16SrDNA实时荧光定量PCR检测DPV强毒感染鸭气管及消化道大肠杆菌动力学

参照Genebank大肠埃希氏菌属(E.coli)16SrDNA保守基因序列设计并合成定量PCR引物及针对大肠杆菌属的Taqman探针,以E.coli标准菌株的PCR扩增产物作为阳性模板制作标准曲线,建立检测E.coli的定量PCR方法。该法Mg2+最佳工作浓度5 mmol/L,能够定量和特异检测E.coli而对肠道优势菌群的代表种葡萄球菌、双歧杆菌呈阴性。检测E.coli的灵敏度可达3个/mL。利用该法对DPV强毒感染鸭急性病例的气管和消化道E.coli检测,结果表明:气管E.coli数量普遍低于对照。食道波动巨大,普遍高于对照;十二指肠、空肠变化不明显;回肠波动巨大,总体高于对照;盲肠显著低于对照;直肠与对照差异不显著。DPV感染致死鸭的气管E.coli数量显著低于对照;食道和十二指肠极显著高于对照;空肠、回肠和盲肠均略高于对照;直肠略低于对照。     

Determination of drift potential of different flat fan nozzles on a boom sprayer using a test bench

This study's objective was to evaluate the functionality of an ad hoc test bench for spray drift measurement with boom sprayers, using it for evaluating different nozzles according to drift risk. The repeatability of results was evaluated by conducting similar tests at two different laboratories. Drift potential values (DPV) obtained showed an interesting effect of Venturi flat fan nozzles on drift reduction, in comparison with conventional flat fan nozzles (reference nozzle was XR 11003). Newly designed flat fan nozzles reduced the risk of drift. Reasonably relations between 10th-percentile, D[v,0.1], 50th-percentile or Volume Median Diameter, D[v,0.5], 90th-percentile, D[v,0.9], V₁₀₀ and DPV were observed in all cases, with R² values of 0.58, 0.65, 0.66 and 0.72, respectively. The lowest drift values were achieved with TTI and TD Spray Max nozzles; they were significantly lower than those obtained for IDK and AIXR ones. Results indicated that the drift test bench can be used as an alternative to the official standard procedure for drift measurements on boom sprayers (e.g. ISO 22866), as it is able to discriminate the influence of different boom settings (especially nozzle types) on drift. Further studies could be useful in order to prove that the classification of nozzles according to drift risk obtained using the test bench is comparable to the nozzle classifications obtained applying the ISO 22866 test method.     

鸭瘟病毒UL47基因克隆及其分子特性分析

通过测定本实验室构建的鸭瘟病毒(Duck plague virus,DPV)DNA基因文库中重组质粒的DNA序列,结合NCBI的ORF Finder和Blast工具分析得到了该病毒UL47基因的ORF。采用PCR扩增出了UL47基因并将其克隆到pMD18-T载体上,经PCR和酶切鉴定以及进一步的核酸斑点杂交试验证实该基因即为DPV UL47基因。利用生物信息学软件ProtScale、SignalP3.0、Scansite、TMpred、Prosite、DNAStar以及在线的EMBOSS等分析了UL47基因的分子特性。结果显示,该基因大小为2 367bp,编码788aa,而且与GenBank上多种α疱疹病毒同源蛋白的核酸和氨基酸序列具有较高的同源性。系统进化树分析表明,DPV UL47与禽类疱疹病毒(α-疱疹病毒)的进化关系最近。密码子偏爱性结果显示,DPV UL47编码相同氨基酸的不同密码子使用频率差异较大,UL47基因密码子使用模式更接近真核生物。研究结果为进一步开展DPV UL47基因功能研究奠定了基础。     

鸭瘟强毒川W株的分离、鉴定及毒力测定

从四川成都某鸭场分离得到一株疑似鸭瘟病毒，经细胞培养、中和实验、PCR鉴定。证明分离株为鸭瘟病毒，毒力测定及动物实验表明其可能为强毒株。试验结果提示鸭瘟病毒毒力变异和增强都会使弱毒疫苗的免疫保护作用下降。     

Rapid diagnosis of duck plagues virus infection by loop-mediated isothermal amplification

Duck virus enteritis is a serious disease among farmed and free-living ducks (Anatidae) and a constant threat to the commercial duck industry in China. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed to rapidly detect and diagnose duck plague virus (DPV) in both farmed and wild waterfowl, and compared with polymerase chain reaction (PCR) method and real-time PCR method in accuracy, sensitivity and specificity. A set of four specific primers was successfully designed to recognize six distinct genomic sequences of UL6 protein from DPV, including one forward inner primer, one back inner primer and two outer primers. The optimum reaction temperature and time were verified to be 61.5 °C and 60 min, respectively. Comparative experiments showed that LAMP assay was a simple, rapid, accurate, sensitive and specific method for detecting DPV, and was superior to PCR assay in sensitivity and specificity for DNA amplification. In addition, challenge tests indicated the newly developed LAMP method was more sensitive for the diagnosis of DPV infection than virus isolation and PCR. LAMP assay would be a good alternative method for on-farm disease diagnosis.