牡蛎疱疹病毒(OsHV-1)间接原位杂交PCR检测方法的建立与初步应用

为建立实现牡蛎疱疹病毒(OsHV-1)在贝类宿主组织中的精确定位方法,基于原位杂交PCR (ISPCR)技术建立了OsHV-1的间接原位杂交PCR (indirect ISPCR)检测方法,并利用该方法对OsHV-1在魁蚶主要器官的分布情况进行了检测和分析。首先选择适合原位杂交PCR的引物,在载玻片上实现对原位固定靶组织内病毒DNA的稳定、特异扩增,然后与使用相同引物制作的地高辛标记核酸探针进行原位杂交,最后通过免疫酶标技术显示样本内OsHV-1的组织分布。为了达到最佳检测效果,对间接ISPCR的扩增循环数以及组织消化的蛋白酶K浓度进行优化。结果显示,最适PCR扩增循环数为20,蛋白酶K浓度为20μg/mL。利用经优化的间接ISPCR检测方法,对OsHV-1在魁蚶外套膜、鳃、肝胰腺、斧足和闭壳肌5个样本中的组织分布情况和亲嗜性进行检测和分析。病毒阳性信号主要分布于魁蚶外套膜结缔组织中浸润的血细胞和成纤维细胞、肝胰腺和鳃浸润的血细胞、斧足和闭壳肌中坏死肌肉细胞的细胞核中。本研究建立的间接ISPCR检测方法具有灵敏、特异和精确定位的优点,通过组织切片上显示的病毒核酸阳性信号,能判别OsHV-1在不同组织部位的分布特点和受感染的细胞类型;适用于OsHV-1感染的确诊、病毒对不同组织器官的亲嗜性、病毒侵染途径和致病机理等相关研究。     

凡纳滨对虾商业苗种抗WSSV性能比较

白斑综合征病毒(WSSV)一直是甲壳类生物的高致病性病原。为了解市场上不同凡纳滨对虾(Litopenaeus vannamei)商业苗种病原携带情况及其抗WSSV性能,本研究收集了6个品牌的凡纳滨对虾商业苗种(分别简称为海南Z、海南S、广州P、广州Z、黄骅R和东营M),先进行包括WSSV在内的8种病原的检测,然后,采用单尾定量口饲感染方式进行抗WSSV性能测试,并比较各组苗种感染WSSV后的平均存活时间、存活率以及累积死亡率的差异。结果显示,6个商业苗种都不携带WSSV,部分苗种检测有潜在虾肝肠胞虫(EHP)和偷死野田村病毒(CMNV)。各苗种感染WSSV后,平均存活时间从长到短依次为海南Z、广州P、黄骅R、海南S、广州Z、东营M。东营M感染WSSV后第4天达到死亡高峰,而海南Z在第6~7天到达死亡高峰,比东营M晚了2~3d。感染实验结束后,海南Z和广州P存活率最高,同为72.5%,而东营M和黄骅R的存活率最低。本研究表明,海南Z和广州P抗WSSV性能最强,研究结果可为凡纳滨对虾抗病新品种的选育提供基础数据。     

Detection efficiency and habitat use to inform inventory and monitoring efforts: juvenile coho salmon in the Knik River basin,Alaska

Imperfect detection associated with sampling gear presents challenges for wildlife inventory and monitoring efforts. We examined occupancy dynamics and habitat use of juvenile coho salmon, Oncorhynchus kisutch, in shallow lake environments over a summer and early fall season in the Knik River area of south central Alaska using models which control for and estimate sampling gear detection efficiency. In addition, we present statements for the probability that observed absences at a survey site or from a survey area (a collection of sites) are true absences given some amount of sampling effort and analysts' beliefs about site occupancy and sampling gear detection efficiency which can be used to guide inventory and monitoring efforts for juvenile salmon or other wildlife and plant species. Occupancy modelling results demonstrate that minnow traps were effective at sampling juvenile coho in shallow lake environments, with a mean probability of detection across the study period of 0.68 (i.e., probability of detecting the presence of juvenile coho given that they are present at a trap site; SE = 0.03). Juvenile coho salmon migrated into shallow water lakes in late summer and early fall, presumably to seek out overwinter habitat. N‐mixture modelling examination of habitat use demonstrated that once in shallow lake environments, juvenile coho were widely distributed across a range of microhabitats, with some evidence for preference for shallower depths and warmer water temperatures.     

Enhanced detection of infectious salmon anaemia virus using a low‐speed centrifugation technique in three fish cell lines

Infectious salmon anaemia (ISA), caused by ISA virus (ISAV), is a serious disease of farmed Atlantic salmon, Salmo salar L. Recently, molecular‐ and immunofluorescent‐based techniques have become powerful diagnostic tools for ISAV detection, but culture‐based techniques remain the gold standard. A disadvantage of ISAV culture is that the incubation time required before cytopathic effect (CPE) is observed in cell monolayers. To decrease time until CPE is observed, a low‐speed centrifugation technique was applied to existing standard operating procedures for ISAV culture in three fish cell lines. Time until CPE observation was compared in CHSE, SHK and ASK cells, treated or not treated with low‐speed centrifugation after inoculation with ISAV. Low‐speed centrifugation treatment significantly enhanced observable cell infection. Compared to control cells, the length of time until ISAV CPE observation decreased in centrifuged ASK and CHSE cells. Low‐speed centrifugation was also incorporated into a modified clinical shell vial assay. At 48 h post‐inoculation with approximately 20 viral particles, ISAV was detected by an immunofluorescence antibody test in treated ASK and SHK1 cells but not in control cells. Finally, this enhanced viral adsorption assay performed in ASK cells demonstrated higher sensitivity than a real‐time RT‐PCR assay performed on RNA isolated from ISAV‐spiked salmon kidney homogenates.     

Study of Photo and Thermoluminescence Properties to Identify Irradiated Dried-Fishery Products

ABSTRACT

Radiation-specific luminescence properties in irradiated dried-fishery products (pollack, little pollack, clams, and shrimp) were investigated at different dose levels (0–10 kGy). Photostimulated luminescence (PSL) analysis of whole samples was effective for dried clams and shrimp, while low PSL sensitivity was observed in dried pollack and little pollack. Thermoluminescence (TL) analysis was conducted after isolation of silicate minerals, in which the two methods of mineral isolation—density separation and acid hydrolysis—were compared. Irradiated samples were easily distinguishable through the strong TL glow curves, with maximum peaks in temperature range of 150–250°C. TL ratios (TL₁/TL₂) were < 0.1 for nonirradiated samples, while > 0.1 for irradiated. There was a clear effect of applied irradiation dose, with a negligible effect of the method used for mineral separation. The mineral composition showed that feldspar and quartz minerals were mainly responsible for the well-characterized luminescence behavior upon irradiation.     

斑节对虾白斑综合症病毒部分基因组文库及核酸探针检测法

通过分离纯化白斑综合症病毒（ＷＳＳＶ）粒子,抽提病毒ＤＮＡ。用限制性内切酶ＥｃｏＲⅠ或ＳａｌⅠ酶切后,克隆入质粒ｐＢｌｕｅｓｃｒｉｐｔⅡＫＳ中,从而建立了ＷＳＳＶ部分基因组文库。估计ＷＳＳＶ基因组ＤＮＡ在１６５ｋｂ以上。将ＷＳＳＶ ＥｃｏＲⅠ克隆片段标记制备为探针。进行Ｓｏｕｔｈｅｒｎ杂交、打点杂交和原位杂交,其结果证明了克隆片段对ＷＳＳＶ特异,并为检测ＷＳＳＶ提供了方法。通过对部分基因组文库序列     

基于固定样地的林分生长模型研建

基于杭州市开展的森林资源与生态状况固定样地监测,以建德市为例,建立了无人为干扰小班主要测树因子更新模型,更新了县级森林资源小班数据库,实现了县市森林资源联动监测。     

Intelligence control of on-line dynamic gray cloth inspecting machine system module design. II. Defects inspecting module design

This study aimed to establish a set of gray cloth defect inspection module using image processing technique, so as to develop a full intelligent online dynamic gray cloth defect automatic inspection system. Gray cloth defects to be recognized in this study included holes, stains, warp missing, spider web and weft missing. First use wavelet transform and co-occurrence matrix to find features of gray cloth defect image, next, use back-propagation neural network (BPNN) to make gray cloth defect classification and data output. BPNN was capable of solving nonlinear problems, thus assisted in enhancing defect recognition effect. As every defect to be inspected in this study varied in size and shape, so advantage of BPNN could be used as aid more than else. This study primarily utilized image processing technique to inspect gray cloth defects, not only in a faster speed than common visual inspection, but also eliminating arbitrary factors of inspectors in body and psychology during inspection, resulting in absolute objectivity. Finally, tension control module built in Part 1 and gray cloth defect inspection module built in this study were integrated, and a full intelligent online dynamic gray cloth defect automatic inspection system established. As validated by experiment result, the system established in this study could successfully recognize gray cloth defects, with total recognition rate amounting to 92.5 %.     

食品营养与检测课程学生学习能动性提升研究

如何提高学生的学习能动性一直是高校教师关注的课题。分析食品营养与检测课程教学中提升学生学习能动性的必要性和可行性,探讨教师课堂用语、多媒体课件、互动式教学、探究式教学等方式对于激发学生学习能动性的重要作用。