Duplex PCR 快速检测松材线虫

利用duplex PCR技术对松材线虫(Bursaphelenchus xylophilus)与拟松材线虫(B. mucronatus)的rDNA部分核苷酸序列扩增.根据松材线虫与拟松材线虫的ITS1序列区别,设计出特异性引物,检测松材线虫的存在;在5.8S,28S保守序列区设计通用引物,检测伞滑刃线虫的存在.     

Establishment of a Duplex Real-time RT-PCR Assay for the Differential Detection of Nipha Virus and Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus

To establish a rapid,sensitive and specific assay for the differential detection of Nipah virus (NiV) and highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV),a duplex Real-time RT-PCR was developed with specific primers and probes targeting to the special sequences of NiV M gene and HP-PRRSV nsp2 gene by optimization of reaction conditions.The performance of the assay was linear ranging from 4.6×10¹ to 4.6×10⁷ copies/μL for RNA standard control of NiV M (NiV-M-RNA) and from 4.1×10¹ to 4.1×10⁸ copies/μL for RNA standard control of HP-PRRSV nsp2 (HP-PRRSV-nsp2-RNA),and detection limits of the assay was 46 copies for the NiV-M-RNA and 4.1 copies for the HP-PRRSV-nsp2-RNA,respectively.The coefficients of variation (CVs) of both inter-assay and intra-assay repeatability were less than 2.0%,showing good repeatability.The assay was able to specifically detect NiV and HP-PRRSV simultaneously without cross-reaction with classical swine fever virus (CSFV),porcine epidemic diarrhea virus (PEDV),swine influenza virus (SIV),porcine parvovirus (PPV),pseudorabies virus (PRV) and porcine circovirus type 2 (PCV2).Of the 236 samples from pigs for both NiV and HP-PRRSV detection by the established assay,all the samples were negative for NiV,8 samples were HP-PRRSV positive.In conclusion,this assay offers a useful approach for the differential detection of NiV and HP-PRRSV in clinical specimens from the pigs.     

猪圆环病毒2型/3型双重荧光定量PCR检测方法

为了能够同时快速地检测和鉴别猪圆环病毒2型和3型(PCV2和PCV3),参考GenBank中已发表的PCV2和PCV3基因组序列,针对其保守区分别设计了2对特异性引物,经优化反应体系和条件,建立了能快速检测和鉴别PCV2/PCV3双重荧光定量PCR方法.结果 表明,PCV2和PCV3的R2分别为0.999、0.9993,E值分别为3.5731、3.3734.该方法能同时特异地检测PCV2和PCV3,而对其他5种猪病原均未检测到荧光信号;PCV2和PCV3的最低检测值分别为41.1 copies·μL-1、27.0 copies·μL-1;批内和批间变异系数均小于1％.临床样本检测结果显示,PCV2和PCV3的阳性率分别为62.12％(41/66)、48.48％(32/66),二者混合感染的阳性率为46.96％(31/66).表明该方法具有敏感、特异和可靠等特点,该方法为PCV2和PCV3单独或者混合感染的早期诊断、定量检测及其流行病学调查提供了可行的技术支持.     

复式潜流湿地净化生活污水的试验研究

研究了新型复式潜流人工湿地对生活污水的净化效果。在不同水力负荷、季节、曝气方式等条件下经过小试试验,分析了该湿地对污染物净化效果的影响。结果表明,该系统出水水质稳定,达到《城镇污水处理厂污染物排放标准》(GB 18918-2002)的一级A标准。在水力负荷184 mm·d^-1条件下COD、NH₃-N去除率最大分别可达87.2%、68.9%。冬季低温条件下对各类污染物去除率仍大于20%。正交试验分析得知,最佳运行条件是气温28.6℃、水力负荷0.184 m³·m^-2·d^-1、水力停留时间2.4 d。对比试验表明,采用预曝气方式对湿地净化效果明显优于厌氧处理。     

现代双腔分动化油器的设计趋势与设计方法

从分析国外先进的双腔分动化油器入手，分析了现代双腔分动化油器的设计趋势和设计方法。     

DOPPLER MEASUREMENT OF SPLANCHNIC BLOOD FLOW DURING DIGESTION IN UNSEDATED NORMAL DOGS

The purpose of this study was to measure splanchnic blood flow during digestion in unsedated dogs by using duplex Doppler sonography. The study population consisted of 12 healthy dogs. Blood flow in the cranial mesenteric artery, the celiac artery, and the aorta was measured before a test meal and at 20, 60, and 90 minutes after eating. The following measurements were made or calculated: vessel diameter, peak systolic velocity, end diastolic velocity, mean velocity, resistive index, pulsatility index, and flow volume. There was a significant postprandial decrease in the resistive and pulsatility indices in both the cranial mesenteric (preprandial RI = 0.867, postprandial RI = 0.796, preprandial PI = 3.033, postprandial PI = 2.173) and the celiac (preprandial RI = 0.854, postprandial RI = 0.769, preprandial PI = 2.639, postprandial PI = 1.930) arteries. In both vessels the end diastolic velocity, the mean velocity, and the flow volume increased significantly postprandially. These changes occurred significantly earlier in the celiac artery than in the cranial mesenteric artery. The findings most likely correspond to postprandial splanchnic vasodilation. Doppler ultrasound provide a good methode of detecting changes in postprandial splanchnic blood flow in the dog.     

一步法复合RT-PCR鉴别ND强毒与AI病毒的研究

根据新城疫病毒（NDV）融合蛋白和禽流感病毒（AIV）核蛋白的核酸序列设计2对引物,对NDV和AIV的特异扩增片段分别为156bp和219bp,建立了同时检测强毒力NDV和AIV核酸的一步法复合RT—PCR。试验中未能检出弱毒力NDV,IBV,IBDV,REV,REOV及SPF鸡肌肉组织的RNA,从不同年代分离的16个NDV强毒株全为阳性而NDV弱毒株全为阴性。经验证,复合引物与单一引物的扩增效率一致,能够检出NDV和H;亚型AIV的最低病毒量分别为10^3.7EID。和10^3.9EID50。试验结果表明,此次建立的一步法复合RT—PCR特异、敏感,适用于强毒力NDV和AIV．感染的快速鉴别。     

利用RAPD技术对碧桃种质资源的分析

利用RAPD技术,采用从200个随机引物中筛选的22个随机引物对8个碧桃类型的基因组DNA进行扩增,通过扩增的180个位点的谱带的聚类,分析供试碧桃的系统发育,并运用特殊谱带,建立了碧桃的分子检索表,并提出了重点保存的碧桃种质。     

猪伪狂犬病毒和猪细小病毒双重PCR方法的建立及初步应用

目的本研究旨在建立一种适用于临床样品和动物源性生物制品中猪伪狂犬病毒和猪细小病毒同时检测的双重PCR技术。方法针对猪伪狂犬病毒(PRV)的gE基因和猪细小病毒(PPV)的VP2基因的保守区域分别设计引物。结果经条件优化后,所建立的双重PCR方法能特异性地检测出样品中的PRV(581bp)和PPV(202bp)。结论本方法具有良好的特异性、敏感性和稳定性,适用于临床样品中对PRV和PPV的同时检测,也可用于猪源性生物制品的检测。     

ESTIMATION OF PORTAL BLOOD FLOW USING DUPLEX REAL-TIME AND PULSED DOPPLER ULTRASOUND IMAGING IN THE DOG

A duplex ultrasound system incorporating a pulsed wave Doppler ultrasound probe with conventional B-mode real-time imaging was used to evaluate portal vein blood flow in eight normal dogs. Adequate visualization of the cranial abdominal vessels was obtained from the right lateral 11th or 12th intercostal space. Doppler spectral analysis showed non-pulsatile flow with a wide range of linear flow velocities across the vessel lumen typical of laminar blood flow. Results for portal vein blood flows were 49.8 ± 13.5 ml/min/kg body weight (mean ± SD) with a range of 37.8 - 76.8 ml/min/kg body weight. These values overestimate portal blood flow by approximately 2 times when compared with published studies using invasive techniques. This overestimation is primarily due to the use of the maximal flow velocity in the blood flow calculations.