牛分枝杆菌特异性PCR检测方法的建立及初步应用

根据已发表的牛分枝杆菌的pncA的基因序列，设计和合成了一对可扩增294bp目的片段的引物，建立了特异性检测牛分枝杆菌的PCR方法。对牛分枝杆菌国际参考株和国内分离株成功扩增出294bp的特异性基因片段；对人结核分枝杆菌、副结核分枝杆菌、鸟胞内分枝杆菌和草分枝杆菌DNA的PCR扩增结果均为阴性。本PCR方法检测的敏感度可达到50pg。对10份牛分枝杆菌培养阳性和10份阴性样品的DNA分别进行了PCR检测，结果10份阳性样品中有9份样品为PCR扩增阳性，阳性符合率为90％（9／10）；而10份阴性样品则PCR扩增全部为阴性，阴性符合率为100％（10／10）。本方法可做为牛分枝杆菌的快速检测和流行病学调查的工具。     

两种早熟禾超氧物歧化酶活性测定的适宜条件

以冷地早熟禾 (Poacrymophila)和草地早熟禾 (P .pratensis)为材料 ,探讨光照反应时间、酶浓度以及酶反应体系等对NBT光化还原反应的影响。随着光照反应时间的延长 ,最大光还原值直线上升。 2 5min后光化还原值逐渐趋于稳定。光照反应开始后 ,酶液对NBT光化还原反应速率的抑制百分率迅速升高 ,在 15～ 2 5min时 ,酶液对NBT光化还原反应的抑制百分率保持稳定 ,2 5min后下降速率较大。光照反应时间对SOD酶活性测定的影响较大 ,确定为 2 5min较为适宜。酶浓度在 4 0～ 6 0mg/mL时光化还原值变化速率较一致。测定中反应体系NBT、核黄素的加样量以及光照强度在一定范围内时光化还原值较为稳定     

兽用疫苗支原体污染的PCR检测技术的建立及应用

根据支原体的16 S rRNA基因设计特异性引物,以兽用疫苗中常见的支原体DNA为模板进行PCR试验,优化反应体系,建立了兽用疫苗支原体污染的PCR 检测方法,并对市场上的兽用疫苗随机抽查检测,其检出率为24.4%(22/90)。结果表明该方法特异性强,灵敏度高,有望成为兽用疫苗支原体污染的检测方法之一。     

猪细小病毒PCR检测与分离研究

根据猪细小病毒(PPV)结构蛋白VP2基因序列,设计合成1对引物,建立了检测PPV的PCR方法。检测14份临床组织,检出阳性11份,阳性检出率为79.5%。阳性样品扩增产物用EcoRⅠ酶切得到了预期的结果,对PCR产物进行测序,测序结果与已发表的PPV基因核苷酸序列比较,同源性达99%~100%,所编码的氨基酸同源性为93%~100%。从PCR检测阳性的一份病料组织中分离出1株PPV。试验证明,建立的PPVPCR检测方法特异性强、敏感性高,适用于临床样品的快速检测。     

小鹅瘟PCR诊断方法的建立和初步应用

根据发表的鹅细小病毒B株VP3基因序列 ,设计合成了 1对寡聚核苷酸引物。以国内分离的小鹅瘟病毒为摸板 ,筛选了 (PCR)最佳反应条件 ,并进行了敏感性、特异性和初步应用试验。结果表明 ,在 4 0 μL体积中 ,PCR最佳系统组成为 2uTaq酶、0 5mol/LdNTP和 2 0 pmol引物 ;最佳反应参数为 96℃变性 4 0s ,5 4℃退火 1min ,72℃延伸 1min ,30个循环。本方法可检出 2 5× 10 -1 5EID50 小鹅瘟病毒 ,并且仅能从小鹅瘟病毒的鹅胚培养物中扩增出与设计值大小相同的 375bp核苷酸片断 ,对照病毒扩增结果为阴性。通过对 6份临床病料的检查 ,2份呈阳性 ,其中 1份来自有典型小鹅瘟症状的雏鹅 ,1份来自无典型小鹅瘟症状的雏鹅。由此说明 ,本试验所建立的诊断小鹅瘟的PCR方法具有潜在的临床应用价值     

VSV的RT-PCR快速检测方法的建立

选取水泡性口炎病毒 N基因序列 ,设计 1对引物 ,建立检测水泡性口炎病毒的 RT- PCR方法。对VSV各毒株进行检测 ,结果均为阳性 ,而对反刍动物病毒性疾病相关病毒进行检测 ,结果均为阴性。结果表明所建立的 RT- PCR技术可用于水泡性口炎的诊断和流行病学调查     

Real-time PCR genotyping assay for feline erythrocyte pyruvate kinase deficiency and mutant allele frequency in purebred cats in Japan

Erythrocyte pyruvate kinase (PK) deficiency is an inherited glycolytic erythroenzymopathy caused by mutations of the PKLR gene. A causative mutation of the feline PKLR gene was originally identified in Abyssinian and Somali cats in the U.S.A. In the present study, a TaqMan probe-based real-time PCR genotyping assay was developed and evaluated for rapid genotyping and large-scale screening for this mutation. Furthermore, a genotyping survey was carried out in a population of four popular purebred cats in Japan to determine the current mutant allele frequency. The assay clearly displayed all genotypes of feline PK deficiency, indicating its suitability for large-scale survey as well as diagnosis. The survey demonstrated that the mutant allele frequency in Abyssinian and Somali cats was high enough to warrant measures to control and prevent the disease. The mutant allele frequency was relatively low in Bengal and American Shorthair cats; however, the testing should still be carried out to prevent the spread of the disease. In addition, PK deficiency should always be considered in the differential diagnosis of anemia in purebred cats in Japan as well as worldwide.     

Evaluation of biomarkers of kidney injury following 4% succinylated gelatin and 6% hydroxyethyl starch 130/0.4 administration in a canine hemorrhagic shock model

Objective : To investigate the association between synthetic colloids and biomarkers of acute kidney injury (AKI) in dogs with hemorrhagic shock. Design : Experimental interventional study. Setting : University. Animals : Twenty‐four healthy ex‐racing Greyhounds. Interventions : Anesthetized Greyhounds subjected to hemorrhage for 60 min were resuscitated with 20 mL/kg of fresh whole blood (FWB), 6% hydroxyethyl starch (HES) 130/0.4, 4% succinylated gelatin (GELO), or 80 mL/kg of isotonic crystalloid (CRYST) over 20 min (n = 6 per treatment). Concentrations of biomarkers of AKI were measured at baseline, end of hemorrhage, and at 40 (T60), 100 (T120), and 160 (T180) min after fluid bolus. Biomarkers included neutrophil gelatinase‐associated lipocalin in urine and serum (uNGAL; sNGAL), and urine cystatin C (uCYSC), kidney injury molecule‐1 (uKIM), clusterin (uCLUST), osteopontin, gamma‐glutamyl transferase, monocyte chemoattractant protein‐1 (uMCP), interleukin‐6, interleukin‐8, protein (uPROT), hyaluronan, and F₂‐isoprostanes. Renal histology was scored for tubular injury and microvesiculation. Biomarker fold‐change from baseline was compared between groups using mixed effects models (Bonferroni–Holm corrected P<0.05). Frequencies of histology scores were compared by Fisher's exact test. Measurements and main results : In dogs treated with GELO, uNGAL fold‐change was markedly greater compared with all other groups at T60, T120, and T180 (all P<0.001), and uCYSC was greater at T60 compared with CRYST (P<0.001), and at T120 and T180 compared with all other groups (all P<0.001). Smaller, albeit significant, between‐group differences in uKIM, uCLUST, uMCP, and urine protein concentration were observed across the FWB, GELO, and HES groups, compared with CRYST. The GELO group more frequently had marked tubular microvesiculation than the other groups (P = 0.015) although tubular injury scores were comparable. Conclusion : In dogs with hemorrhagic shock, GELO was associated with greater magnitude increases in urine biomarkers of AKI and more frequent marked tubular microvesiculation, compared with FWB, CRYST, and HES.