反转录─套式PCR方法检测鸡传染性法氏囊病病毒

采用蛋白酶 Ｋ 消化,酚、氯仿抽提的方法提取法氏囊匀浆和细胞培养液中的鸡传染性法氏囊病病毒（ Ｉ Ｂ Ｄ Ｖ）基因组 Ｒ Ｎ Ａ,用特异的寡核苷酸引物对其 Ｖ Ｐ２ 高变区进行反转录套式 Ｐ Ｃ Ｒ 扩增。应用该方法从一个法氏囊匀浆中即可特异地检出 Ｉ Ｂ Ｄ Ｖ Ｒ Ｎ Ａ,得到的扩增产物可用于分子流行病学的进一步分析,而从感染材料的处理到扩增结果的电泳检测,在两个工作日之内可轻松完成。本实验为法氏囊病病毒的诊断和分子流行病学的分析提供了一种快速、简便、可靠的手段。     

IBDV逆转录—聚合酶链反应和核酸杂交检测方法的研究

以建立的传染性法氏囊病病毒（ＩＢＤＶ）逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）和核酸杂交检测方法，检测了细胞培养物、病理组织、粪便和饲料中的ＩＢＤＶ，并与ＩＢＤＶ夹心ＥＬＩＳＡ、琼脂扩散试验（ＩＤ）和病毒分离方法进行了平行比较试验。结果表明，ＲＴ－ＰＣＲ和核酸杂交检测方法具有高度的敏感性和特异性，是深入研究ＩＢＤ流行病学，特别是传播途径的新手段     

鸡传染性法氏囊病病毒基因组A片段cDNA序列分析

用鸡胚成纤维细胞繁殖鸡传染性法氏囊病病毒（IBDV）天水毒株。用提纯的病毒颗粒提取基因组RNA。根据已报道的英国52／70株的序列设计引物，用反转录－聚合酶链式反应（RT－PCR）进行cDNA扩增，获得1558bp和1590bp两个部分重叠的片段。结果表明，所克隆片段为3099的IBDV大开放读框。经与已报道的多个毒株相应序列比较后发现，核苷酸同源性介于97.4%-99.8%之间，推导的氨基酸同源性介于98.1%-99.5%。     

Digoxigenin-labeled nucleic acid probe for the detection of infectious bursal disease virus in infected cells.

A cDNA probe was synthesized from the VP-4 region of a virulent field isolate of infectious bursal disease virus (IBDV). The probe was labeled during synthesis with a non-radioactive steroid hapten, digoxigenin. The probe was used to develop a hybridization assay to detect the presence of IBDV in infected cell-culture and tissue suspensions from the bursa of Fabricius of infected chickens. The test was rapid, reproducible, and sensitive, and it could detect four serologic subtypes of IBDV, including the GLS-5 isolate.     

Molecular and phenotypic characterization of infectious bursal disease virus isolates

Two infectious bursal disease viruses (IBDVs 1174 and V1) were isolated from IBDV-vaccinated broiler flocks in California and Georgia. These flocks had a history of subclinical immunosuppression. These isolates are commonly used in IBDV progeny challenge studies at Auburn, AL, as well as vaccine manufacturer's vaccine efficacy studies, because they come from populated poultry-producing states, and are requested by poultry veterinarians from those states. Nested polymerase chain reaction (PCR) generated viral genome products for sequencing. A 491-bp segment from the VP2 gene, covering the hypervariable region, from each isolate was analyzed and compared with previously sequenced isolates. Sequence analysis showed that they were more closely related to the Delaware (Del) E antigenic variant than they are to the Animal Health Plant Inspection Service (APHIS) standard, both at the nucleotide level (96%, 97%) and at the amino acid level (94%, 97%). Both isolates had the glutamine to lysine shift in amino acid 249 which has been reported to be critical in binding the virus neutralizing Mab B69. Phenotypic studies showed that both isolates produced rapid atrophy of the bursae and weight loss, without the edematous bursal phase, in 2-wk-old commercial broilers having antibody against IBDV. A progeny challenge study showed both isolates produced more atrophy of the bursae (less percentage of protection) than the Del E isolate. Molecular and phenotypic data of these important IBDV isolates help in the improved detection and control of this continually changing and important viral pathogen of chickens.     

应用RT／PCR—SSCP法分析传染性法氏囊病病毒的变异性

根据传染性法氏囊病病毒（IBDV）cDNA序列，在病毒VP2区域设计1对引物，应用RT／PCR－SSCP方法对4个不同时间及不同地域从传染性法氏囊病（IBD）病鸡法氏囊组织中分离的IBDV分离物进行了分析，发现4个IBDV分离物的SSCP图谱均存在明显差异，本试验表明，IBDV变异在我国普遍存在，SSCP方法可用于IBDV的变异性分析。     

鸡传染性法氏囊病病毒适应于Vero细胞的初步研究

将国内６个省市（广东、巾东、河北、辽宁、、新疆和北京）的７株鸡传染性法氏囊病（ＩＢＤ）的法氏囊组织处理后，直接在Ｖｅｒｏ细胞上盲传３～４代，均能不同程度的产生特征性细胞病变效应（ＣＰＥ），并通过选用具有广谱性的抗法氏囊病病毒的单克隆抗体（ＩＢＩ）Ｖ－ＭｃＡｂ）采用间接免疫荧光（ＩＦＡ）和碱性磷酸酶—抗碱性磷酸酶桥联酶显色技术（ＡＰＡＡＰ）的方法对７株分离毒的Ｖｅｒｏ第８代细胞进行鉴定，又用逆转—聚合酶链式反应（ＲＴ－ＰＣＲ）对７株分离毒的第１４代Ｖｅｒｏ细胞毒进行进一步证实，结果表明这７株ＩＢＤ囊毒均适应于Ｖｅｒｏ细胞上，这是国内首次报道将组织毒直接适应于Ｖｅｒｏ传代细胞上。     

Occurrence and expression of p53 suppressor gene and c‐Myc oncogene in dog eyelid tumors

Purpose To detect the occurrence and expression of the suppressor gene p53 and of the oncogene c‐Myc in eyelid tumors of dogs using the PCR, RT‐PCR, PCR‐ELISA and RT‐PCR‐ELISA techniques. These genes have not been described in dog eyelid tumors before. Methods Nine samples of eyelid or third eyelid epithelial tumors were obtained from the archives of the Department of Veterinary Pathology. Tumor diagnosis was confirmed by evaluation of hematoxylin‐eosin stained sections, and immunohistochemistry for cytokeratin AE1/AE3 and vimentin V9. A canine mammary tumor was used for positive control. Agarose gel electrophoresis, PCR‐ELISA and RT‐PCR‐ELISA were used to detect p53 and c‐Myc genes. Results The occurrence of p53 was detected in most of the eyelid tumors and third eyelid tumors studied (88.8%, n = 8) and was expressed in 75% of the positive samples, as indicated by ELISA. The c‐Myc gene was found in 77.7% (n = 7) of the samples and was expressed in eight samples. Conclusions Eyelid and third eyelid tumors of dogs express both the p53 and the c‐Myc genes as shown by PCR and RT‐PCR. However, PCR ELISA and RT‐PCR ELISA were more efficient in assessing occurrence and expression of these genes because they identified amplified products that were not detected by agarose gel electrophoresis.     

传染性法氏囊病病毒VP2基因高变区序列分析

根据传染性法氏囊病病毒（ＩＢＤＶ）ＶＰ２基因ＣＤＮＡ序列,在ＶＰ２基因高变区设计一对引物,用ＲＴ－ＰＣＲ方法扩增ＩＢＤＶ分离株ＪＳ３和ＪＳ４。将扩增片段克隆后以双脱氧链末端终止法测定核苷酸旬。ＪＳ３和ＪＳ４的同源性最高达９８％。与已发表的ｖｖＩＢＤＶ,ＩＢＤＶ变异要ＢＤＶ经典株为ＩＢＤＶ弱毒株核苷酸序列的同尖拨天９２￣９８％之间,根据ＩＢＤＶ的大ＯＲＦ推导出该片段蛋白的氨基酸序更,ＪＳ３和ＪＳ４的