PCR检测猪伪狂犬病病毒方法的研究

根据编码猪伪狂犬病病毒gH基因保守序列，设计合成一对引物，通过改进病毒核酸提取方法和优化PCR反应条件，成功的从猪伪狂犬病毒感染的细胞中扩增出预期的355bp片段。而猪繁殖与呼吸综合征病毒、猪细小病毒、猪瘟病毒、猪乙型脑炎病毒和正常细胞均未扩增出相应的片段，经NaeⅠ酶切鉴定，证实了该扩增片段的特异性；敏感性实验表明，该体系可检测到0．48Pg的猪伪狂犬病毒DNA。本方法的建立使猪伪狂犬病病毒的检测更为快速、简便、经济、实用。     

PCR制备地高辛标记的探针检测禽流感病毒核酸

用聚合酶链反应（PCR）技术,制备了广东禽流感无致病力分离株A/goose/China/24/96(H7N3）核蛋白基因片段（NPc）的地高辛标记的cDNA探针。建立并优化了检测禽流感病毒核酸的探针杂交法,探针杂交法能鉴别出非免疫鸡胚和SPF鸡胚尿囊液中的病毒,攻毒后第3天的SPF和非免疫鸡泄殖腔拭子中AIV的最大检出率为1/10,对临床样品中的AIV的最大检出率为1/7,而直接HA和HI法及AGP试验检不出临床样品的AIV。该探针具有较好的特异性和敏感性,为从分子水平探讨AIV的发病机理、临床早期快速诊断提供了新的研究手段。     

饲料中牛羊源性成分PCR方法定性检测能力验证计划的实施和结果研究

"饲料中牛羊源性成分PCR方法定性检测"是由国家认证认可监督管理委员会(CNCA)组织的A类能力验证计划.通过对本次能力验证计划的实施和对结果的分析研究,了解了国内饲料中牛羊源性成分PCR检测领域的整体水平以及各实验室在检测中存在的差异.     

Development of a polymerase chain reaction method for diagnosing Babesia gibsoni infection in dogs

A pair of oligonucleotide primers were designed according to the nucleotide sequence of the P18 gene of Babesia gibsoni (B. gibsoni), NRCPD strain, and were used to detect parasite DNA from blood samples of B. gibsoni-infected dogs by polymerase chain reaction (PCR). PCR was specific for B. gibsoni since no amplification was detected with DNA from B. Canis or normal dog leucocytes. PCR was sensitive enough to detect parasite DNA from 2.5 microl of blood samples with a parasitemia of 0.000002%. PCR detected parasite DNA from 2 to 222 days post-infection in sequential blood samples derived from a dog experimentally infected with B. gibsoni. The detection of B. gibsoni DNA by PCR was much earlier than the detection of antibodies to B. gibsoni in blood samples by the indirect fluorescent antibody test (IFAT) or that of the parasite itself in Giemsa-stained thin blood smear film examined by microscopy. In addition, 28 field samples collected from dogs in Kansai area, Japan, were tested for B. gibsoni infection. Nine samples were positive in blood smears, 9 samples were positive by IFAT and 11 samples were positive for B. gibsoni DNA by PCR. The nucleotide sequences of PCR products from all 11 samples found positive by PCR were completely identical to that of the P18 gene of the B. gibsoni, NRCPD strain. These results suggest that PCR provides a useful diagnostic tool for the detection of B. gibsoni infection in dogs.     

鸡传染性喉气管炎病毒PCR诊断方法的建立与应用

根据GenBank登录的传染性喉气管炎病毒(ILTV)的TK基因序列设计并合成1对特异性引物,以ILTV疫苗株DNA为模板,建立了检测ILTV TK基因的PCR方法。应用该方法能从临床分离毒株和疫苗株中扩增到长为427 bp的目的片段;但不能从新城疫病毒(NDV)、传染性法氏囊病毒(IBDV)、禽呼肠孤病毒(ARV)、减蛋综合征病毒(EDSV)、H9亚型禽流感病毒(H9-AIV)、传染性支气管炎病毒(IBV)、大肠杆菌以及金黄色葡萄球菌等病原中扩增出阳性条带;敏感性试验表明其DNA最小检出量为4.9 ng;应用该方法和病毒分离法对2份临床病例和人工感染鸡的检测,两者符合率为100%。上述结果表明该PCR方法具有良好的特异性和敏感性,可用于传染性喉气管炎病毒鉴定和临床诊断。     

应用双重PCR方法检测羊支原体肺炎病原

通过对丝状支原体山羊亚种(M.mycoides subsp.capri,Mmc)特异性引物MmcF/MmcR和绵羊肺炎支原体(M.ovipneumontiae,Mo)特异性引物LmF/LmR退火温度、引物浓度比例等条件的选择,建立了一个可以同时检测Mmc和Mo的双重PCR方法。该方法可同时扩增出Mmc 195 bp和Mo 361 bp目的片段,但对其他病原菌不能扩增出任何条带,具有良好的特异性。敏感性试验表明,该方法能够分别检测出0.1ng的Mmc DNA和0.01 ng的Mo DNA,或同时检测出1ng Mmc和1ng Mo混合的DNA。用该双重PCR方法可对实验室保存的4株绵羊肺炎支原体和2株丝状支原体山羊亚种进行准确鉴定,并可从临床病料中检测出相应支原体,表明建立的双重PCR方法可用于Mmc和Mo的快速鉴定、实验室诊断和病原学调查。     

Species diagnosis of a tissue using the polymerase chain reaction

The unambiguous identification of a biological specimen can deliver invaluable evidence to solve criminal cases. In this case the origin of a heart had to be clarified. Using the polymerase chain reaction technique and species-specific primer pairs for two genes it was clearly shown that this tissue was not from a human but from a pig.     

牛传染性鼻气管炎病毒LUXTM新型荧光PCR检测方法的建立

本研究针对牛传染性鼻气管炎病毒（Infectious bovine rhinotracheitis virus，IBRV）高度保守的gC基因设计单标记并具有自身荧光淬灭功能的LUX^TM引物，建立L刚新型实时荧光PCR方法用于快速检测IBRV。该方法对四株IBRV细胞培养物的检测均呈典型阳性反应，而对其它动物疱疹病毒以及健康牛组织DNA和细胞对照的检测结果为阴性，检测时间包括核酸提取仅需1h～2h。试验表明，LUX^TM荧光PCR法对IBRV细胞增殖病毒液的检测敏感性可达0．04TCID50，比病毒分离敏感性至少提高10倍；对10倍系列稀释的纯化IBRV核酸样品，L刚荧光PCR的检测敏感性比常规PCR可提高10^3倍。将病毒液添加到健康牛精液和血液样品中，该荧光PCR可检测到牛冻存精液中40TCID50牛抗凝全血、血清和临床精液中0．04TCID50的病毒，说明对临床样品的检测有效。本研究所建立的LUXTM荧光PCR方法快速敏感，适合应用于活牛及其遗传物质的进出口检疫、养牛业疾病防控等领域对IBRV的快速检测。     

Detection of porcine circovirus type 1 in commercial pig vaccines using polymerase chain reaction

The absence of extraneous viruses is a requirement in the quality control of vaccines for veterinary use in the European Pharmacopoeia. A polymerase chain reaction (PCR) assay for the detection of porcine circovirus type 1 (PCV1) and type 2 (PCV2) was evaluated in 18 commercial porcine vaccines. Since vaccine components may contain PCR enhancers or inhibitors, 13 of the studied vaccines (used as diluents) were subsequently spiked with different dilutions of PCV2 and tested by PCR. Although PCV2 DNA was not detected in any of the vaccines tested, PCV1 was detected in 2/18 vaccines (11%). Eleven out of 13 PCV2 spiked vaccines showed a positive PCR result. The lack of amplification observed in two spiked vaccines suggested that use of the PCR assay to detect PCV2 could depend on vaccine composition. The results of this exploratory study have demonstrated that PCR is a rapid and fairly sensitive method for the detection of porcine circoviruses as extraneous agents in vaccine products and can be used in the quality control of pig vaccines. The study has also indicated the need for optimising the sensitivity of PCR methods for PCV genome detection in vaccine products.     

多重聚合酶链反应快速检测鉴别鸡毒支原体强毒株和弱毒疫苗株的研究

根据鸡毒支原体强毒株和弱毒疫苗株基因组的结构特点，设计合成了二对引物XZ1，XZ2和XZ45、XZ46，建立了一种同时检测鉴别MG野毒株和弱毒疫苗株的多重PCR技术。试验结果表明，用这两对引物对MG强毒株和弱毒疫苗侏进行多重PCR，强毒株只扩增出732bp一条带，而弱毒疫苗株则可同时扩增出732bp、524bp二条带，而对其他种类鸡支原体和其它禽病病原的扩增不出现任何条带，结果均为阴性；敏感性测定结果表明，该多重PCR最低能检出1Pg的MG强毒株和弱毒疫苗株的DNA模板。     

Development of a polymerase chain reaction method for diagnosis of Babesia ovis infection in sheep and goats

In this study, a pair of oligonucleotide primers were designed according to the nucleotide sequence of the small subunit ribosomal RNA (ssu rRNA) gene of Babesia ovis isolated from sheep in eastern Turkey. The primers were used to detect parasite DNA from blood samples of B. ovis-infected sheep and goats by polymerase chain reaction (PCR). A 549-bp DNA fragment was specifically amplified from blood samples from sheep and goats, naturally infected with B. ovis. No PCR products resulted from Babesia motasi, T. ovis, Theileria sp. OT1, Theileria sp. OT3, T. lestoquardi, B. canis, B. microti,T. annulata or normal sheep leucocytes DNA using these specific primers. B. ovis-infected erythrocytes with 1% parasitemia were subjected to 10-fold serial dilutions (from 10(-1) to 10(-9)) using an uninfected sheep erythrocytes, and DNA was extracted from each diluted sample for testing the sensitivity of the PCR. The PCR was sensitive enough to detect parasite DNA from the dilution of 10(-5) with 0.00001% parasitemia. This is more sensitive than examining 200 fields under light microscopy. In addition, 98 field samples collected from small ruminanats in eastern Turkey were tested for B. ovis infection. Four samples were positive Babesia spp. in blood smears, 21 samples were positive for B. ovis DNA by PCR. These results indicate that the PCR provides a useful diagnostic tool for the detection of B. ovis infection in sheep and goats.     

Development of a nested polymerase chain reaction method to detect oncogenic Marek's disease virus from feather tips.

For the easy survey of Marek's disease virus (MDV), feather tip-derived DNA from MDV-infected chickens can be used because feather tips are easy to collect and feather follicle epithelium is known to be the only site of productive replication of cell-free MDV. To develop a diagnostic method to differentiate highly virulent strains of MDV from the attenuated MDV vaccine strain, CVI988, which is widely used, nested polymerase chain reaction (PCR) was performed to detect a segment of the meq gene in feather tip samples of chickens experimentally infected with MDV. In chickens infected with Md5, a strain of oncogenic MDV, the meq gene was consistently detected, whereas the L-meq gene, in which a 180-base pair (180-bp) sequence is inserted into the meq gene, was detected in CVI988-infected chickens. Moreover, the meq gene was mainly detected even in chickens co-infected with both Md5 and CVI988. These results suggest that this method is appropriate for the surveillance of the highly virulent MDV infection in the field.     

Development of a shuttle polymerase chain reaction for the detection of bovine herpesvirus 2

Three different polymerase chain reaction (PCR) protocols were evaluated for their ability to detect bovine herpesvirus 2 (BoHV-2): single-step PCR with 3 reaction stages (denaturation, annealing and extension), 2 reaction stages (denaturation and annealing/extension; shuttle PCR), and semi-nested PCR with 3 reaction stages. All the PCR protocols showed the same sensitivity (detection limit of 0.4 TCID(50)). A non-specific band sometimes appeared in mock cell DNA at annealing temperatures below 64 degrees C. The shuttle PCR was found to be superior to the other protocols under consideration because of the speed of its application. Furthermore, no non-specific band was detected in DNAs of eight other DNA viruses. Thus, the shuttle PCR seems to be an excellent diagnostic tool for BoHV-2 infections.     

PCR检测感染早期小鼠血液中旋毛虫幼虫的研究

为了研究PCR检测感染小鼠血液中旋毛虫DNA的敏感性,应用旋毛虫1.6 kb重复序列为扩增靶序列对旋毛虫(T1)、乡土旋毛虫(T2)、布氏旋毛虫(T3)、伪旋毛虫(T4)和南方旋毛虫(T7)肌幼虫DNA进行PCR扩增,并检测小鼠感染20、100、300条T1肌幼虫后不同时间的外周血.结果表明,T1、T4和T7肌幼虫可扩增出特异性目的条带(510 bp),而T2和T3无扩增产物;1、0.04和0.02条T1、T4和T7肌幼虫均能扩增到清晰的目的条带(510 bp).20条幼虫感染小鼠后5 d～6 d,PCR阳性率均为7.69%;100条幼虫感染小鼠后5 d～12 d可检出旋毛虫DNA,其中感染后5 d～7 d的阳性率分别为30.77%、38.46%及30.77%;300条幼虫感染小鼠后5 d～15 d可检出旋毛虫DNA,感染后7 d的阳性率为61.54%,感染后6 d与8 d～10 d的阳性率均为53.85%. 3组旋毛虫感染小鼠PCR阳性率间的差异有统计学意义(p<0.01),PCR阳性率随感染剂量的增加而升高(p<0.01),100条与300条感染小鼠感染后不同时间的PCR阳性率与检测时间有相关性(p<0.01).以上实验结果表明PCR检测感染小鼠血液中旋毛虫DNA的敏感性与感染程度和检测时间有关,对感染早期旋毛虫抗体阴性宿主有一定诊断价值.     

Sexing of newly-hatched chicks using DNA isolated from chorio-allantoic membrane samples by polymerase chain reaction in Denizli chicken

1. The aim of the present study was to determine the sex of newly-hatched chicks of Denizli chicken, a local Turkish breed, by polymerase chain reaction (PCR) using DNA extracted from the chorioallantoic membrane (CAM). 2. Fertilised eggs were incubated individually and a total of 20 CAM samples were collected following the hatching process. DNA was isolated from the CAM samples and PCR was performed using W-repeat (W) and 18 S ribosomal gene (R) primers. 3. Screening of the PCR products by agarose gel electrophoresis revealed that males have a single band (256 bp) and females have an extra second band (415 bp) as expected. 4. The present study describes a reliable, rapid, and simple multiplex PCR protocol that can be put into use to sex local breeds of chicken in which phenotypic sexing is impossible, using DNA isolated from the CAM that is discarded and remains attached to the egg shell following the hatching process.     

Development and application of a multiplex polymerase chain reaction for avian respiratory agents

A multiplex polymerase chain reaction (PCR) was developed and optimized to simultaneously detect 6 avian respiratory pathogens. Six sets of specific oligonucleotide primers for infectious bronchitis virus (IBV), avian influenza virus (AIV), infectious laryngotracheitis virus (ILTV), Newcastle disease virus (NDV), Mycoplasma gallisepticum (MG), and Mycoplasma synoviae (MS) were used respectively in the test. With the use of agarose gel electrophoresis for detection of the PCR-amplified DNA products, the sensitivity of detection was between 10 pg for IBV, AIV, MG, and ILTV and 100 pg for NDV and MS after 35 cycles of PCR. Similar sensitivity of these primers was achieved with chickens experimentally infected with respiratory pathogens. In experimental infections, the multiplex PCR was able to detect all the infected chickens in each group at I and 2 wk postinfection as compared with serologic tests at 2 wk postinfection that confirmed the presence of specific antibodies. The multiplex PCR was also able to detect and differentiate coinfections with two or more pathogens. No specific DNA amplification for respiratory avian pathogens was observed among noninoculated birds kept separately as a negative control group.     

聚合酶链式反应检测猪细小病毒方法的研究

本研究依据编码猪细小病毒结构蛋白VP2基因序列,合成了一对寡核苷酸引物,通过减少病毒核酸的提取时间、费用及优化聚合酶链式反应(PCR)的条件,成功地从猪细小病毒(PPV)感染的细胞中扩增出预期的158 bp片段,经EcoRⅠ酶切鉴定,证实了该扩增片段的特异性.经敏感性试验测定,PCR的最低检出量为0.008病毒血凝(HA)单位.这些结果表明本试验建立的PCR对PPV的检测人有快速、简便、经济、灵敏度高和特异性强的特点.     

Evaluation of a 23S rDNA polymerase chain reaction assay for identification of Serpulina intermedia, and strain typing using pulsed-field gel electrophoresis

A polymerase chain reaction assay, amplifying a 1027 base pair portion of the 23S rDNA gene, was evaluated for identification of the intestinal spirochaete Serpulina intermedia. A total of 34 strains of S. intermedia isolated from pigs and chickens and 195 strains of other related species were tested. The optimised assay correctly identified all the S. intermedia strains, but generated 11 false positive reactions, giving a test sensitivity of 100% and a test specificity of 94.3%. The false positive reactions were generated from strains of four different species of intestinal spirochaetes, and the product was of the original predicted size. This suggests that the primer sites selected on the 23S rRNA gene were not completely specific for S. intermedia. Pulsed-field gel electrophoresis was then developed to investigate diversity amongst the S. intermedia strains. All strains tested had distinct DNA banding patterns using Mlu1, although three isolates from chickens on the same farm appeared closely related. The collection exhibited considerable genetic diversity, and strains from pigs and chickens were distributed in clusters throughout the dendrogram produced. The most closely related porcine and avian strains shared only 62% similarity.     

RT-PCR检测猪瘟病毒方法的建立与应用

建立RT PCR检测猪瘟病毒的方法。根据已发表的猪瘟病毒E2基因 (囊膜糖蛋白gP55基因 )序列 ,设计合成了一对特异性引物 ,扩增片段的大小为 50 7bp ,用RT PCR技术对石门系标准株和 1 0株分离株进行检测。结果这对引物对标准株和 1 0株分离株均能扩增出与预期大小相符 50 7bpRT PCR产物 ,而对其他 6种猪病病原核酸的扩增结果为阴性。该RT PCR可检出 1 0 0pg的猪瘟病毒RNA模板 ,对人工感染猪不同组织样品进行检测 ,结果对白细胞抽提的核酸样品检出率最高为 1 0 0 % (2 4 / 2 4 ) ,其次为扁桃体、脾、肾 ,检出率为 83 3 % (2 0 / 2 4 ) ,再者为淋巴结 ,检出率为66 7% (1 6/ 2 4 )。对送检的 1 9份疑似猪瘟的病死猪病料组织进行RT PCR检测 ,结果有 1 6份样品为猪瘟病毒阳性。兔体交叉反应试验结果RT PCR阳性的 1 6份病料中 ,有 1 4份样品被判为含有猪瘟病毒 ,其他病料兔体交叉反应试验结果全为阴性     

Development of a polymerase chain reaction for the detection of Mycoplasma felis in domestic cats

Mycoplasma felis is associated with conjunctivitis and respiratory disease in domestic cats. Currently no rapid diagnostic test is available for the detection of M. felis in clinical samples that does not rely on prior cultivation of the organism. The objective of this study was to determine whether a polymerase chain reaction (PCR) based upon the 16S/23S rRNA intergenic spacer sequence is suitable for the identification of M. felis directly in feline clinical samples. The high conservation between the 16S/23S rRNA intergenic spacers (IGS) of differing isolates of M. felis was established by sequence analysis and a PCR was developed to this region by comparison to IGS of other mycoplasmas. The PCR was found to be highly specific for M. felis and further PCR analysis on clinical samples showed the PCR to be highly sensitive and more rapid than the other methods of identification currently available.