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.     

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。     

Early Detection of Brucella Canis via Quantitative Polymerase Chain Reaction Analysis

Canine brucellosis is a reportable zoonotic disease that can lead to canine reproductive losses and human infection through contact with infected urine or other genitourinary secretions. Although many locations require testing and euthanasia of positive dogs, current diagnosis is limited by the time required for seroconversion, for example, presence of B. canis‐specific antibodies. The goal of this study was to determine the diagnostic ability of Brucella canis‐specific quantitative polymerase chain reaction (qPCR) assay to detect B. canis in field samples prior to serological positivity for faster diagnosis and prevention of transmission within kennels or in households. Two kennels, one of which was located in the owner's home, were sampled following observation of suggestive clinical signs and positive serology of at least one dog. Specimens obtained were comparatively analysed via serology and qPCR analysis. 107 dogs were analysed for B. canis infection via qPCR: 105 via whole‐blood samples, 65 via vaginal swab, six via urine and seven via genitourinary tract tissue taken at necropsy. Forty‐five dogs were found to be infected with canine brucellosis via qPCR, of which 22 (48.89%) were seropositive. A statistically significant number (P = 0.0228) of qPCR‐positive dogs, 5/25 (20.00%), seroconverted within a 30‐day interval after initial serologic testing. As compared to serology, qPCR analysis of DNA from vaginal swabs had a sensitivity of 92.31% and specificity of 51.92%, and qPCR analysis of DNA from whole‐blood samples had a sensitivity of 16.67% and specificity of 100%. B. canis outer membrane protein 25 DNA qPCR from non‐invasive vaginal swab and urine samples provided early detection of B. canis infection in dogs prior to detection of antibodies. This assay provides a critical tool to decrease zoonotic spread of canine brucellosis, its associated clinical presentation(s), and emotional and economic repercussions.     

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.     

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。     

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。     

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.     

荧光PCR法检测鸡肉中沙门氏菌的研究

作者采用合成的基因探针和引物，对2株阳性标准菌株、2株野生菌株和1个无菌去离子水进行检测，结果准确可靠，整个试验过程在30 h内完成。可见，该法是一种特异、敏感、简便、快速的沙门氏菌检测方法，对快速通关将发挥积极的作用。     

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。     

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。     

Comparison of the Polymerase Chain Reaction and Culture for the Detection of Feline Chlamydia psittaci in Untreated and Doxycycline-Treated Experimentally Infected Cats

The diagnostic sensitivity of the polymerase chain reaction (PCR) was compared with that of culture on conjunctival swabs over the course of infection in 4 doxycycline-treated and 4 untreated cats that were experimentally infected with feline Chlamydia psittaci. Treated cats were given 25 mg (5 mg/kg) of doxycycline orally twice daily for 3 weeks from day 6 after challenge. Clinical signs improved within 3 days of institution of treatment. Culture remained positive for 1 day and PCR remained positive for up to 5 days after treatment was commenced. No recurrence of clinical signs occurred and the organism could not be detected by either PCR or culture for 2 weeks after cessation of therapy. In the 4 untreated cats, conjunctival swabs were taken daily to day 14 and every 2nd weekday to day 64 after challenge. PCR was significantly more sensitive than culture in untreated cats overall (PCR 85.7%, culture 72.9%, P approximately 0) and for cats with clinical signs (PCR 89.2%, culture 79.2%, P = .008). PCR and culture had equivalent sensitivity (100%) for cats showing clinical signs in the 1st month of infection, whereas PCR was considerably more sensitive than culture for cats showing clinical signs in the 2nd month (PCR 72.9%, culture 47.9%, P = .028). Organisms were not detected by PCR in blood or any tissue collected from treated or untreated cats at postmortem. Thus, effective treatment of chlamydiosis in cats is possible with much shorter treatment regimens than currently recommended, and PCR is the more sensitive diagnostic method in chronically infected cats.     

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。     

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.     

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.     

禽腺病毒血清4型感染鸡组织中NLRP3基因转录水平的实时荧光定量PCR分析

旨在分析禽腺病毒血清4型(FAdV-4)感染鸡组织中NLRP3基因的转录水平,本研究设计鸡NLRP3特异性引物,利用RT-PCR扩增NLRP3基因180 bp片段并克隆至pMD-18T载体,制备重组质粒pMD-18T-NL-RP3.以pMD-18T-NLRP3质粒作为标准品进行荧光定量PCR并建立标准曲线.通过反应条件...     

应用聚合酶链式反应检测禽痘病毒田间分离株及疫苗株中网状内皮组织增殖病毒LTR片断,env及re1基因

本文利用聚合酶链式反应对来自澳大利亚昆士兰州、维多利亚州和新南维尔士州的４株禽痘病毒田间分离株及二疫苗株中网状内皮组织增殖病毒的ＬＴＲ片断、ｅｎｖ及ｒｅｌ基因因进行了检测。所有４株痘病毒分离株均为ＬＴＲ片断、ｅｎｖ阳性,曾被ＰＣＲ证实为ＬＴＲ阳性的一疫苗株在本实验中也为ＬＴＲ、ｅｎｖ阳性,另一疫苗株为ＬＴＲ、ｅｎｖ阴性,所有上述禽痘病毒株均为ｒｅｌ阴性。     

应用RT-PCR方法从进境南美白对虾亲虾中检出桃拉综合征病毒(Taura syndrome virus)

应用PCR和RT—PCR技术，对一批进境的南美白对虾亲虾进行了多种病毒性病原的检疫。结果检出桃拉综合征病毒。临床观察也表明，该批亲虾体表散布大量黑色病灶，为TSV感染引起的TS典型病变。     

Evaluation of 3 Serological Tests for Early Detection Of Leptospira‐specific Antibodies in Experimentally Infected Dogs

Background

Leptospirosis in dogs is a disease of global importance. Early detection and appropriate therapeutic intervention are necessary to resolve infection and prevent zoonotic transmission. However, its diagnosis is hindered by nonspecific clinical signs and lack of rapid diagnostic tests of early infection. Recently, 2 rapid point‐of‐care tests (WITNESS Lepto [WITNESS Lepto, Zoetis LLC, Kalamazoo, MI, USA] and SNAP Lepto [SNAP Lepto, IDEXX Laboratories, Westbrook, ME, USA]) for detection of Leptospira‐specific antibodies in canine sera were developed.

Hypothesis

Immunoglobulin M‐based WITNESS Lepto containing multiple detection antigens can detect Leptospira‐specific antibodies to common leptospiral serovars earlier in the course of infection as compared to microscopic agglutination test (MAT) and SNAP Lepto.

Animals

Four groups of 8 6‐ to 8‐month‐old male Beagle dogs were used.

Methods

Thirty‐two healthy seronegative dogs were inoculated experimentally with serovars Canicola, Grippotyphosa, Icterohaemorrhagiae, and Pomona (8 dogs/serovar). Acute‐phase sera were collected at regular intervals and monitored for Leptospira‐specific antibodies by WITNESS Lepto, MAT, and SNAP Lepto.

Results

Seroconversion was detected in all dogs by day 10 by WITNESS Lepto and in 30 of 32 dogs by day 14 by MAT. The SNAP Lepto test detected seroconversion in 3 dogs during the 2 weeks postchallenge.

Conclusions

Immunoglobulin M‐based WITNESS Lepto detected immune responses specific to multiple leptospiral serovars early in the course of infection and identified seroconversion in all animals earlier than did the gold standard MAT. The SNAP Lepto test displayed considerably lower and inconsistent performance during the study period. At the point‐of‐care, WITNESS Lepto should be the test of choice for rapid and reliable screening of acutely ill dogs suspected to have leptospirosis.