非洲猪瘟病毒的快速检测方法研究进展

非洲猪瘟是由非洲猪瘟病毒感染引起的一种高度接触性传染病,对养猪业危害严重。世界动物卫生组织将其列为法定呈报的动物疫病,我国将其列为一类动物传染病。由于此病目前无有效疫苗进行预防,为防止疫病的传播,可靠、快速的早期诊断对于实施严格的卫生和生物安全措施必不可少。本文就非洲猪瘟的病原学检测和血清学抗体检测技术及其应用进行了综述,以期为非洲猪瘟的快速诊断提供技术参考。     

A visual on-site method for African swine fever virus detection in raw pig tissues

The spread of African swine fever virus (ASFV) leads to high mortality in domestic pigs, and has caused huge losses in the global pork industry. Therefore, the rapid detection of ASFV is essential for the prevention and control of the epidemic. Herein, a denaturation bubble-mediated strand exchange amplification (SEA) with colorimetric detection strategy for the rapid detection of ASFV was established and the results were visible with the naked eyes. The entire detection process based on isothermal amplification was performed on a portable metal bath only. And the whole process including rapid extraction and visual detection was finished within 50 min. The limit of detection of this method reached to 10⁴ copies/μl of plasmid containing p72 gene. And the ASFV was successfully detected in eight different tissue and seven types of pig feed which revealed a strong anti-interference ability in the presence of complex matrix. Through comparing 94 field positive and negative ASFV samples, excellent performance of SEA assay with 92.86% sensitivity and 100% specificity relative to PCR was observed. The developed method could be used for on-site detection of ASFV with simplicity, rapidity, and visuality, especially suitable for pork industry food safety and ASFV control in resource-limited areas.     

低盐腊肉在加工过程中的菌相变化初探

为了解低盐腊肉在加工过程中的微生物变化,采用选择性培养基对低盐腊肉加工过程中的主要微生物菌相进行分离和计数。结果发现,在低盐腊肉中的微生物总数为2.58×103cfu/g,优势菌群是葡萄球菌和微球菌,它们的数量较之其它微生物的数量高了一个数量级。     

非洲猪瘟病毒核酸荧光定量PCR检测方法研究

目的 建立非洲猪瘟病毒核酸的荧光定量PCR检测方法, 并与微滴式数字PCR检测方法的优缺点进行比较。方法 根据非洲猪瘟病毒保守基因VP72和K205设计合成引物和探针, 探究荧光定量PCR检测方法的反应条件和方法的特异性、线性、灵敏性和重复性, 及其与微滴式数字PCR检测方法灵敏度差异。结果 本研究建立的非洲猪瘟病毒核酸荧光定量PCR检测方法检出限达到0.1 fg/反应, 在0.1~500.0 fg/反应范围内呈现良好的线性关系, 检测重复性变异系数均低于1%, 不与猪瘟病毒、猪蓝耳病毒发生交叉反应。结论 本研究建立的荧光定量PCR法灵敏度高、特异性强, 适用于猪肉及其制品中非洲猪瘟病毒的检测。     

锌铜法加工无铅皮蛋技术

以新鲜鸭蛋为试材,采用浸泡法工艺,研究NaOH浓度及金属盐种类与用量对皮蛋加工品质的影响,探讨锌法、铜法和锌铜混合法加工皮蛋的可行性.结果表明:单独使用Zn<,2+>、Cu<,2+>,时,浸液NaOH浓度对皮蛋加工具有重大影响,其中最适浓度为4.5%;锌法和铜法都能加工成各具特点的合格皮蛋,锌铜盐混合能产生协同效应,一定程度缩短加工时间,且加工品质优良;选用腌制液 NaOH 浓度4.5%,Zn<'2+>添加量0.3%,Cu<'2+>添加量0.05%～0.075%腌制25 d左右出缸,皮蛋具有较好品质.     

猪戊型肝炎病毒检测技术的研究进展

猪戊型肝炎是一种新的人畜共患病,其病原戊型肝炎病毒(hepatitis E virus,HEV)是无囊膜的RNA病毒,主要通过消化道进行传播。研究发现,世界上许多国家的猪群中普遍存在HEV抗体及猪戊型肝炎病毒RNA携带。与患病猪群密切接触、食用未煮熟的猪肉或猪肉制品、被污染的水源及水产品,这些因素都可增加戊型肝炎的患病风险。猪作为戊型肝炎病毒的宿主,不仅对公共卫生和食品安全构成威胁,同时对人类戊型肝炎流行病学有很大的影响。本文简要介绍了HEV的病原学以及抗原抗体检测方法,包括血清学、免疫学及分子生物学方法等,并对国内外开展的戊型肝炎检测技术的研究进展进行综述。     

复合亚氯酸钠灭活烟草花叶病毒的机理分析

为揭示复合亚氯酸钠（500 mg/L）杀灭烟草花叶病毒（Tobacco mosaic virus,TMV）的机理,采用荧光定量RT-PCR检测药剂处理后的病毒CP基因和Rep基因表达量,并利用间接酶联免疫吸附法测定了TMV CP的破坏程度;同时接种心叶烟,通过枯斑数统计分析了TMV的侵染活性;利用大片段步移RT-PCR检测TMV相关基因片段的损伤,并使用透射电镜观察药剂处理后病毒粒子被破坏的情况。结果显示：(1)药剂处理30 min可以完全抑制TMV CP基因的表达,处理60 min可完全抑制TMV Rep基因的表达。此时,TMV CP被完全破坏,病毒也完全丧失了对枯斑寄主心叶烟的侵染能力。(2)药剂处理30 min时,TMV的183kDa-1、183kDa-2、183kDa-3、183kDa-4、54kDa、CP&MP基因编码区等6个区域中的183kDa-2、183kDa-4和CP&MP区域首先被损伤,但接种叶叶脉和接种叶叶柄上的这3个区域可以正常扩增。处理45 min时,接种叶叶脉、接种叶叶柄和接种叶上部叶片的183kDa-1、183kDa-3和54kDa区域可以正...     

Inactivation of avian influenza virus by heat and high hydrostatic pressure

Avian influenza viruses threaten the life of domestic terrestrial poultry and contaminate poultry meat and eggs. Recently, these viruses rarely infected humans but had a high mortality rate in Southeast Asia, the Middle East, and Egypt. Thereby, these viruses caused high economic costs for production of poultry and health protection. We inactivated a highly pathogenic avian influenza A virus of subtype H7N7 in cell culture medium and chicken meat by heat and high hydrostatic pressure. Because heat and pressure inactivation curves of the H7N7 virus showed deviations from first-order kinetics, a reaction order of 1.1 had to be selected. A mathematical inactivation model has been developed that is valid between 10 and 60 degrees C and up to 500 MPa, allowing the prediction of the reduction in virus titer in response to pressure, temperature, and treatment time. Incubation at 63 degrees C for 2 min and 500 MPa at 15 degrees C for 15 s inactivated more than 10(5) PFU/ml, respectively. Thus, we suggest high-pressure treatment of poultry and its products to avoid the possible health threat by highly pathogenic avian influenza viruses.     

分段碱调“无金属添加”水晶鸡皮蛋的控制技术

以鲜鸡蛋为原料腌制鸡蛋皮蛋,使用不同阶段改变料液中碱的浓度的控制技术,实现全过程“无金属添加”,并分析腌制过程中料液碱浓度、鸡皮蛋流变性、色差、质构特性等随着时间的变化,优化鸡皮蛋加工工艺,达到鸡皮蛋外观色浅透明度高的感官效果。结果表明:料液碱浓度越高,相对蛋清碱浓度变化越大,第一阶段料液碱浓度为3%、4%、5%时鸡皮蛋结构凝胶速度最快;鸡皮蛋颜色变化、质构特性与料液碱浓度关系显著,白度值在料液碱浓度4%更换为1%后腌制30 d时达到峰值,感官接受度最好,碱浓度对鸡皮蛋硬度影响较大,内聚性、粘性等无明显差异。结论:无金属添加水晶鸡皮蛋腌制最佳工艺为:鸡蛋以浓度为4%的NaOH溶液腌制14 d后,更换料液浓度为1%的NaOH溶液再次腌制25 d。     

浅谈非洲猪瘟的流行现状及防控措施

非洲猪瘟(ASF)是由非洲猪瘟病毒引起的一种急性烈性高接触性传染病,其发病过程从急性、亚急性、慢性发病不等,其临诊特征是高热、皮肤发绀,各内脏器官出血,同时伴有呼吸障碍和神经症状,发病率和死亡率几乎可高达100%。该病在临床上与其他败血性、出血性疾病极其类似其死亡的几率都相对较高;非洲猪瘟的出现严重的影响了生猪行业的发展,已经对养猪业产生极大的危害,从饲料加工、生猪养殖、生猪屠宰、肉品零售到肉制品加工全产业链均受到了严重的影响。简要的分析了非洲猪瘟发生的起源、流传以及在国内的现状,剖析了应该采取何种手段进行防控,来抑制住非洲猪瘟的扩散。     

Effect of curing salt and phosphate on the activity of porcine muscle proteases

The effect of curing salt on the activity of porcine muscle proteases was evaluated, within the salt concentration range found in the manufacturing process of Spanish cured ham. Salt (NaCl) acts as a strong inhibitor of proteolytic activity; sodium nitrate and potassium nitrate do not affect cathepsin D activity; cathepsin L is inhibited at levels of the salts not found in cured ham, and Ca-dependent proteolytic activity is enhanced by nitrate concentrations below 800 ppm.

The appearance of phosphate precipitates in several areas of ham has led to the study of phosphate effect on enzymatic activity. Results show that phosphate is an inhibitor of proteolytic activity.     

Inactivation of glucocorticoids by 11beta-hydroxysteroid dehydrogenase enzymes increases during the meiotic maturation of porcine oocytes

Recent reports have shown that glucocorticoids can modulate oocyte maturation in both teleost fish and mammals. Within potential target cells, the actions of physiological glucocorticoids are modulated by 11beta-hydroxysteroid dehydrogenase (HSD11B) isoenzymes that catalyse the interconversion of cortisol and cortisone. Hence, the objective of this study was to establish whether HSD11B enzymes mediate cortisol-cortisone metabolism in porcine oocytes and, if so, whether the rate of glucocorticoid metabolism changes during oocyte maturation. Enzyme activities were measured in cumulus-oocyte complexes (COCs) and denuded oocytes (DOs) using radiometric conversion assays. While COCs and DOs oxidised cortisol to inert cortisone, there was no detectable regeneration of cortisol from cortisone. The rate of cortisol oxidation was higher in expanded COCs than in compact COCs containing germinal vesicle (GV) stage oocytes (111+/-6 vs 2041+/-115 fmol cortisone/oocyte.24 h; P<0.001). Likewise, HSD11B activities were 17+/-1 fold higher in DOs from expanded COCs than in those from compact COCs (P<0.001). When GV stage oocytes were subject to a 48 h in vitro maturation protocol, the enzyme activities were significantly increased from 146+/-18 to 1857+/-276 fmol cortisone/oocyte.24 h in GV versus MII stage oocytes respectively (P<0.001). Cortisol metabolism was inhibited by established pharmacological inhibitors of HSD11B (glycyrrhetinic acid and carbenoxolone), and by porcine follicular and ovarian cyst fluid. We conclude that an HSD11B enzyme (or enzymes) functions within porcine oocytes to oxidise cortisol, and that this enzymatic inactivation of cortisol increases during oocyte maturation.     

盐溶性猪肝蛋白提取及功能特性和结构分析

目的：优化盐溶性猪肝蛋白（salt soluble liver proteins,SSLP）的制备工艺,提高猪肝副产物利用率。方法：以提取率为指标,分析NaCl浓度、pH、提取时间、固液比对SSLP提取率的影响,通过正交旋转优化试验确定最佳制备工艺。以水溶性猪肝蛋白（water soluble liver proteins,WSLP）为对照,对SSLP的功能特性（溶解度、起泡性、泡沫稳定性、乳化性、乳化稳定性等）进行对比分析,并通过傅里叶红外光谱,低场核磁共振和差式扫描量热法分析其结构变化。结果：SSLP最佳制备工艺为NaCl浓度0.4 mol/L,pH 8,提取时间12.95 h,料液比（m_猪肝∶V_缓冲液）1∶4.6 （g/mL）,提取率为10.60%。FT-IR分析结果显示SSLP特征官能团吸收峰明显,二级结构中β-转角相对含量39.91%,α-螺旋相对含量13.55%,β-折叠相对含量21.32%,无规卷曲相对含量25.23%;LF-NMR分析结果表明SSLP结合水高,保水性更高;DSC结果表明SSLP的变性温度为78.68 ℃。结论：在最佳工艺条件下制备的SSLP的溶解度、起泡性、乳化性、乳化稳定性均优于WSLP,是开发猪肝蛋白产品的优质来源。     

Inactivation of hepatitis A virus and a calicivirus by high hydrostatic pressure

Potential application of high hydrostatic pressure processing (HPP) as a method for virus inactivation was evaluated. A 7-log10 PFU/ml hepatitis A virus (HAV) stock, in tissue culture medium, was reduced to nondetectable levels after exposure to more than 450 MPa of pressure for 5 min. Titers of HAV were reduced in a time- and pressure-dependent manner between 300 and 450 MPa. In contrast, poliovirus titer was unaffected by a 5-min treatment at 600 MPa. Dilution of HAV in seawater increased the pressure resistance of HAV, suggesting a protective effect of salts on virus inactivation. RNase protection experiments indicated that viral capsids may remain intact during pressure treatment, suggesting that inactivation was due to subtle alterations of viral capsid proteins. A 7-log10 tissue culture infectious dose for 50% of the cultures per ml of feline calicivirus, a Norwalk virus surrogate, was completely inactivated after 5-min treatments with 275 MPa or more. These data show that HAV and a Norwalk virus surrogate can be inactivated by HPP and suggest that HPP may be capable of rendering potentially contaminated raw shellfish free of infectious viruses.     

口蹄疫、水泡性口炎和猪水泡病多重荧光定量RT-PCR检测方法的建立

目的建立同时检测口蹄疫病毒、水泡性口炎病毒和猪水泡病病毒的多重荧光RT-PCR检测方法。方法根据口蹄疫病毒3D蛋白编码基因、水泡性口炎病毒N蛋白编码基因和猪水泡病病毒VP1蛋白编码基因的高保守区设计特异性引物和探针,对3种动物病毒进行多重荧光定量RT-PCR扩增。结果经过扩增,可以同时检测口蹄疫病毒、水泡性口炎病毒和猪水泡病病毒,而其他参试病原均无扩增信号,显示其良好的特异性。对口蹄疫病毒、水泡性口炎病毒、猪水泡病病毒的最低检测限分别达到101、102、102个质粒拷贝浓度。结论本方法灵敏度高,特异性良好,可实现多种病毒混合感染的同时检测。     

Presence of hepatitis E virus in a naturally infected swine herd from nursery to slaughter

Hepatitis E virus has recently been recognized as having zoonotic potential and could be transmitted from pig to human. Pigs are identified as a potential animal reservoir and HEV is highly prevalent in the swine population around the world. In this study, the presence of HEV was investigated in 51 subjects reared on a simulated commercial farm setting from the age of 2 weeks up to slaughter. Samples were collected on four occasions: at 2, 8, and 18 weeks and between 22-29 weeks of age. Anti-HEV IgG in plasma samples, presence of HEV RNA in plasma samples and feces were monitored. At 2 weeks of age, HEV RNA was detected in feces of 6 subjects (11.8%) but not in their plasma. At 8 weeks, HEV was detected in feces of 27 subjects (52.9%) and in plasma of one subject. At 18 weeks, HEV was detected in feces of 44 subjects (86.2%) and in plasma of 24 subjects (47.1%). At slaughter time (22-29 weeks), HEV was present in plasma of 6 subjects (11.8%) and in stools of 21 subjects (41.2%). Spread of the virus inside the population was evaluated by comparison of means (paired t-test, P<0.05) of anti-HEV IgG ELISA results from the 4 bleedings. Significant differences were noted between the results of populations at 8 and 18 weeks and also between 18 and 22 to 29 weeks indicating an immune response to the virus. Based on the comparison of a 304 nucleotides sequence of the 5' ORF 2 gene, all amplified fragments clustered in genotype 3a.     

Inactivation of foot-and-mouth disease virus in various bovine tissues used for the production of natural sausage casings

Bovine intestines, bladders and oesophagus are used for the production of natural casings ("beef casings") as edible sausage containers. Derived from cattle experimentally infected with FMDV (initial dosage 10(4) TCID(50)/mL, strain A Iran 97), these beef casings were treated with sodium chloride (NaCl) or phosphate supplemented salt (P-salt). In addition, different in-vitro experiments using beef casings were done on a small scale with other FMDV strains (A Turkey 06, C-Oberbayern and O(1) Manisa) as "proof of principle". Based on the combined results of the in-vivo and in-vitro experiments, it can be concluded that the storage period of 30 days at 20 °C in NaCl is sufficiently effective to inactivate a possible contamination with FMDV in beef casings and that the usage of P-salt does not clearly enhance the inactivation of FMDV infectivity. Storage of salted beef casings at about 20 °C for 30 days is already part of the Standard Operating Procedures (included in HACCP) of the international casing industry and can therefore be considered as a protective measure for the international trade in natural casings.     

Chemistry of swine liver and products of liver processing. 3. The Maillard reaction of a model and of preserved liverwurst

The storage life of industrial-made tinned liver sausage has more effect on the decrease in glucose and total carbohydrate content than the corresponding sterilisation program. The addition of glucose and casein shows greater effect as respects the Maillard-reaction on tinned model than on minced liver.     

低钠盐对皮蛋的钠钾含量、硬度及蛋白质二级结构的影响

以新鲜鸭蛋为原料,采用清料法腌制皮蛋。探讨低钠盐替代食盐对皮蛋的钠钾含量、凝胶的硬度、蛋清蛋白质二级结构、感官品质等的影响,旨在为降低皮蛋中的钠含量提供新的思路,为加工低钠皮蛋提供新的方向。结果表明,100%低钠盐组比食盐组蛋清钠含量降低了21%,蛋黄钠含量降低了30%。不同比例的低钠盐替代组与食盐组对比,皮蛋凝胶的硬度、蛋清蛋白质二级结构的含量均无显著性差异(p>0.05)。70%低钠盐替代食盐制得的皮蛋,感官品质良好。