禽流感RT-PCR诊断法的建立

根据禽流感病毒（ＡＩＶ）Ａ／Ｃｈｉｃｋｅｎ／Ｂｒｅｓｌｉｎ／１９０２（Ｈ７Ｎ７）株的血凝素（ＨＡ）基因参考序列设计合成一对Ｈ７ＨＡ特异引物,并应用ＳＤＳ－蛋白酶Ｋ法提取病毒ＲＮＡ,建立了逆转录－聚合酶链式反应（ＲＴ－ＰＣＲ）检测ＡＩＶＲＮＡ的方法。应用此法对鸡胚培养的ＡＩＶ尿囊液、ＳＰＦ感染鸡粪便进行了检测,并与琼脂扩散试验和电镜技术作了对比。特异性试验以新城疫病毒（ＮＤＶ）、传染性法氏囊病病毒（ＩＢＤＶ）、传染性支气管炎病毒（ＩＢＶ）、减蛋综合征病毒（ＥＤＳ－７６Ｖ）等的感染鸡胚尿囊液作为对照。结果表明,ＡＩＶ尿囊液ＲＴ－ＰＣＲ全部阳性,ＳＰＦ感染鸡粪便８７份,ＲＴ－ＰＣＲ检出６９份阳性,样品处理浓缩后琼脂扩散检出１１份,电镜检测了２３份样品,仅检出２份阳性。非特异性对照样品经ＲＴ－ＰＣＲ检测全部阴性。将ＡＩＶ感染鸡胚尿囊液作１０倍系列连续稀释,可检出稀释度为１０－２的病毒尿囊液。ＲＴ－ＰＣＲ最早检出时间为感染后第３天,而琼扩和电镜均是７天。该法具有高度的特异性和灵敏性,且节约时间（只需８小时左右）,可从分子水平上对ＡＩＶ进行早期快速诊断和流行病学调查     

Comparison of PCR methods for diagnosis of canine visceral leishmaniasis in conjunctival swab samples

Four PCR assays for detection of Leishmania DNA in conjunctival swab samples were compared. All methods had two steps: a first amplification followed by hybridization or by a new amplification (nested or seminested). Two methods (kDNA PCR-hybridization and kDNA snPCR) used primers targeted to the minicircles of kinetoplast DNA (kDNA) and the other two methods to the coding (LnPCR) and intergenic noncoding regions (ITS-1 nPCR) of ribosomal rRNA genes. kDNA PCR-hybridization was positive for 22/23 dogs (95.6%) and for 40/46 samples (86.9%), considering the right and the left conjunctivas. kDNA snPCR was positive for 21/23 dogs (91.3%) and for 40/46 samples (86.9%). The ITS-1 nPCR and LnPCR were both able to detect the parasites in 17/23 dogs (73.9%) and 29/46 (63%) and 30/46 (65.2%) samples, respectively. The positivities of the kDNA based methods were significantly higher; however the choice of the best method will depend on the kind of information required with the diagnosis.     

聚合酶链反应（PCR）鉴定猪肉方法的建立及应用

依据猪小卫星ＤＮＡ序列设计了１对引物,建立了ＰＣＲ鉴定生、熟猪肉的方法。应用本方法特异地扩增出预期的猪３０７ｂｐ的ＤＮＡ片段,其检测敏感度对生猪肉达３８．６ｆｇＤＮＡ,对熟猪肉和高压猪肉为０．３７５ｆｇ全基因组ＤＮＡ。运用该引物仅可扩增生猪ＤＮＡ的单一片段,而对马、牛、羊等１２种动物肉的ＤＮＡ扩增则呈阴性。应用本法对６３份生、熟猪肉进行鉴定,正确率为１００％,对各种样品检测,均可在６ｈ内完成。     

PCR法对伪狂犬病病猪不同部位的检测及敏感性试验

应用ＰＣＲ方法对ＰＲＶ感染细胞及自然发病猪不同组织样品进行检测,感染细胞毒最低检出量为１０５个ＴＣＩＤ５０病料组织样品最低取样为０１ｍｇ时,仍能得到阳性结果其敏感性显著高于微量血清中和试验。ＰＲＶ在自然发病猪体内分布很广,脑、三叉神经节（三叉Ｎ节）、嗅球、扁桃体、心脏、肝、脾、肺、肾均有ＰＲＶ的存在,ＰＲＶ检出率最高的组织为三叉神经节,其检出率为１００％。其它对照病毒及传代细胞ＰＣＲ反应产物电泳结果均为阴性。     

用PCR技术检测牛胎儿弯曲杆菌

在国内首次用PCR技术检测牛胎儿弯曲杆菌(Campylobacterfetus),试验结果表明,灭菌生理盐水中的胎儿弯曲杆菌胎儿亚种或性病亚种均可检出;而空肠弯曲杆菌、葡萄球菌、链球菌、大肠杆菌等均为阴性。本法特异性和灵敏度很高,甚至可检出样品中的一个菌体。应用前景良好。     

建立全套式PCR对牛胚胎性别鉴定的方法

选择公牛Y染色体上的特异DNA序列和公母牛共有的DNA序列片段设计并合成引物,通过全套式PCR反应对公、母牛静脉血及牛胚胎进行特异性条带鉴定。结果表明,公牛出现322bp的性控条带和584bp的质控条带,而母牛仅出现584bp的质控条带。静脉血样与实际性别相符率为100％,显微切割下的5～8个胚胎细胞亦出现与静脉血样相同的特异性条带。这表明该种全套式PCR的鉴定方法准确、严谨、简便,具有很高的灵敏性和稳定性。     

应用PCR-限制性内切酶技术快速检测副结核分枝杆菌

根据副结核分枝杆菌的IS900序列,设计相应的引物,建立检测副结核分枝杆菌的PCR技术。利用该方法,可从副结核分枝杆菌中扩增出PCR产物,扩增产物具有高度特异性,长度为388bp,经限制性内切酶Saμ3AI酶切,证实该扩增产物为副结核分枝杆菌的片段。表明此项技术具有快速、敏感和特异性强等特点,通过PCR扩增IS900基因(副结核分枝杆菌独有的基因),可快速的应用于诊断反刍动物的副结核病和乳及乳制品中副结核分枝杆菌的鉴定。     

应用PCR检测单核细胞增多症李氏菌的研究

以二对位于单核细胞增多症李氏菌β－溶血素基因内的２６－ｂｐ寡核苷酸为引物，用ＰＣＲ对单核细胞增多症李氏菌进行了检测，结果所有株单核细胞增多症李氏菌均扩增出１８６－ｂＰ的特异条带，其它种李氏菌和细菌均呈阴性反应。本方法可测出模拟肉样中少至９２个细菌，模拟奶样中少至１８个细菌，其特异性和敏感性不受其它污染杂菌ＤＮＡ的影响。对市售猪肉和牛奶的检测结果表明，ＰＣＲ法优于传统的分离培养法，具有快速、简便和敏     

Molecular Evidence for Persistence of Anaplasma phagocytophilum in the Absence of Clinical Abnormalities in Horses after Recovery from Acute Experimental Infection

Background: Anaplasma phagocytophilum infects several mammalian species, and can persist in sheep, dogs, and calves. However, whether this organism persists in horses or induces long-term clinical abnormalities is not known.
Objectives: To evaluate whether A. phagocytophilum can persist in horses and to document clinical findings for 3 months after complete recovery from acute disease.
Animals: Five clinically normal adult horses that had recovered spontaneously from experimentally induced acute disease caused by a Swedish equine isolate of A. phagocytophilum .
Methods: Horses were monitored for up to 129 days post inoculation (PI) by daily clinical examination and at least alternate day blood sampling for evidence of A. phagocytophilum on polymerase chain reaction (PCR) and blood smears. All horses were euthanized and underwent postmortem examination.
Results: All horses were periodically PCR positive after recovery from acute infection. Before day 66 PI 2 horses were persistently PCR negative whereas 3 horses were intermittently PCR positive. Subsequently, 4 of 5 horses were intermittently PCR positive, particularly after stress mimicking interventions. One animal was positive immediately before postmortem examination. Clinical abnormalities related to persistence of anaplasma were not observed. No specific changes were found at postmortem examination, and all sampled tissues from all horses were negative on PCR for A. phagocytophilum .
Conclusions and Clinical Importance: Infection with A. phagocytophilum can persist in the horse for at least 129 days. However, the continued presence of the organism is not associated with detectable clinical or pathological abnormalities.