaction assay for detection and quantitation of virulent Rhodococcus equi

OBJECTIVE: To evaluate a real-time quantitative polymerase chain reaction (QPCR) assay in the detection and quantitation of virulent Rhodococcus equi. SAMPLE POPULATION: 1 virulent, 2 intermediately virulent, and 2 avirulent strains of R. equi and 16 isolates of bacteria genetically related to R. equi. PROCEDURE: The QPCR assay was evaluated for detection and quantitation of the virulence-associated gene (vapA) of R. equi in pure culture and in samples of tracheobronchial fluid, which were inoculated with known numbers of virulent R. equi. Results were compared with those derived via quantitative microbial culture and standard polymerase chain reaction methods. RESULTS: The QPCR assay detected the vapA gene in pure culture of R. equi and in tracheobronchial fluid samples that contained as few as 20 CFUs of virulent R. equi/mL and accurately quantitated virulent R. equi to 10(3) CFUs/mL of fluid. The assay was highly specific for detection of the vapA gene of virulent R. equi and was more sensitive than standard polymerase chain reaction for detection of R. equi in tracheobronchial fluid. CONCLUSIONS AND CLINICAL RELEVANCE: The QPCR assay appears to be a rapid and reliable method for detecting and quantitating virulent R. equi. The accuracy of the QPCR assay is comparable to that of quantitative microbial culture. The increased sensitivity of the QPCR method in detection of virulent R. equi should facilitate rapid and accurate diagnosis of R. equi pneumonia in foals.     

Clinical application of a polymerase chain reaction assay in the diagnosis of pneumonia caused by Rhodococcus equi in a horse

Diagnosis of pneumonia caused by Rhodococcus equi can be made more rapidly by use of a polymerase chain reaction (PCR) assay than by use of conventional bacteriologic culture techniques. Use of a PCR assay aids in the differentiation between virulent and avirulent strains of R equi, and the assay may be used to identify R equi in feces and soil of breeding farms.     

Evaluation of a multiplex polymerase chain reaction assay for simultaneous detection of Rhodococcus equi and the vapA gene

OBJECTIVE: To evaluate sensitivity and specificity of a multiplex polymerase chain reaction (PCR) assay for simultaneous detection of Rhodococcus equi and differentiation of strains that contain the virulence-associated gene (vapA) from strains that do not. SAMPLE POPULATION: 187 isolates of R equi from equine and nonequine tissue and environmental specimens and 27 isolates of bacterial species genetically or morphologically similar to R equi. PROCEDURE: The multiplex PCR assay included 3 gene targets: a universal 311-bp bacterial 16S ribosomal RNA amplicon (positive internal control), a 959-bp R equi-specific target in the cholesterol oxidase gene (choE), and a 564-bp amplicon of the vapA gene. Duplicate multiplex PCR assays for these targets and confirmatory singleplex PCR assays for vapA and choE were performed for each R equi isolate. An additional PCR assay was used to examine isolates for the vapB gene. RESULTS: Results of duplicate multiplex and singleplex PCR assays were correlated in all instances, revealing high specificity and reliability (reproducibility) of the vapA multiplex assay. Of the pulmonary isolates from horses with suspected R equi pneumonia, 97.4% (76/78) yielded positive results for vapA. Seven of 50 (14%) human isolates of R equi yielded positive results for vapA. Six human R equi isolates and 1 porcine isolate yielded positive results for vapB. No isolates with vapA and vapB genes were detected. CONCLUSIONS AND CLINICAL RELEVANCE: The multiplex PCR assay is a sensitive and specific method for simultaneous confirmation of species identity and detection of the vapA gene. The assay appeared to be a useful tool for microbiologic and epidemiologic diagnosis and research.     

Multiplex polymerase chain reaction for identification and differentiation of Streptococcus equi subsp. zooepidemicus and Streptococcus equi subsp. equi

The closely related streptococcal species Streptococcus equi subsp. zooepidemicus and S. equi subsp. equi were identified by polymerase chain reaction using oligonucleotide primers designed according to species-specific parts of the superoxide dismutase A encoding gene sodA. A further differentiation of both subspecies could be performed by amplification of the genes seeH and seeI encoding the exotoxins SeeH and SeeI, respectively, which could be detected for S. equi subsp. equi but not for S. equi subsp. zooepidemicus. A further simplification of the identification and differentiation of both subspecies was conducted by sodA-seeI multiplex polymerase chain reaction.     

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.     

Detection of rhodococcus equi by microbiological culture and by polymerase chain reaction in samples of tracheobronchial secretions of foals

The goal of the present study was to investigate whether new PCR-methods would improve diagnostic of R. equi. In a first step, sensitivity and specificity of the PCR-methods in respect to the"gold standard" microbiological culture were determined. Secondly, sensitivity and specificity of both microbiological methods were evaluated in respect to the clinical diagnosis. The tracheobronchial secretions of 48 foals with pulmonary abscesses and of 37 healthy foals were evaluated by bacteriological culture as well as by four PCR-methods: aceA-, ideR-, vapA- and VP-PCR. In respect to the"gold standard" microbiological culture, the sensitivity of most PCR methods lay between 63.9 and 69.4 % except the vapA-PCR (27.8 %).The specificity of all PCR methods in this comparison was between 98 to 100 %. In this analysis, clinical diagnosis had a low sensitivity (66.7 %) and a low specificity (51.0 %). In respect to the clinical diagnosis, microbiological culture sensitivity was 50.0 % and specificity 67.7 9%. In this analysis, sensitivity rates of aceA-, ideR and VP-PCR methods lay between 33.3 and 37.5 %, sensitivity of the vapA-PCR was lower (10.4 %).The specificity of all PCR methods ranged from 78.4 to 86.5 %. In conclusion, these results show that the diagnostic potential of the microbiological methods"Culture" and "PCR" is different and that for the diagnosis of R. equi-pneumonia in foals the combination of microbiological culture with PCR should be used for examination of samples of the airways of foals.     

Detection of phocine distemper virus using the polymerase chain reaction

During the fatal seal epizootics in the North and Baltic Seas in summer 1988 a virus was isolated which was shown to be the causal agent. It was subsequently classified as morbillivirus by neutralization assays, reaction with monoclonal antibodies and nucleic acid hybridization studies. The virus (tentatively called Phocine Distemper Virus, PDV) is difficult to grow in culture making rapid diagnosis difficult. We have used the Polymerase Chain Reaction (PCR) as an alternative and fast method to detect the presence of virus-specific nucleic acid and we describe here the amplification of cell culture derived PDV RNA in a "one-tube" reaction using heterologous (Rinderpest Virus cDNA derived) F gene primers. The resulting 370 bp DNA fragment was shown to be morbillivirus derived by Southern blot hybridization using cloned RPV F gene as probe.     

副溶血性弧菌PCR检测方法的建立

为了建立适于基层单位应用的针对副溶血性弧菌检测的特异PCR方法,检测副溶血性弧菌的基因序列.针对该菌的属特异性基因t1基因进行引物设计,扩增片段大小为449 bp.运用该PCR法可特异性的从副溶血性弧菌ATCC17802标准株中扩增出目的基因片段,与GenBank上发表的序列同源性为100%.而经常污染海产品的溶藻弧菌,嗜水气单胞茵标准株J-1,铜绿假单胞茵标准株ATCC27853结果均为阴性.该PCR法最低检出菌体DNA量为10-2ng以及最低检出菌数为5.7 × 103 CFU/mL.对临床上送检的35份样品进行菌体DNA PCR方法检测并同时与国标GB/T4789.7-2003中使用的检测方法进行比对,两种检测方法的结果完全符合,与国标中使用的检测方法相比可大大缩短鉴定时间.因此该PCR法能快速鉴定当前副溶血性弧菌流行群,有利于流行病学溯源等研究.     

Detection of mammalian and avian hepadnaviruses by the polymerase chain reaction.

The hepadnavirus family contains a number of related viruses able to infect a variety of animal species. In the present study, we have used the polymerase chain reaction and oligonucleotide primers to a conserved region of the viral replicase gene of hepadnaviruses to identify viral sequences in de novo tissues in three well-characterized hepadnavirus systems: the woodchuck, ground squirrel and Pekin duck. We did not detect related hepadnavirus sequences in liver specimens from tree squirrels putatively infected with the tree squirrel hepatitis virus, or in liver specimens from horses with hepatitis (serum sickness), or from dogs with chronic active hepatitis or hepatocellular carcinoma.     

Detection of natural infection of Boophilus microplus with Babesia equi and Babesia caballi in Brazilian horses using nested polymerase chain reaction

The potential role of Boophilus microplus as a natural tick vector of Babesia equi and Babesia caballi in Brazilian horses was assessed using nested polymerase chain reaction (PCR)-based marker assay. B. equi merozoite-specific 218bp gene fragment was detected in almost 96% of horse blood samples, and 45.3-62.5% of females, eggs, larvae, and nymphs of B. microplus collected from 47 horses at Campo Grande in the State of Matto Grosso, Brazil. Except for the partially-fed female ticks, the B. caballi-specific 430bp gene fragment was amplified from horse blood samples, and all developmental stages. Parasite DNA from both species was detected in horse blood samples and B. microplus, with the preponderance of B. equi DNA. No DNA samples were positive solely for B. caballi parasite. Only 32% of the Giemsa-stained thin blood smears were positive for Babesia parasites, as against detection of B. equi parasite DNA in 95.7% of the blood samples by nested PCR. We have obtained molecular evidence that strengthens earlier experimental and ultrastructural studies in Brazil incriminating B. microplus as a natural vector of B. equi, and possibly of B. caballi. The detection of B. equi and B. caballi DNA in eggs and larvae of B. microplus is likewise suggestive of the possibility of both transovarial and transstadial parasite transmission in this tick vector.     

Use of repetitive sequence-based polymerase chain reaction for molecular epidemiologic analysis of Streptococcus equi subspecies equi

OBJECTIVE: To determine whether repetitive sequence-based polymerase chain reaction (rep-PCR) could be used to differentiate Streptococcus equi isolates, to examine S equi isolates from throughout the world, and to determine whether a horse had > 1 subtype of S equi during an outbreak of disease. SAMPLE POPULATION: An initial group of 32 S equi isolates, 63 S equi isolates from various geographic areas, and 17 S equi isolates obtained during outbreaks of disease. PROCEDURE: An aliquot of S equi genomic DNA was amplified, using enterobacterial repetitive intergenic consensus primers. Gel electrophoresis was performed on 1.5% agarose gels, and a computed-assisted program was used to compare rep-PCR results. RESULTS: Use of these primers to analyze 100 ng of S equi genomic DNA resulted in patterns of 6 to 14 bands. The 32 initial isolates were separated into 7 rep-PCR subtypes. There were 30 rep-PCR subtypes found among 29 S equi isolates obtained from Minnesota, Michigan, Canada, and Australia and 34 S equi isolates obtained from Kentucky and other sources. Furthermore, the same clone was identified in several horses during an outbreak of disease. Infected horses on the same farm all had a single clone of S equi. CONCLUSION AND CLINICAL RELEVANCE: Analysis of these results suggests that rep-PCR is useful for delineating S equi into rep-PCR subtypes. Results revealed that isolates with the same geographic source or similar date of collection did not always have the same rep-PCR subtype. A single clone of S equi usually predominated during an outbreak of disease.     

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.     

应用PCR技术检测鹅细小病毒

根据鹅细小病毒GPVH1株结构蛋白VP1与VP3编码基因非重叠核苷酸序列设计了一对引物GPR1 /GPR2 ,用这对引物对感染器官内的GPR和接种鹅胚增殖的GPV进行PCR扩增 ,其结果引物GRP1 /GPR2能特异性地扩增GPVVP1 VP3区段核苷酸序列 ,扩增出与设计核苷酸片段大小相符的 0 6kb的序列 ,而对照的鹅副粘病毒cDNA及鸭瘟病毒 (DPR)DNA对照组均出现阴性结果。     

Detection of leptospires in bovine semen by polymerase chain reaction

OBJECTIVE: In view of the considerable importance of venereal transmission of bovine leptospirosis, the objective of the present study was to compare the polymerase chain reaction (PCR), culture/isolation and serology to detect leptospire infection in bovine semen. DESIGN: Blood for serologic examination and semen for bacterial culture and PCR were collected from 20 bulls at artificial insemination centres in Brazil. Each animal was sampled twice for serology. RESULT: Forty-five percent (9/20) of the serum samples collected showed agglutinin titers to serovar hardjo in the first sample and 25% (5/20) had agglutinin titers to serovar hardjo in the second sample. Eighty percent (16/20) of semen samples were positive by PCR. Leptospires could not be isolated from any of the semen samples examined. CONCLUSION: Polymerase chain reaction can be a method of great potential for the detection of leptospires at artificial insemination centres.     

Evaluation of the Brucella abortus species-specific polymerase chain reaction assay, an improved version of the Brucella AMOS polymerase chain reaction assay for cattle.

In a blind test, 344 samples representing 80 bacterial isolates were analyzed by the Brucella abortus species-specific polymerase chain reaction (BaSS PCR) assay for the identification and discrimination of B. abortus field strains (wild-type biovars 1, 2, and 4) from 1) B. abortus vaccine strains, 2) other Brucella species, and 3) non-Brucella bacteria. Identical samples were tested in 2 laboratories. Half the samples were fully viable, and half were bacteria that had been killed by methanol fixation. The results in 1 laboratory correctly identified 100% of the samples, resulting in a predictive value of 100% for all categories and 100% sensitivity and specificity under the prescribed conditions. The second laboratory misidentified 31 samples, resulting in a range of 66.7-100% sensitivity, 93.2-99.7% specificity, and 77.3-98.2% predictive values depending on the category. There was no significant difference in viable versus fixed bacteria for either laboratory. Subsequent review of the protocol indicated that contamination was the likely cause of 26 of the 31 erroneous identifications. The results show that the BaSS PCR assay has the potential to be a very reliable screening tool for B. abortus identification. However, the data also provide a cautionary reminder of the importance of preventing contamination in diagnostic PCR.     

Detection of Brucella melitensis in semen using the polymerase chain reaction assay

An evaluation of the polymerase chain reaction (PCR) for detection of Brucella melitensis DNA in bovine and ovine semen was performed. Since semen contains different components that inhibit PCR amplification, a protocol was used to purify Brucella-DNA from bovine and ovine semen samples prior to conducting amplification of the targeted DNA. When separated fractions of naturally Brucella contaminated semen were analyzed by the PCR, most of B. melitensis DNA were present in the seminal fluid and non-sperm fractions.The PCR examination results for detection of B. melitensis DNA in different semen fractions were compared with the results for traditional cultural methods of Brucella from semen. The PCR was more sensitive than the traditional cultural methods since it detected Brucella-DNA in 12 (10%) out of 120 semen samples while direct culture detected only 7 (5.8%) in the same semen samples. The limit of detection by PCR was 100 CFU/ml of semen. In addition, the results of PCR were available in one day, whereas isolation and identification of Brucella organisms required days or even weeks. The PCR may be used as a supplementary test for detection of B. melitensis in semen.