为建立反刍动物埃立克体(E.ruminantium)准确定量的微滴数字PCR方法(ddPCR),本研究以E.rum-inantium pCS20基因为靶基因,选取保守区域设计引物和探针,建立了 E.ruminantium ddPCR方法.利用本研究建立的ddPCR方法检测E.ruminantium、牛巴贝斯虫、牛双芽巴贝斯虫、环形泰勒虫、无形体、犬埃立克体、牛埃立克体、马埃立克体和立氏埃立克体等,结果显示仅E.ruminantium出现特异性扩增,而与其他寄生虫均无交叉反应,特异性强;将pUC57-pCS20重组质粒标准品10倍倍比稀释(1×10-1 拷贝/μL～1×105拷贝/μL)后,利用该ddPCR方法检测,结果显示,该方法对重组质粒标准品的检测限为1.8拷贝/μL,比相应荧光定量PCR方法的敏感性高10倍,本实验建立的ddPCR方法敏感性高;批内和批间重复性试验结果变异系数(CV)均小于2％,重复性好.利用建立的ddPCR方法对50只钝眼蜱样品检测,结果显示,阳性样品反刍动物埃立克体核酸的最低拷贝数为26拷贝/μL,ddPCR和荧光定量PCR检测试剂盒的检出率均为16.0％(8/50).本研究首次建立的ddPCR方法敏感性高、特异性强、重复性好,可作为E.ruminantium准确定量检测的有效手段.

为快速、准确地检测反刍动物埃立克体,本研究以反刍动物埃立克体pCS20为靶基因设计特异性引物和探针,建立了 TaqMan和Eva Green荧光定量PCR方法,对其反应的特异性、敏感性和重复性进行了分析,并与OIE推荐的套式PCR方法一起对临床样品进行检测.结果显示,本方法特异性强,与牛巴贝斯虫、牛双芽巴贝斯虫、环形泰勒虫、犬埃立克体、牛埃立克体、马埃立克体和立氏埃立克体无交叉反应;TaqMan和Eva Green荧光定量PCR对pCS20质粒标准品的最低检测限分别为17.4拷贝·μL-1和1.74拷贝·μL-1,标准曲线相关系数大于0.99,组内和组间CV均小于1.5％.对420只钝眼蜱样本的检测显示,TaqMan和Eva Green荧光定量PCR的检出率分别为25.48％和29.29％,与套式PCR检测方法相比,敏感性更高.本研究为反刍动物埃立克体的检测和流行病学调查提供了一种快速、准确的检测方法.

为建立4种肉类(牛肉、猪肉、鸡肉和马肉)成分的鉴别方法,本试验基于动物线粒体细胞色素b基因,设计1条通用正向引物CF,以牛、猪、鸡和马的特定DNA序列设计4条特异性反向引物R,优化反应条件,建立了多重PCR检测方法.该方法对牛、猪、鸡和马肉的检测灵敏度为10×10-3ng/μL,具有良好的特异性.对80份市售肉制品样本进行检测,检出掺假样本31份,与本实验室常用的荧光定量PCR试剂盒检测结果一致.本试验建立的多重PCR方法能够有效地对牛、猪、鸡和马源性成分进行快速检测.

为建立一种简单、快速的炭疽杆菌(Bacillus anthracis)分子检测方法,本研究基于炭疽杆菌BA5345基因保守序列,设计合成1对重组酶聚合酶扩增(RPA)引物,通过对反应时间的优化,建立39℃恒温水浴锅检测炭疽杆菌的RPA方法.结果显示,所建立的RPA方法在39℃水浴锅中恒温反应30 min,能够特异性的检测炭疽杆菌;以重组质粒pUC57-BA5345作为模板,RPA方法对其检测下限为102拷贝/μL,与荧光定量PCR检测试剂盒检测下限一致,比常规PCR方法敏感性高100倍;RPA方法对污染皮毛临床样品的阳性检出率为65.71％,略低于荧光定量PCR试剂盒的检出率(71.43％),明显高于常规PCR的检出率(42.86％).本研究建立的RPA方法操作简单、反应快速,结果确实可靠,适用于炭疽杆菌的快速检测.

随着电子商务的飞速发展,进出境邮寄物已成为当前商品流动的主要途径.对温州口岸进出境邮寄物检验检疫的现状进行调研,从检验检疫工作、社会环境两个层面剖析其存在的问题,提出建立“全申报+人+机+犬+黑名单”五位一体的国际邮寄物检验检疫查验模式,可有效解决进出境邮寄物携带动植物疫情疫病、有害生物、传染病、有毒有害物质等非传统安全隐患问题.

Based on the pet food industry in Zhejiang province's export industry management,business management, supervision and management of the status of the official investigation,understand the health status of safety problems in order to further understand the situation,eventually proposed to promote enterprises to improve safety and health conditions, inspection and quarantine departments to enhance the level of checks and service solutions and measures.

The gel strength and sensory evaluation are important quality indexes of surimi products. In this paper,taking the low surimi as raw material,the effect of ingredients is studied such as soy protein isolate,potato starch and oil. The results show that the optimum additions are as follows:surimi products 60%,soy protein isolate 10%,potato starch 13%,vegetable oil 3.5%.

A pair of primers were designed according to the sequence of specific Brucella gene of Outer membrane protein(OMP31),the OMP31 gene was amplified by PCR and cloned into pMD18-T vector,and then the gene sequence was detected.The results showed that the target gene band at a length 602 bp was amplified by PCR,with a homology of 99.7% to the gene sequence reported in GenBank.The experiments had proved that PCR assay possessed a high specificity,DNA bands couldn't amplified in other bacterial except Brucella.And the sensitivity test results indicated that PCR assay was more sensitive,which could detect OMP31 with only 0.9 pg/μL DNA.The successfully construction of the PCR assay provides strongly technical support for detection,identification and epidemiological investigations of bovine brucellosis.

According to the sequences of specific Brucella gene of outer membrane protein(OMP31) and Mycobacterium paratuberculosis gene of C-2 chromosome(ISMav2) available in the GenBank,two pairs of primers were designed to establish a rapid duplex PCR assay for differentiation of Brucella and M.paratuberculosis.Results showed that the duplex PCR assay possessed a high specificity and sensitivity,with 602 bp and 246 bp amplicons amplified from Brucella and M.paratuberculosis,respectively.And none pro-ducts were found from 7 other strains(Babesia bigemina,Escherichia coli,Salmonella typhimurium,Toxoplasma gondii,Streptococcus,Sphaerotilus bovis,the above DNA mixture).Sensitivity of genomic DNA detection of Brucella and M.paratuberculosis were 1.92 pg and 2.51 pg respectively,with the detection limit in artificially contaminated beef as low as 6×104 CFU/mL and 7×104 CFU/mL,respectively.The duplex PCR assay was successfully constructed which will provide strongly technical support for the detection,identification and epidemiological investigations of bovine brucellosis and paratuberculosis.

A pair of primers were designed according to the sequence of specific Mycobacterium paratuberculosis gene of C-2 chromosome(ISMav2),the ISMav2 gene was amplified by PCR and cloned into pMD18-T vector and then the gene sequence was detected.The results showed that the target gene band at a length 246 bp was amplified by PCR,with a homology of 99.6% to the gene sequence reported in GenBank.The experiments had proved that PCR assay possessed a high specificity,DNA bands couldn't be amplified in other bacterial except Mycobacterium paratuberculosis.And the sensitivity test results indicated that PCR assay was more sensitive,which could detect ISMav2 with only 1 pg DNA.The successfully construction of the PCR assay provides strongly technical support for detection,identification and epidemiological investigations of bovine paratuberculosis.