Establishment and Preliminary Application of Nested PCR Method for Detection of Porcine Proliferative Enteropathy

Porcine proliferative enteropathy (PPE), caused by Lawsonia intraeellularis (LI), was a kind of swine contagious infectious disease. In order to establish a nested PCR method to detect LI, two pairs of primers were designed according to the genome of LI, and the nested PCR was established for detection of LI in ileum or feces of pigs. The sequencing result analysis showed that the homologies of the amplified nucleotide sequences to the LI references sequence were above 99%. The nested PCR method established in this study was rapid, easy and sensitive, could be used in clinical diagnosis of PPE.     

应用巢式RT-PCR检测水仙退化病毒1

水仙退化病毒（Narcissus degeneration virus，NDV）是水仙上发生较为严重的病毒之一。本文根据 NDV 已报道的外壳蛋白基因序列，设计特异性引物，以感病水仙的总 RNA 为模板，建立了快速检测 NDV 的巢式 RT-PCR 方法。结果表明，巢式 RT-PCR 具有良好的特异性和灵敏度，能够从感染 NDV 的水仙样品上扩增出与预期大小相符的特异性目的条带，同时与其他病毒无交叉反应；灵敏度测定结果表示，巢式 RT-PCR 灵敏度较高，是普通 RT-PCR 的104倍，当 RNA 稀释到109倍时仍能被检测到。本文建立的巢式 RT-PCR 为水仙上 NDV 的快速检测提供了技术参考依据。     

A nested‐PCR method for rapid detection of Sclerotinia sclerotiorum on petals of oilseed rape (Brassica napus)

This study established a quick and accurate method to detect petal infection of oilseed rape (Brassica napus) by Sclerotinia sclerotiorum using a nested‐PCR technique. DNA samples were extracted from each petal using a microwave method, followed by two rounds of PCR amplification. The first‐round PCR amplification was performed using the universal fungal primer pair ITS4/ITS5, and the second‐round amplification with a specific primer pair XJJ21/XJJ222, which was designed using the single‐nucleotide polymorphisms among nuclear rDNA ITS sequences of Sclerotinia spp., Botrytis spp. and other selected fungi. The established technique is rapid and inexpensive, and has a high degree of specificity and sensitivity. This assay can distinguish Sclerotinia spp. from other fungi, including Botrytis cinerea, a closely related and frequent cohabitant on oilseed rape petals, and can detect 50 fg genomic DNA, five ascospores of S. sclerotiorumin vitro or 50 ascospores of S. sclerotiorum on one petal in approximately 6 h, even in the presence of a high background of oilseed rape DNA. This technique was successfully applied in detecting natural petal infections.     

国外引进甘蔗材料白叶病植原体巢式PCR检测及其序列分析

为有效防止引起重要危险性检疫病害甘蔗白叶病(SCWL)的植原体病原随引进甘蔗材料侵入我国,确保我国甘蔗生产安全,本研究对从缅甸、菲律宾、法国和泰国引进的22个甘蔗材料进行了SCWL植原体巢式PCR检测,并对阳性样品的巢式PCR产物进行了测序及序列分析。结果表明,18个甘蔗材料呈SCWL植原体阳性,阳性检出率为81.8%。所有SCWL阳性样品的16S-23S基因间隔区片段长210bp,与GenBank中已有的其他SCWL植原体分离物(登录号HQ917068、AB646271)的同源性为99.8%~100%,并在系统发育树中聚为一个类群。根据SCWL的巢氏PCR检测及其序列分析结果,对呈阳性的材料及时进行了集中销毁处理。     

砂田抑制蒸发功能随覆砂年限的演变规律

【目的】研究压砂覆盖(砂田)保温及抑制蒸发功能随砂田退化程度的演变规律。【方法】基于多年Landsat卫星数据,使用辐射传输方程法反演香山地区砂田地表温度(LST),结合田间地表温度监测,对比砂田与裸地的地表温度变化,分析了砂田抑制土壤蒸发的机理,并探讨了砂田的功效与砂田使用年限的关系。【结果】砂田在LST-NDVI梯形空间中贴近于暖边,其土壤水分比耕地少,接近于干土层。因此,砂田可以隔离辐射与土壤表层,从而减小潜热通量,抑制土壤蒸发。砂田昼夜温差明显比裸地大,且对于西瓜等作物,砂田的有效地表积温也比裸地提高了10%。【结论】砂田退化过程可分为纯砾石阶段、砂土混合阶段和砂土连通阶段,从砂田的保温及抑制蒸发功能来看,砂田的有效使用年限为25～30 a。     

谷子锈菌巢式PCR检测体系的建立

<正>由粟单胞锈菌(Uromyces setariae-italicae)引起的谷子锈病是一种危害严重的真菌性病害,该病可在短期内大面积流行,通常导致减产10%~30%。谷子锈病病原菌U.setariae-italicae,属担子菌亚门(Basidiomycotina)、柄锈菌科(Pucciniaceae)、单胞锈菌属(Uromyces),是一种多孢型转     

Excel应用于方差分析的实训教学探究

针对国内应用型本科湖南文理学院院校概率统计课程的实训教学越来越多地注重Excel作为初学者操作平台的现状,就非平衡数据和嵌套结构的数据在Excel中实现方差分析的过程进行探究。突出以组内矫正数为主线整理数据后简化平方和分解公式的理念,使得Excel中进行方差分析时,数据整理流程完全格式化,平方和的分解步骤更为简洁,自由度分解、F检验等步骤不再繁琐,为非统计专业本科阶段借助Excel电子表格率先实现概率统计课程实训教学的宽口径模式提供借鉴。     

奶牛隐孢子虫Nested PCR—RFLP方法的建立及初步应用

为快速检测并准确鉴别奶牛隐孢子虫种,以隐孢子虫18S rRNA基因的特殊区域为基础,设计内、外引物,并根据软件分析确定相应的内切酶EcoT141,采用Nested PCR-RFLP方法进行虫种种型鉴别分析。在Nested PCR两次PCR反应中,以微小隐孢子虫（Cryptos poridium parvum,C．p）和安氏隐孢子虫（Cryptosporidium andersoni,C．an）卵囊提取的DNA为模板,均能扩增出长约800bp和500bp的明亮条带,且特异性强,其他虫种不能扩增出条带,该方法最低可检测到5个卵囊／g粪便;对于由内引物扩增出的500bp的条带,C.an的PCR产物能被内切酶EcoT141酶切,酶切后的片段分别为416bp和92bp,C.p的PCR产物不能被此酶酶切。用所建立的Nested PCR-RFLP法对上海奶牛389头和进口奶牛200头的共计589份粪样进行检测,Nested PCR的结果表明上海奶牛和进口奶牛的隐孢子虫阳性率分别为19．02％和3．5％,RFLP的结果表明上海奶牛感染的主要是Cp和C．an,进口奶牛感染的主要是C．p。研究结果表明,本研究建立的检测奶牛粪便中的隐孢子虫的NestedPCR-RFLP法,可用于奶牛隐孢子虫流行病学调查并有效鉴别奶牛隐孢子虫种。