PCR技术检测饲料中沙门氏菌的应用研究

用聚合酶反映(PCR)技术检测饲料中沙门氏菌。对已知被沙门氏菌污染的动物饲料的培养物均能检出阳性,说明此方法对检测饲料中沙门氏菌具有特异性高、灵敏、快速等特点,适用于快速和准确地检测饲料中沙门氏菌的需要。     

用PCR技术检测动物产品中沙门氏菌的研究

用聚合酶链反应（ＰＣＲ）技术检测沙门氏菌，结果表明，对各种不同血清型的沙门氏菌和已知被该菌污染的鱼粉和动物产品的肉汤培养物均检出阳性，而其他的枸橼酸杆菌、奇异变形杆菌，大肠艾希氏杆菌、缓慢爱德华氏菌和嗜水气单胞菌等非沙门氏菌类，均为阴性。证明本方法特异性高且有灵敏、快速等特点，适用于口岸检疫进出境动物产品快速、准确验放的需要。     

应用聚合酶链反应技术检测沙门氏菌

利用聚合酶链反应（ＰＣＲ）检测沙门氏菌,使用了２对引物ＨＩ和ｐｈｏＰ。ＨＩ引物对各长２０ＮＴ,根据鞭毛Ｉ相抗原基因自行设计,扩增序列长２６９ｂｐ;ｐｈｏＰ引物对各长２１ＮＴ,根据ｐｈｏＰ／ｐｈｏＱ基因设计,扩增片段长２９９ｂｐ。ＰＣＲ采用５０μＬ反应体系。ｄＮＴＰＳ各１００μｍｏｌ／Ｌ,引物各１μｍｏｌ／Ｌ,Ｍｇ２＋２．５ｍｍｏｌ／Ｌ,酶２Ｕ。三温段ＰＣＲ循环条件为：９７℃预变性７ｍｉｎ;９４℃变性６０ｓ、５５℃复性４０ｓ、７２℃延伸６０ｓ,３５个循环;７２℃延伸７ｍｉｎ。检测８株标准阳性菌,结果都出现了ＨＩ引物和ｐｈｏＰ引物特异带,标准阴性菌３株只出现ｐｈｏＰ特异带,说明ＨＩ引物特异性很强。     

沙门氏菌PCR诊断试剂盒的研制及初步应用

采用酚提取法和裂解法制备聚酶链反应（ＰＣＲ）模板,并制备了ＰＣＲ诊断试剂盒,用于沙门氏菌。结果,粪便样品只能采用酚提取法制备模板,而血液样品可采用裂解法,灵敏度高达５个沙门氏菌ＤＮＡ水平,最佳反应时间为变性、退火各４０ｓ,延伸５０ｓ。用该分水岭 工发病和自然发病动物血液、粪便中的沙门氏菌,阳性纺均比２法高,且培养法阳性的样品ＰＣＲ法均为阳性,整个检测过程仅需６－８ｈ。     

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。     

沙门氏菌临床检测比较研究

比较研究了临床检测沙门氏菌的三种不同方法，采用聚合酶链反应技术，玻片免疫凝集试验和生化检测方法对３０份样品检出阳性率分别８３％，７３％和３０％，同时讨论了三种方法的优缺点。     

饲料中沙门氏菌的检测

本实验通过常规分离培养鉴定技术,对某饲料厂随机抽取的30份鱼粉样品进行了沙门氏菌的分离鉴定;结果从这些份样品中分离到四株沙门氏菌菌株,分别为鼠伤寒沙门氏菌2株,鸭沙门氏菌1株,肠炎沙门氏菌1株,阳性率为13.3%。     

饲料中沙门氏菌的检测技术研究

配合饲料及动物性饲料如鱼粉、骨粉等是动物摄取蛋白质、钙、磷的主要来源，也适合沙门氏菌及其他致病菌的生存。国内文献已多次报道因沙门氏菌污染饲料而导致畜禽大量死亡的事例，给畜牧业造成巨大的经济损失。沙门氏菌是最常见的人畜共患病原菌之一，能引起人和动物肠热症、胃肠炎、败血症等。因此必须在饲料原料、饲料加工生产中严格控制沙门氏菌，准确检测出饲料中的沙门氏菌是质量控制的一个重要环节。沙门氏菌共有2500以上的血清型，目前对该菌的检测主要有常规检测、免疫酶试验、免疫扩散法、乳胶凝集试验、荧光抗体法、酶联免疫吸附试验及PCR等方法。     

畜禽饲料中沙门氏菌的检测

2005年3～9月,对318份畜禽饲料样品进行沙门氏菌实验室检测,通过预增菌、选择性增菌和选择性分离培养及生化试验、血清学鉴定等检验,结果有4份样品检出沙门氏菌,检出阳性率为1.26%,其中3份是猪浓缩饲料、1份是鱼粉.     

应用PCR技术检测鹅细小病毒

根据鹅细小病毒GPVH1株结构蛋白VP1与VP3编码基因非重叠核苷酸序列设计了一对引物GPR1 /GPR2 ,用这对引物对感染器官内的GPR和接种鹅胚增殖的GPV进行PCR扩增 ,其结果引物GRP1 /GPR2能特异性地扩增GPVVP1 VP3区段核苷酸序列 ,扩增出与设计核苷酸片段大小相符的 0 6kb的序列 ,而对照的鹅副粘病毒cDNA及鸭瘟病毒 (DPR)DNA对照组均出现阴性结果。     

Application of the polymerase chain reaction (PCR) in veterinary diagnostic virology

The polymerase chain reaction has become an important diagnostic tool for the veterinary virologist. Conventional methods for detecting viral diseases can be laborious or ineffective. In many cases PCR can provide a rapid and accurate test. In this article we explain the basic principles of PCR and supply a reference list of its uses in diagnostic veterinary virology.Abbreviations BLV bovine leukaemia virus - BVDV bovine viral diarrhoea virus - DNA deoxyribonucleic acid - ds double-stranded - ELISA enzyme-linked immunosorbent assay - PCR polymerase chain reaction - RNA ribonucleic acid - ss single-stranded - TK thymidine kinase     

动物饲料中沙门氏菌检测技术的研究进展

本文就饲料中沙门氏菌污染现状,微生物学方法及生化鉴定方法、分子生物学诊断方法在沙门氏菌检测中的应用进行综述。     

Use of nested polymerase chain reaction (PCR) for detection of retroviruses from formalin-fixed, paraffin-embedded uveal melanomas in cats

Thirty-six formalin-fixed, paraffin-embedded enucleated globes from cats with a diagnosis of diffuse anterior uveal melanoma were obtained. Sections of tumor were excised, deparaffinized, and subjected to nested polymerase chain reaction (PCR) to identify proviral DNA sequences from the feline leukemia virus (FeLV)–feline sarcoma virus (FeSV; 36 eyes), and the feline immunodeficiency virus (FIV; 18 eyes). All samples tested were negative for FIV DNA. Three samples were positive for FeLV–FeSV DNA. This is the first reported evidence of a possible link between naturally occurring feline anterior uveal melanoma and the presence of FeLV–FeSV DNA.     

RT-PCR技术诊断猪瘟的应用研究

应用反转录—聚合酶链反应(RT-PCR)对猪瘟进行诊断应用研究。应用RT-PCR况对来自广西不同地区的135份疑似猪瘟病料进行检测，以份诊断为阳性，阳性率62．2％。从百色、柳州地区等采集的健康猪扁桃体和淋巴结共276份，经RT-PCR检测，37份为阳性，阳性率为13．4％。其中健康猪扁桃体带毒较高，246份扁桃体中有35份阳性，占14．2％。采自柳州健康猪的26份淋巴结材料全为阴性，只有邕宁县的1份健猪淋巴结阳性。结果表明，RT-PCR技术可应用于猪瘟的临床诊断。     

A polymerase chain reaction (PCR) assay for the detection of bovine herpesvirus 1 (BHV1) in selectively digested whole bovine semen

A PCR assay for the detection of bovine herpesvirus type 1 (BHV1) DNA in selectively digested whole bovine semen was developed and evaluated. A brief treatment with proteinase-K was used to lyse free virus, virus present in non-sperm cells and virus adhering to the spermatozoa. Genomic bovine DNA was not released by this treatment. Primers and probes were based on the nucleotide sequence of the gD gene. BHV1 virus-spiked split samples were used as positive controls and the PCR products were detected by eye in ethidium bromide-stained agarose gels. Sequentially collected non-extended semen samples from experimentally infected bulls were used to compare this assay with virus isolation. Of a total of 162 ejaculates, 51 were found positive by virus isolation, whereas PCR detected BHV1 DNA in 73. PCR detected BHV1 DNA for a longer period after infection and reaction. Apart from its superior sensitivity, this PCR assay also has the advantage of being a relatively simple procedure, providing results within 24 h.Abbreviations AI artificial insemination - BHV1 bovine herpesvirus type 1 - PCR polymerase chain reaction - TCID₅₀ tissue culture infective dose, 50%     

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。     

Real-time polymerase chain reaction: a novel molecular diagnostic tool for equine infectious diseases

The focus of rapid diagnosis of infectious disease of horses in the last decade has shifted from the conventional laboratory techniques of antigen detection, microscopy, and culture to molecular diagnosis of infectious agents. Equine practitioners must be able to interpret the use, limitations, and results of molecular diagnostic techniques, as they are increasingly integrated into routine microbiology laboratory protocols. Polymerase chain reaction (PCR) is the best-known and most successfully implemented diagnostic molecular technology to date. It can detect slow-growing, difficult-to-cultivate, or uncultivatable microorganisms and can be used in situations in which clinical microbiology diagnostic procedures are inadequate, time-consuming, difficult, expensive, or hazardous to laboratory staff. Inherent technical limitations of PCR are present, but they are reduced in laboratories that use standardized protocols, conduct rigid validation protocols, and adhere to appropriate quality-control procedures. Improvements in PCR, especially probe-based real-time PCR, have broadened its diagnostic capabilities in clinical infectious diseases to complement and even surpass traditional methods in some situations. Furthermore, real-time PCR is capable of quantitation, allowing discrimination of clinically relevant infections characterized by pathogen replication and high pathogen loads from chronic latent infections. Automation of all components of PCR is now possible, which will decrease the risk of generating false-positive results due to contamination. The novel real-time PCR strategy and clinical applications in equine infectious diseases will be the subject of this review.     

RT-PCR技术检测猪瘟病毒的应用研究

本试验应用反转录-聚合酶链式反应(RT-PCR)对猪瘟进行诊断应用研究.应用RT-PCR对来自广西不同地区的135份疑似猪瘟病料进行检测,84份诊断为阳性,阳性率62.2%.从百色、柳州地区等采集的健康猪扁桃体和淋巴结共276份,经RT-PCR检测,37份为阳性,阳性率为13.4%.其中健康猪扁桃体带毒较高,246份扁桃体中有35份阳性,占14.2%.采自柳州健康猪的26份淋巴结材料全为阴性,只有邕宁县的1份健猪淋巴结阳性.结果表明,RT-PCR技术可应用于猪瘟的临床诊断.