Animal-derived food safety incidents caused by zoonotic diseases occur frequently. In order to control and ensure the safety of pork consumption from its raw material source and to control the cost of pork and its products consumption, this study constructed a triple real-time fluorescent quantitative PCR (qPCR) method that can simultaneously detect hepatitis E virus (HEV), porcine epidemic diarrhea virus (PEDV), and porcine δ corona virus (PDCoV) in pork and its products. The results showed that the triple qPCR method could only amplify the specific gene fragments of three target viruses, which had high specificity. The minimum detection limits of the three viruses (HEV, PEDV, and PDCoV) were 6.02, 6.98 and 6.92 copies/μL respectively. The coefficient of variation (CV%) within and between groups ranged from 0.10% to 3.0%, with good repeatability. The established method was applied to detect 248 samples of pork products and exports and 282 raw pig manure swabs, and parallel detection was also conducted using corresponding virus detection methods for comparison. The detection rates of porcine epidemic diarrhea virus, porcine deltacoronavirus, and hepatitis E virus in pork products and exports were all 0%, which was consistent with the results of the corresponding virus detection methods. The detection rates of the three viruses (PEDV, PDCoV, and HEV) using this method were 1.06%, 3.19% and 0.35%, respectively. The detection rates of the standard method for these three viruses were 1.06%, 3.19% and 0%, respectively. The study showed that the established triplex qPCR detection method can accurately and rapidly detect three viruses in pork and its products or pig samples, providing technical support for ensuring the market circulation of fresh pork and its products and blocking the transmission of viral foodborne diseases.

Hepatitis E virus(HEV)is a universal foodborne pathogen that infects swine,deer,rabbits,camels,and other animals.Direct contact with infected pigs and other animals or consumption of contaminated food(uncooked or undercooked)poses a risk of HEV infection in humans.This article summarizes the etiology and epidemiology of HEV,its prevalence in various animal species,and the contamination of meat and meat products,the aquatic environment,seafood,vegetables,and other crops.Therefore,people can better understand the risk of foodborne HEV transmission and provide a scientific basis for preventing and controlling its prevalence.

随着我国养猪业的发展和进步,对于疾病的鉴别诊断和大数据分析提出了更高的要求和目标.为建立一种同时检测7种猪(Sus scrofa)繁殖障碍性疫病的方法,促进高通量检测技术在动物病原检测中的发展和应用,本研究针对猪伪狂犬病毒(Pseudorabies virus,PRV)、猪日本乙型脑炎病毒(Japanese encephalitis virus,JEV)、猪瘟病毒(Classical swine fever virus,CSFV)、猪圆环病毒2型(Porcine circovirus type 2,PCV-2)、猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)、猪细小病毒(Porcine parvovirus,PPV)、非洲猪瘟病毒(African classical swine fever virus,ASFV)7种猪繁殖障碍性疫病病原,设计特异性引物、探针,经反应条件优化、探针与微球偶联和杂交捕获反应,成功建立1种同时检测7种猪疫病的液相芯片检测方法,并将该方法进行临床应用.结果显示,微球与探针偶联效率较高,在液相环境中能实现对特异性目的基因捕获检测,该方法检测灵敏性较高,可同时检测到7种特异性病原的最低检测限度为103 copies/μL;该方法检测特异性好,与猪其他常见病原无交叉反应,重复性试验中组内和组间检测数据一致性较强.利用建立的方法对临床采集四川规模化猪场疑似发病的65份样品和ASFV阳性对照核酸进行检测和验证,共检出49份阳性样品,阳性率达75.38％(49/65),混合感染检出阳性率为23.08％(15/65),经单重PCR复检后结果一致,证实建立的液相芯片检测方法可应用于临床检测.本研究为猪病多种病原快速鉴别诊断提供有力的技术储备,同时为高通量检测技术的进一步发展提供参考.

犬冠状病毒在世界范围内多有流行,其潜伏期1～8d.本病传染迅速,数日内可蔓延全群.且随着日龄的增长,死亡率降低,是威胁犬类健康的重大传染性疾病之一.本文探讨犬冠状病毒的流行病学、临床症状、诊断及有效防治.旨在为犬冠状病毒的综合防治提供一些参考思路.

新型冠状病毒在全球范围内大流行,冷链生鲜食品及外包装作为新型冠状病毒潜在远程传播载体,增加了病毒通过"物传人"的感染风险.非热杀菌技术是一类新型杀菌技术,无需热能消耗即可杀灭食品中有害或致病微生物,避免了传统热杀菌技术传热相对较慢和对杀菌对象产生热损伤等缺点,将该技术应用于生鲜食品中不仅能有效阻断病毒传播,还能在食品保鲜和延长货架期方面发挥积极作用.本文主要介绍了适用于冷链环节中生鲜食品表面及外包装的非热杀菌技术,包括化学消毒剂、紫外线辐射、臭氧消毒、低温等离子体等,并从不同非热杀菌技术的工作机制、对病原体的灭活作用、对冷链过程中生鲜食品的保鲜效果进行阐述,以期为非热杀菌技术在冷链生鲜食品中的杀菌消毒应用提供理论指导,为保障冷链生鲜食品安全提供一定参考.

宠物(猫、犬)作为人类的伴侣动物,正突飞猛进的增长,越来越多的进入社会家庭,尤其老人、单身和丁克家庭.新冠病毒传染性极强,猫、犬可带毒但不表现症状,病人或无症状阳性人员因密接宠物,有可能传染给猫、犬,通过猫、犬的活动又污染环境或可传染给其他人,对宠物新冠抗原的检测是有效防控新冠疫情的重要举措,对公共卫生安全具有重要意义.本试验采用红色乳胶微球标记新型冠状病毒N蛋白单抗,建立宠物新冠病毒抗原红乳胶免疫试剂盒.试验结果表明该试剂盒的灵敏度高(150 pg/mL),特异性强(与其他19病毒或病菌抗原无交叉反应),操作简便,快捷,15 min出结果,具有良好的稳定性和重复性,可用于宠物(猫、犬)新冠病毒抗原的快速检查.

Objective To establish a rapid detection method for multiple recombinant enzyme polymerase amplification(RPA)of African swine fever virus(ASFV),Porcine Delta coronavirus(PDCoV),and Seneca virus type A(SVA).Methods RPA detection was carried out using recombinant plasmid as a template,and reaction conditions such as temperature,time,and primer probe ratio were optimized.Multiple RPA detection methods were established,which were applied to the detection of actual samples,and the accuracy of the established method was evaluated by comparing with the results of real-time fluorescence polymerase chain reaction(qPCR).Results Simultaneous detection of the three viruses can be achieved within 20 min with good specificity.Sensitivity to ASFV,PDCoV,and SVA was 940,770,and 570 copies/μL,respectively.The accuracy rates for actual sample detection were 93.33％,100.00％,and 100.00％,respectively.Conclusion The established multiple RPA method is fast and convenient,and it is suitable for on-site preliminary screening of ASFV,PDCoV,and SVA in pork and pork products.

DNA methylation modification can regulate gene expression without changing the genome sequence, which helps organisms to rapidly adapt to new environments. However, few studies have been reported in non-model mammals. Giant panda (Ailuropoda melanoleuca) is a flagship species for global biodiversity conservation. Wildness and reintroduction of giant pandas are the important content of giant pandas’ protection. However, it is unclear how wildness training affects the epigenetics of giant pandas, and we lack the means to assess the adaptive capacity of wildness training giant pandas. We comparatively analyzed genome-level methylation differences in captive giant pandas with and without wildness training to determine whether methylation modification played a role in the adaptive response of wildness training pandas. The whole genome DNA methylation sequencing results showed that genomic cytosine methylation ratio of all samples was 5.35%–5.49%, and the methylation ratio of the CpG site was the highest. Differential methylation analysis identified 544 differentially methylated genes (DMGs). The results of KEGG pathway enrichment of DMGs showed that VAV3, PLCG2, TEC and PTPRC participated in multiple immune-related pathways, and may participate in the immune response of wildness training giant pandas by regulating adaptive immune cells. A large number of DMGs enriched in GO terms may also be related to the regulation of immune activation during wildness training of giant pandas. Promoter differentially methylation analysis identified 1,199 genes with differential methylation at promoter regions. Genes with low methylation level at promoter regions and high expression such as, CCL5, P2Y13, GZMA, ANP32A, VWF, MYOZ1, NME7, MRPS31 and TPM1 were important in environmental adaptation for wildness training giant pandas. The methylation and expression patterns of these genes indicated that wildness training giant pandas have strong immunity, blood coagulation, athletic abilities and disease resistance. The adaptive response of giant pandas undergoing wildness training may be regulated by their negatively related promoter methylation. We are the first to describe the DNA methylation profile of giant panda blood tissue and our results indicated methylation modification is involved in the adaptation of captive giant pandas when undergoing wildness training. Our study also provided potential monitoring indicators for the successful reintroduction of valuable and threatened animals to the wild.

由严重急性呼吸系统综合症冠状病毒2型(Severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)引起的新型冠状病毒病(Coronavirus disease 2019,COVID-19)对全人类的健康造成了严重威胁.除人类以外,动物感染SARS-CoV-2的情况也不断被发现,包括与人类活动密切相关的犬、猫等伴侣动物[1-3].本文结合该领域的最新研究和报道,以犬、猫为重点,概括了伴侣动物感染SARS-CoV-2的情况和机制,以便为下一步研究提供一些思路.

The giant panda (Ailuropoda melanoleuca) is a global flagship species for biodiversity conservation. As the time for captive giant pandas to be released into the wild matures, wildness training is provided to allow adaptation to their natural environment. It is assumed that changes in the immune system would be integral in this adaptation from captive to wild, where many more pathogens would be encountered in their natural habitats. Therefore, this study aims to determine the expression changes of immune-related genes and their potential as immunoassay markers for adaptation monitoring in wildness training giant pandas, and then to understand the adaptation strategy of wildness training giant pandas to the wild environment, thereby improving the success rate of panda reintroduction. We obtained 300 differentially expressed genes (DEGs) by RNA-seq, with 239 up-regulated and 61 down-regulated DEGs in wildness training giant pandas compared to captive pandas. Functional enrichment analysis indicated that up-regulated DEGs were enriched in several immune-related terms and pathways. There were 21 immune-related DEGs, in which most of them were up-regulated in wildness training giant pandas, including several critical innate and cellular immune genes. IL1R2 was the most significantly up-regulated gene and is a signature of homeostasis within the immune system. In the protein–protein interaction (PPI) analysis, CXCL8, CXCL10, and CCL5 were identified as the hub immune genes. Our results suggested that wildness training giant pandas have stronger innate and cellular immunity than captive giant pandas, and we proposed that a gene set of CXCL8, CXCL10, CCL5, CD3D, NFKBIA, TBX21, IL12RB2, and IL1R2 may serve as potential immunoassay markers to monitor and assess the immune status of wildness training giant pandas. Our study offers the first insight into immune alterations of wildness training giant pandas, paving the way for monitoring and evaluating the immune status of giant pandas when reintroducing them into the wild.

大熊猫Ailuropoda melanoleuca是独特的食肉目Carnivora动物,幼年早期以母乳为食,成年却严格以竹子为食.为探究microRNA(miRNA)对大熊猫幼年早期及成年营养利用及代谢的调控作用,对2只幼年早期和2只成年大熊猫的胰腺组织进行了small RNA测序分析,鉴定了202个保守miRNA和8个新miRNA.保守miRNA差异表达分析中,在成年组中显著上调39个、显著下调38个.成年大熊猫胰腺组织中miR-27a、miR-370和miR-122的差异表达与脂质代谢有关;miR-1上调有利于其氨基酸代谢水平的提高;miR-363、miR-382、miR-494及miR-127下调促进了其能量代谢过程,这些均与mRNA分析结果一致.此外,鉴定出36个仅在幼年早期个体中表达的miRNA,其功能与蛋白质和脂质等大分子化合物的合成有关.这些结果表明,miRNA可能与大熊猫营养利用及代谢调控有关.

为了进一步分析谷氨酸消旋酶的结构和生物信息功能,试验以γ-P GA生产菌株地衣芽孢杆菌(Bacillus licheniformis)DY136为材料,PCR扩增其谷氨酸消旋酶基因(racE),纯化racE基因进行原核表达,并运用多种在线生物信息学分析软件对该基因编码蛋白质的理化性质、亲/疏水性、信号肽、结构域等进行分析.结果表明:PCR扩增racE基因条带大小为819 bp,对racE基因进行大肠杆菌原核表达,通过构建表达载体和优化表达条件,成功表达了大小为30.15 ku的重组融合蛋白.racE蛋白编码272个氨基酸,氨基酸序列与NCBI中公布的地衣芽孢孢杆菌racE蛋白(GenBank登录号为KAA0845975.1)氨基酸同源性为99.62％.racE蛋白理化性质稳定,是亲水蛋白,无跨膜结构域,不含有信号肽,α-螺旋是其主要的二级结构,主要定位于细胞质中(73.90％).说明racE基因保守且相对稳定,在谷氨酸的合成中发挥重要作用.

为了建立一种能同时检测GⅠ型诺如病毒(GⅠ-NV)、GⅡ型诺如病毒(GⅡ-NV)和A群轮状病毒(A-RV)的三重荧光定量PCR检测方法,本试验根据GⅠ-NV和GⅡ-NV的开放阅读框1(Orfl)与开放阅读框2(Orf2)基因之间保守序列、A-RV的非结构蛋白3(Nsp3)基因保守序列,分别设计合成3对特异性引物和3个TaqMan探针,人工合成含有目标序列的基因,制备重组质粒标准品,绘制标准曲线,并对该检测方法的特异性、灵敏度、重复性进行评估.同时通过检测收集的109份动物源食品样品,对建立的方法与商品化试剂盒进行检测比对.结果显示:该方法可以特异性地区分GⅠ-NV、GⅡ-NV和A-RV,而与其他病原无交叉反应;该方法的最低检出限均为1×102copies/μL的质粒标准品,具有较高的敏感性;组内和组间重复性试验结果显示,变异系数均小于5％,重复性较好;该方法与商品化的试剂盒检测结果符合率为100％.结果表明,本试验建立的检测方法能够快速、准确区分GⅠ-NV、GⅡ-NV和A-RV.

为了解进口美国种猪中获取的猪圆环病毒3型(PCV3)毒株全基因组序列及遗传变异情况,根据GenBank中收录的PCV3基因组序列,设计2对特异性引物,对确诊感染PCV3的病死猪组织进行全基因组序列扩增、拼接、测序和序列分析.结果显示,获得的PCV3美国毒株(命名为PCV3_USA2021,GenBank登录号OL799306)全基因组序列长度约为2000 bp,与国内外不同地区的38个PCV3参考毒株同源性为98.7％～99.8％.其中,PCV3_USA2021株开放阅读框ORF1和ORF2与国内外38个参考毒株同源性分别为99.1％～99.9％和98.0％～100％.构建的全基因组系统进化树显示,该毒株为PCV3a亚型,与美国毒株(PCV3-US_SD2016)亲缘关系最近.本研究为PCV3的遗传变异、流行病学分析提供了参考,也为相关疫苗的研制打下了基础.

The influences of ecological protection redlines on urban sustainable development can significantly vary from place to place with distinct ecological functions and human activity interference. However, recent research lacks the evaluation of ecological functions and human activity interference in ecological protection redline areas near urban areas. This article presents an evaluation method consisting of two evaluation criteria systems: an ecological function evaluation criteria system based on conserving water resources and soil and maintaining biodiversity and a human activity interfer- ence evaluation criteria system based on the human activity impact index, population and road network density, and the intensity of tourism activity and livelihood sewage discharge. This evaluation method was verified in Zoige County, China, by using remote sensing data to evaluate ecological functions and human activity interference in the ecological redline area for an urban environment. This evaluation method is one of the preliminary studies of urban sustainable develop- ment planning, and the result could provide a basis for the formulation of urban sustainable development strategy.

为确保从挪威进口至四川的冰鲜大西洋鲑鱼的卫生与安全,防止有害生物因子传入我国.本试验参照OIE及相关国内行业标准推荐的水生动物疾病诊断手册中分子生物学检测方法,采用TaqMan荧光定量RT-PCR结合HE基因中高度多态区域(HPR)克隆测序等诊断方法,对从挪威进口至四川成都的某批次抽检去内脏冰鲜大西洋鲑鱼进行传染性鲑鱼贫血症病毒(ISAV)的检测.结果 显示,该进口大西洋鲑鱼鱼肉中检出HPR缺失型ISAV,且其HPR区间存在51 bp的碱基缺失,基于HPR的序列分析结果提示其与分离自挪威的ISAV9和9/93 HPR缺失型ISAV的亲缘关系最近.结果 表明,这是我国国内首例通过分子生物学方法,从进口大西洋鲑鱼鱼肉中检出HPR缺失型ISAV的案例,该结果警示各大水生动物检疫机构应重视对进口的去内脏冰鲜大西洋鲑鱼中ISAV的筛查,严防该病毒进入我国.

为探究蜡样芽孢杆菌PAS38影响肉鸡免疫的分子机制,将60羽7日龄白羽肉鸡分为2组,对照组饲喂基础日粮,试验组饲喂含蜡样芽孢杆菌PAS381×106 cfu/g的基础日粮.于42日龄时从每个组随机取6只肉鸡,解剖采取脾脏并提取总RNA、反转录为cDNA.然后分别采用SSH和ACSSH技术构建脾脏差异基因文库并筛选免疫相关基因.结果显示,SSH筛选得到119个差异基因,其中上调基因63个,下调基因56个,包括9个免疫调节基因;ACSSH筛选得到5个差异基因,都为下调基因,无免疫调节基因.结果表明,饲喂蜡样芽孢杆菌能影响JCHAIN、FTH1、P2RX7等肉鸡脾脏免疫基因的表达;SSH文库明显优于ACSSH文库,ACSSH技术存在一定缺陷.

为了对肉制品中羊肉源性成分进行准确定量,该研究采用微滴数字PCR(ddPCR)技术对羊肉的单拷贝基因进行定量检测,根据基因拷贝数建立肉制品中羊肉源性成分ddPCR的定量检测方法.结果表明,通过向样品中添加牛肉作为内标,建立了基于DNA拷贝数与样品质量间线性关系对羊肉源性成分进行ddPCR内标定量的方法,实现了从靶基因拷贝数到样品质量间的一步转化.该方法在检测出0.01％的羊肉源性成分时,检测结果达0.12 copies/μL,能够对含量5％以上的羊肉源性成分进行准确定量.通过对已知成分的混合样品和市售样品进行检测,显示该方法能够准确检出不同样品中羊肉源性成分含量.因此,该方法在肉及肉制品中羊肉源性成分检测和掺假鉴别方面具有较大应用潜力.

该研究将实时荧光定量PCR技术(real-time fluorescence quantitative PCR,rPCR)与微滴式数字PCR技术(micro-droplet digital PCR,ddPCR)相结合,利用ddPCR建立拷贝数和羊肉质量的函数关系,确立了基于rPCR定量检测羊肉含量的方法.特异性实验结果表明,除绵羊和山羊外,非目标物种DNA未出现特异性扩增;灵敏度实验结果表明,该方法的最低检出限为0.01 ng/μL;通过对已知成分的混合样品和市售样品的检测表明,该方法能够对含量在5％以上的羊肉进行准确定量.因此,该方法在肉制品中羊肉含量检测和掺假鉴别方面具有较好应用潜力.

近年来,世界各地肉制品掺假情况频繁发生.为维护市场稳定,保障食品安全,迫切需要量化肉制品中所含动物成分的质量百分比.本研究建立了一种实时荧光定量PCR定量检测系统,借助微滴式数字PCR技术(droplet digital,ddPCR),有效实现靶向基因拷贝数的绝对定量,利用实时荧光定量PCR技术(real-time quantitative PCR detecting system,q-PCR),将扩增Ct值转化为基因拷贝数,再从基因拷贝数转换为肉的质量,有效实现了Ct值与质量间的转化,从而实现了羊肉中掺假鸡肉的精确定量.实验数据表明,该方法可以很好地应用于掺假羊肉中鸡肉质量百分比的检测,鸡肉的最低定量检测限(LOD)为5％,在5％～80％范围内相对误差小于±9％,相对标准偏差(RSD)小于±20％,定量结果准确、重复性高,可以有效区分生产污染与故意掺假,为肉类掺假的监管工作提供有力的技术参考.