PCR用于鸭瘟病毒诊断的研究

根据鸭瘟病毒UL6和UL7基因序列,设计合成了一对引物,以2株疫苗株、1株强毒株和1株山东分离株DNA为模板,进行PCR扩增,得到预期690bp的目的片段.将扩增的目的片段克隆到pMD18-T载体,经Amp平板筛选,HindⅢ、BamHⅠ双酶切鉴定,获得阳性重组质粒.对重组质粒进行序列测定,与参考序列比较,山东分离株与参考序列的同源性为99.7%,其余3株DPV与参考序列的同源性均为100%.应用PCR可检测人工感染和自然感染鸭瘟的组织中的鸭瘟病毒,表明PCR检测鸭瘟病毒具有很高的特异性、敏感性,该法能够用于鸭瘟急性及亚临床感染的检测与诊断.     

Identification of duck plague virus by polymerase chain reaction

A polymerase chain reaction (PCR) assay was developed for detecting duck plague virus. A 765-bp EcoRI fragment cloned from the genome of the duck plague vaccine (DP-VAC) virus was sequenced for PCR primer development. The fragment sequence was found by GenBank alignment searches to be similar to the 3' ends of an undefined open reading frame and the gene for DNA polymerase protein in other herpesviruses. Three of four primers sets were found to be specific for the DP-VAC virus and 100% (7/7) of field isolates but did not amplify DNA from inclusion body disease of cranes virus. The specificity of one primer set was tested with genome templates from other avian herpesviruses, including those from a golden eagle, bald eagle, great horned owl, snowy owl, peregrine falcon, prairie falcon, pigeon, psittacine, and chicken (infectious laryngotracheitis), but amplicons were not produced. Hence, this PCR test is highly specific for duck plague virus DNA. Two primer sets were able to detect 1 fg of DNA from the duck plague vaccine strain, equivalent to five genome copies. In addition, the ratio of tissue culture infectious doses to genome copies of duck plague vaccine virus from infected duck embryo cells was determined to be 1:100, making the PCR assay 20 times more sensitive than tissue culture for detecting duck plague virus. The speed, sensitivity, and specificity of this PCR provide a greatly improved diagnostic and research tool for studying the epizootiology of duck plague.     

Post-epizootic surveys of waterfowl for duck plague (duck virus enteritis)

Surviving birds from nine duck plague outbreaks in urban and confined waterfowl were sampled for duck plague (DP) virus and DP antibody during 1979-86. Duck plague virus was found in combined oral and cloacal swabs of birds from three outbreaks, and DP-neutralizing antibody was demonstrated in some birds from all nine outbreaks. Greater prevalence of DP antibody and higher titers were found in survivors from confined populations than from free-flying urban populations. Free-flying waterfowl from within 52 km of four DP outbreak sites were also sampled; virus was not found in any birds, but DP antibody was found in urban waterfowl in the vicinity of an outbreak in Potterville, Michigan. No evidence of exposure to or shedding of DP virus in migratory waterfowl was found in two regions where DP appears enzootic in urban and confined waterfowl (Eastern Shore of Maryland and the vicinity of Sacramento, California).     

RT-PCR技术诊断猪瘟的应用研究

应用反转录—聚合酶链反应(RT-PCR)对猪瘟进行诊断应用研究。应用RT-PCR况对来自广西不同地区的135份疑似猪瘟病料进行检测，以份诊断为阳性，阳性率62．2％。从百色、柳州地区等采集的健康猪扁桃体和淋巴结共276份，经RT-PCR检测，37份为阳性，阳性率为13．4％。其中健康猪扁桃体带毒较高，246份扁桃体中有35份阳性，占14．2％。采自柳州健康猪的26份淋巴结材料全为阴性，只有邕宁县的1份健猪淋巴结阳性。结果表明，RT-PCR技术可应用于猪瘟的临床诊断。     

Diagnosis of canine leishmaniasis by a polymerase chain reaction technique

A polymerase chain reaction (PCR) technique for the diagnosis of canine leishmaniasis on bone marrow samples was developed which amplified a 120 bp DNA fragment of the Leishmania kinetoplast DNA, common to all Leishmania species. Forty-five of 46 dogs in which leishmaniasis had been diagnosed were positive with the PCR technique, whereas none of 41 healthy dogs gave a positive result. Fifteen dogs with leishmaniasis that had been treated for six months with N-methylglucamine antimoniate and allopurinol were also investigated. Seven were positive, implying that they remained infected despite the resolution of their clinical signs.     

Vertical transmission of duck plague virus (DPV) by apparently healthy DPV carrier waterfowl

Duck plague virus (DPV) was transmitted vertically in muscovy, pekin, and mallard ducks that were persistently infected with the LA-SD-73, MSN-WI-77, or CO-WI-73 isolates of DPV. The effects of vertical transmission on the fertility and hatchability of eggs laid by DPV carrier ducks varied with the DPV isolate and duck species. Fertility was reduced significantly only in eggs laid by MSN-WI-77 virus carrier pekin and muscovy ducks. The hatchability of eggs laid by DPV carrier mallards and muscovies was significantly reduced from that of uninfected control ducks. All ducklings tested that hatched from eggs laid by DPV carrier waterfowl shed DPV in the feces. The DPV carrier ducklings shed DPV in small amounts. Vertical transmission of DPV in domestic flocks can lower fertility and hatchability. In wild waterfowl, vertical transmission may be a means of virus perpetuation from generation to generation.     

Diagnosis of trichomoniasis in 'virgin' bulls by culture and polymerase chain reaction

The diagnostic test for Tritrichomonas foetus in bulls is microscopic examination of cultured preputial samples. Trichomonads other than T. foetus can be present in a preputial sample. Both a staining technique and a polymerase chain reaction assay were useful in differentiating between T. foetus and another trichomonad observed in samples from virgin bulls.     

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.     

Diagnosis of feline leukaemia virus infection by semi-quantitative real-time polymerase chain reaction

In this paper the design and use of a semi-quantitative real-time polymerase chain reaction assay (RT-PCR) for feline leukaemia virus (FeLV) provirus is described. Its performance is evaluated against established methods of FeLV diagnosis, including virus isolation and enzyme-linked immunoassay (ELISA) in a population of naturally infected cats. The RT-PCR assay is found to have both a high sensitivity (0.92) and specificity (0.99) when examined by expectation maximisation methods and is also able to detect a large number of cats with low FeLV proviral loads that were negative by other conventional test methods.     

鸭瘟的诊断与免疫防治

鸭瘟又名鸭病毒性肠炎（DVE）,是鸭、鹅的一种疱疹病毒感染的急性、接触性传染病。其特征是侵害血管、组织出血、消化道黏膜糜烂、淋巴器官出现病变、实质器官退行性病变。临床主要表现为腿软、走动困难、不愿下水、肿头,故名“大头瘟”,是危害养鸭业的三大疾病之一。     

Identification of the crayfish plague fungus Aphanomyces astaci by polymerase chain reaction and restriction enzyme analysis

To characterise the DNA of the crayfish plague fungus Aphanomyces astaci, Saprolegniales (Oomycetes), primers were developed to amplify a 1050bp segment of the 28S rDNA region. Restriction enzymes were applied to the amplicon obtained, to distinguish A. astaci from 12 fungal species belonging also to the Saprolegniales and five more distantly related fungi. Most of the fungal species included in the study are either known parasites of freshwater crayfish cuticle or can be found in their natural environment. A. astaci DNA was distinguishable from the DNA of other fungal species tested by using the primers developed plus restriction enzymes AluI, HindIII and AvaI.Prior to this study, methods for A. astaci-species determination, e.g. spore production and infection experiments, required a protracted period to yield results; the method described in this study is quicker.     

Diagnosis by polymerase chain reaction of Erysipelas septicemia in a flock of ring-necked pheasants

A flock of 810 pheasants experienced 6.2% mortality over 6 days. Affected birds were weak and lethargic for up to 24 hr before death. Examined birds were thin, and gross lesions consisted of thick opaque crops and cecal cores. Histologically, there was capillariasis of the crop and multifocal ulcerative typhlitis with Heterakis spp. infection, and numerous systemic intravascular monocytes were filled with clusters of blue rod-shaped organisms. The organisms were gram-positive bacilli by Brown and Brenn staining and ultrastructural analysis. Liver bacterial cultures were negative for pathogenic bacteria. Erysipelas septicemia was diagnosed by an Erysipelothrix species-specific polymerase chain reaction method with the substrate DNA isolated from formalin-fixed, paraffin-embedded liver.     

Development of a polymerase chain reaction to detect Vietnamese isolates of duck virus enteritis.

A polymerase chain reaction (PCR) method for the detection of duck virus enteritis (DVE) virus in tissues of infected and affected ducks, and in cell culture was developed. This required us to obtain specific nucleotide sequence information as we could not find any specific data about the genome of the virus. We found the assay to be highly effective in detecting the virus under experimental conditions and to be easily transferred to laboratories in Vietnam where it is being used in studies on the epidemiology of the disease. We have applied this simple and rapid diagnostic method to the detection of DVE isolates grown in cell culture and tissues from infected birds. The assay was also able to differentiate DVE from other avian herpesviruses, such as Marek's disease, infectious laryngotracheitis virus and goose herpesvirus.     

Diagnosis of equid herpesviruses -1 and -4 by polymerase chain reaction.

The polymerase chain reaction (PCR) is a sensitive technique used to detect DNA of viral pathogens. We have applied the technique to the detection of Equid herpesviruses-1 and -4 (EHV-1 and EHV-4) DNA within nasopharyngeal swab samples from horses. Ninety-eight samples from suspected field cases and in-contact horses were analysed. The assays were conducted blind and later decoded and compared with virus isolation data. Our results indicate that PCR is a sensitive and rapid technique for the diagnosis of EHV-1 and EHV-4 infection.     

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.     

Development of a polymerase chain reaction procedure for detection and differentiation of duck and goose circovirus

This article reports the complete nucleotide sequences of four duck circovirus (DuCV) isolates from sick ducks in Taiwan and development of a polymerase chain reaction (PCR) for detection and differentiation of goose circovirus (GoCV) and DuCV. Sequence comparison showed that Taiwanese DuCV isolates had 82.5%-83.8% nucleotide sequence identity to the German and North American DuCV isolates. This is the first report on the presence of DuCV and its associated diseases outside Germany. A PCR test was developed using a universal primer pair based on conserved sequences present in the genomes of GoCV and DuCV. This PCR test could detect and differentiate between GoCV and DuCV by the size of PCR product each virus produced (256 bp for GoCV and 228 bp for DuCV). Application of this PCR test to samples of bursa of Fabricius from sick birds in the field showed that 9 of 26 goose samples contained GoCV, while 13 of 34 duck samples contained DuCV. This PCR test could serve as a fast and sensitive method for detection and differentiation of DuCV and GoCV.