Detection and Differentiation of CAV-1 and CAV-2 by Polymerase Chain Reaction

Canine adenovirus type 1 (CAV-1) and type 2 (CAV-2) can be categorized in the laboratory by haemagglutination and neutralization tests, but they are difficult to differentiate from each other in specimens, especially when infection occurs in the digestive tract. The object of this study was to develop a simple method of detecting and differentiating them. One pair of common primers was designed and synthesized according to the sequences of the E3 and flanking regions and a polymerase chain reaction (PCR) assay was established using these two primers to amplify the virus-specific DNA fragment from clinical specimens as well as from cell cultures. After electrophoresis, under the same amplification conditions, 508 bp and 1030 bp PCR products were observed for CAV-1 and CAV-2, respectively. These were further shown to be adenovirus specific by dot hybridization and sequencing. As only one pair of primers was involved in the PCR procedure, it was faster and easier to perform than any of the other assays used for detecting canine adenovirus, making it applicable in the rapid confirmation of diagnosis and differentiation of the two types of canine adenoviruses.     

用PCR对鸡毒支原体感染的检测

应用已建立的检测 MG和 MS的 PCR方法 ,对人工接种鸡毒支原体后 2～ 2 0 d的 SPF鸡气管棉拭样品和气管、肺、肝、脾、胸肌、腿肌等器官和组织进行了检测 ;对现场采集的样品同样作 PCR检测。结果表明 ,上述人工样品均检测到病原 ,以气管棉拭样品检出最多 ;现场样品PCR的阳性检出率为 1 0 .2 8% ,分离培养的阳性率为 2 .8% ,敏感性前者高于后者。     

PCR鉴定牛肉方法的建立及初步应用

依据牛１．７０９卫星ＤＮＡ序列设计了１对引物，建立了ＰＣＲ鉴定生、熟牛肉的方法。应用本方法特异地扩增出预期的牛２１８ｂｐＤＮＡ片段，其检测敏感度对生牛肉达３３．６ｆｇＤＮＡ，对熟牛肉和高压牛肉为０．３２ｐｇ。运用该引物均可扩增出水牛、牦牛、奶牛、黄牛肉单一的相同大小的ＤＮＡ条带，而对马、山羊、绵羊、骆驼、鹿、猪等１５种动物肉的ＤＮＡ扩增则呈阴性。扩增片段经ＨａｅⅢ酶切分析确认，所得１２９、７９、１０ｂｐ片段与微机分析结果一致。利用本法对１０３份生、熟牛肉及其制品进行鉴定，检出率为１００％。对各种样品检测，均可在６ｈ内完成。     

内蒙古鄂尔多斯地区传统发酵乳制品中乳酸菌优势菌群q-PCR定量分析

采用实时荧光定量聚合酶链反应(real-time quantitive polymerase chain reaction,q-PCR)技术对从内蒙古鄂尔多斯地区采集的28份传统发酵乳制品(7份酸绵羊乳、8份酸山羊乳以及13份酸牛乳)样品中分离到的乳酸菌优势菌群L.helveticus、Leu.mesenteroides及ac.lactis subsp.lactis的数量进行比较分析.结果表明:酸山羊乳和酸牛乳中,3种优势菌属的数量关系为Leu.mesenteroides＞L.helveticus＞ Lac.lactis subsp.lactis;酸绵羊乳中,3种优势菌属数量关系为L.helveticus＞ Leu.mesenteroides＞ Lac.lactis subsp.lactis.     

Usefulness of Polymerase Chain Reaction in Early Detection and Tissue Tropism of Fowl Adenovirus in Experimentally Infected Chicken

Veterinary Research Communications -     

Detection of Fowl Adenovirus Associated With Hydropericardium Hepatitis Syndrome by a Polymerase Chain Reaction

Hydropericardium hepatitis syndrome HHS), previously unknown in the broiler industry, is an emerging disease that causes severe hydropericardium. A polymerase chain reaction PCR) was developed to detect the fowl adenovirus FAV) associated with HHS. The virus from infectedl ivers was purified, with confirmation by electron microscopy and experimental infection. Methods were developed to isolate the viral DNA from purified virus and infected tissues. Available sequence data on the hexon gene of fowl adenoviruses and other adenoviruses were aligned to determine the conserved and variable regions. Primers were constructed from the alignment data. The amplified fragment consisted of the variable region of the hexon gene flanked by conserved primer sites. Optimum conditions were standardized to achieve the amplification of the desired fragment. As expected, the amplified product was found to be of 0.7 kg size. The nucleotide sequence analysis confirmed the specific nature of the product. Amplification of the specific product could be obtained not only from the DNA isolated from the purified virus but also from the total DNA extracted from infected tissues. The PCR was useful for the detection of FAV associated with HHS.     

Human Brucella canis Infection and Subsequent Laboratory Exposures Associated with a Puppy,New York City, 2012

Human Brucella canis infection incidence is unknown. Most identified cases are associated with pet dogs. Laboratory‐acquired infections can occur following contact with Brucella spp. We identified a paediatric B. canis case, the source and other exposed persons. A 3‐year‐old New York City child with fever and dyspnoea was hospitalized for 48 h for bronchiolitis. After her admission, blood culture grew B. canis, she was prescribed anti‐microbials and recovered. B. canis was also isolated from blood of the child's pet dog; these isolates were genetically similar. The dog originated from an Iowa breeding facility which was quarantined after identification of the dog's infection. Additionally, 31 laboratory workers were exposed and subsequently monitored for symptoms; 15 completed post‐exposure prophylaxis. To our knowledge, this is the first report strongly suggesting B. canis zoonotic transmission to a child in the United States, and highlights the need for coordinated control policies to minimize human illness.     

套式反转录—聚合酶链反应检测恙螨及鼠肺内肾综合征出血热病毒RNA

选择覆盖肾综合征出血热病毒（ＨＦＲＳＶ）膜蛋白（ＭＰ）基因的保守区核苷酸序列合成２对引物,采用异硫氰酸胍一步抽提ＲＮＡ,建立了ＲＴ－ＰＣＲ检测鼠体恙螨及游离恙螨体内ＨＦＲＳＶ－ＲＮＡ的方法,扩增产物经凝胶电泳及斑点印迹杂交证实具有特异性。结果显示,ＨＦＲＳＶ抗原阳性鼠体恙螨５０只组、１０只组、游离螨５０只组,ＨＦＲＳＶ抗原阳性鼠肺１０００ｍｇ、５００ｍｇ组经ＲＴ－ＰＣＲ检测为阳性;ＨＦＲＳＶ抗原阳性鼠体恙螨５只组,ＨＦＲＳＶ抗原阴性鼠体恙螨５０只和１０只组,游离螨１０只和５只组,鼠肺ＨＦＲＳＶ抗原阳性１００ｍｇ组均未见有明显扩增带。进一步用套式反转录－聚合酶链反应（ＮｅｓｔｅｄＲＴ－ＰＣＲ）检测,在ＲＴ－ＰＣＲ未测出ＨＦＲＳＶ－ＲＮＡ的各组中均检测有ＨＦＲＳＶ－ＲＮＡ。结果表明ＮｅｓｔｅｄＲＴ－ＰＣＲ具有高特异、高敏感的特点,可用于检测恙螨体内微量ＨＦＲＳＶ－ＲＮＡ,为确认恙螨作为ＨＦＲＳＶ的传播媒介提供了分子生物学证据。     

逆转录—聚合酶链反应（RT—PCR）检测轮状病毒

为了给轮状病毒感染的诊断和流行病学研究提供更为敏感和可靠的手段,选取Ａ组轮状病毒ＶＰ７基因上的２段高度保守序列作为引物,在优化逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）条件的基础上,建立了检测轮状病毒的ＲＴ－ＰＣＲ方法。通过对比试验,确定了ＰＣＲ的最优模式：９４℃变性１ｍｉｎ→５５℃退火１ｍｉｎ→７２℃延伸２ｍｉｎ,３０个循环后再在７２℃下延伸１０ｍｉｎ。用此模式进行了ＲＴ－ＰＣＲ的特异性和敏感性试验。检测的６株轮状病毒分离株（牛ＨＮ－７、ＢＲＶ００７、ＢＲＶ０１４、ＢＲＶ６５５５、猪Ｌｉ９９、Ｎａｎ８６）及２株参考株（牛ＮＣＤＶ、猴ＳＡ１１）,都能扩增出唯一的３４２ｂｐ的目的条带;对猪流行性腹泻病毒（ＰＥＤＶ）及传染性胃肠炎病毒（ＴＧＥＶ）感染猪的粪样、正常ＭＡ１０４细胞检测结果均呈阴性;检测的敏感度可达１ｐｇ水平。对４０份猪、牛、兔的腹泻粪样检测,３０份呈阳性,而用作平行对照的夹心ＥＬＩＳＡ法检测,有２５份呈阳性,两者符合率为８７．５％。两法检测不符的５份粪样的ＰＣＲ扩增产物,用地高辛标记探针进行了斑点杂交,结果均呈阳性,表明ＲＴ－ＰＣＲ法比ＥＬＩＳＡ法敏感性高。     

Identification of Papillomaviruses in Scrapings from Bovine Warts by use of the Polymerase Chain Reaction

Bloch, N., Sutton, R.H., Breen, M. and Spradbrow, P.B., 1997. Identification of papillomaviruses in scrapings from bovine warts by use of the polymerase chain reaction. Veterinary Research Communications, 21 (1), 63-68.     

聚合酶链反应检测4种猪源细胞系中的内源性反转录病毒

为了建立检测猪内源性反转录病毒（porcine endogenous retrovirus,PERV)的特异性方法，根据巳发表的PERV的序列，设计并合成了针对PERV核心蛋白（gag）、多聚酶（pol)、囊膜蛋白（env）基因的3对引物，预期扩增片段分别为361、150、265bp。应用PCR技术检测了PERV在4株猪源细胞中的整合情况。结果表明，在所有被检细胞的基因组中均存在有PERV的前病毒序列，应用RT-PCR检测上述4株猪源细胞中PERV特异性MRNA的表达，结果均为阳性。试验还对建立的上述2种方法的特异性进行了探讨，结果表明试验建立的PERV检测法具有较高的特异性。该方法的建立为进一步研究PERV奠定了基础，还可对异种移植动物模型及异种移植受体进行病原安全性监测。     

猪瘟病毒荧光定量PCR检测方法研究进展

实时荧光定量PCR技术是结合PCR技术、光谱技术和计算机技术发展起来的一种新型分子生物学定量检测技术,其融合了PCR技术的高灵敏性、DNA杂交的高特异性和光谱技术的高精确定量的特点,它不仅实现了对核酸模板的定量,而且具有特异性强、灵敏度高、重复性好、准确性高、自动化程度高、速度快、全封闭反应、无污染等优点,在猪瘟疫苗的定量检测、猪瘟的诊断以及猪瘟病毒强、弱毒株鉴别诊断中得到了广泛应用,显示出了良好的应用前景。论文就荧光定量PCR技术及其在猪瘟诊断上的应用做一简要综述,为我国猪瘟的综合防控提供参考。     

PCR扩增invA基因特异性检测沙门氏菌

建立了扩增invA基因检测沙门氏菌的PCR方法。对收集的50个血清型123株沙门氏菌及7种27株非沙门氏菌进行PCR,2％琼脂糖电泳检查,结果所有沙门氏菌都扩增出了300bp的特异性产物,非沙门氏菌都未扩增出此目的条带。产物的特异性由slot blot杂交进一步证实。通过电泳判定结果,该法可检出扩增体系中10pg染色体DNA及10~2cfu的纽波特沙门氏菌50029。为下步克隆而设计的两个酶切点（Bam HI,Eco RI）对引物的特异性没有影响。本研究为沙门氏菌的检测提供了简洁、敏感、特异的新方法,同时为克隆invA基因做属特异性探针打下了基础。     

Capnocytophaga canimorsus Meningitis: Three Cases and a Review of the Literature

Bacterial meningitis is a disease with a high morbidity and mortality. It may be caused by the zoonotic pathogen Capnocytophaga canimorsus, which is part of the commensal oral flora in dogs and cats. We report three cases of C. canimorsus meningitis in a nationwide cohort study of bacterial meningitis patients and performed a review of the literature. Three episodes of C. canimorsus meningitis were identified in three patients included in a nationwide cohort study from 2006 through 2014. The calculated annual incidence was 0.03 per million adults. When combined with the literature, 33 patients were identified of which 28 were male (85%). The median age was 63 years, and 13 (42%) were immunocompromised, which consisted of alcoholism in 7 (21%). Animal contact could be established in 29 of 30 patients (93%) and consisted of dog bites in 22 of 29 (76%). One patient died (3%) and 8 had neurological sequelae upon discharge (25%), most often hearing loss (n = 6, 19%). Capnocytophaga canimorsus meningitis is associated with dog bites. Although mortality is relatively low, survivors often have neurological sequelae.     

Genomic Typing of Canine Parvovirus Circulating in the State of Rio de Janeiro,Brazil from 1995 to 2001 Using Polymerase Chain Reaction Assay

In this study, the genomic types of canine parvovirus (CPV) circulating in the State of Rio de Janeiro, Brazil, from 1995 to 2001, were investigated using the polymerase chain reaction assay (PCR). A total of 78 faecal samples from gastroenteritic puppies, confirmed as positive for canine parvovirus by haemagglutination/haemagglutination inhibition tests or virus isolation in cell culture (MDCK), were examined. The viral DNA was extracted from faecal samples using a combination of phenol– chloroform and silica–guanidine thiocyanate methods. PCR was carried out with differential pairs of primers to distinguish the old (CPV-2) and new types of virus (CPv-2a or CPV-2b). Specific amplicons were observed for all samples using the primer pair P2ab, which detects CPV-2a and CPV-2b. Seventy-six from a total of 78 samples (97%) were considered as CPV-2b because of their reaction with the primer pair P2b. Thirty samples (30/78) were from previously vaccinated puppies and in 15 of them the enteritis symptoms began from 1 to 12 days after vaccination. PCR confirmed the infection by wild virus (CPV-2b) in 5 of these 15 puppies who had received old-type vaccines. Our results show that CPV-2b was the prevalent type circulating in the State of Rio de Janeiro from 1995 to 2001.     

利用荧光定量PCR进行饲料中牛羊源性成分检测的快速筛选

通过荧光定量PCR进行饲料中牛羊源性成分的快速筛选研究,其可以快速的检测饲料中是否含有牛羊源性成分,避免了常规检测中大量阴性样品造成的巨大工作量,并首次对饲料中牛羊源性成分进行定量分析,推导出定量计算公式,且灵敏度比常规的PCR检测方法高100倍以上。     

锦鲤疱疹病毒荧光定量PCR检测方法的建立

根据锦鲤疱疹病毒(KHV)胸苷激酶(TK)基因的保守序列设计一对引物和相应的TaqMan探针,建立荧光定量PCR方法,用于锦鲤疱疹病毒快速、灵敏、可定量的检测手段。构建并制备实时荧光定量PCR的标准品,对反应体系进行优化,并做特异性,敏感性和重复性试验。结果表明,成功构建荧光定量PCR标准品,建立荧光定量标准曲线,标准曲线的相关系数为0.992,所建立的荧光定量PCR方法特异性强、敏感性高,重复性好。     

丝状支原体丝状亚种SC生物型PCR检测方法的建议

建立了一个可以识别丝状支原体丝状亚种SC生物型（MmmSC)的PCR方法。根据丝状支原体丝状亚种SC生物型（MmmSC)相关核苷酸序列，设计合成了MC1、MC2和SC1、SC2两对引物。MC1、MC2是一对簇特异性引物，用于鉴别丝状支原体族6个成员，对MmmSC、MmmLC扩增出与预期结果相符的462bp片段；而SC1、SC2是针对MnnSC的一对特异性引物，只能对MmmSC扩增出277bp的片段，经过VspI、Bsp143I和Dral三种限制性内切酶鉴定与预期结果相符，而不能扩增出MmmLC型Y－goat代表株，说明具有非常好的特异性。对MmmSC进行的敏感性试验显示SC1、SC2引物能够检测到100个CFU，具有非常高的敏感性。