Real-time qPCR检测安氏隐孢子虫卵囊方法的建立及应用

根据GenBank公布的安氏隐孢子虫SSU rRNA基因序列设计1对引物和TaqMan探针,建立了基于TaqMan探针检测安氏隐孢子虫的实时荧光定量PCR方法,并对奶牛粪便进行了检测.结果显示,设计的探针对检测安氏隐孢子虫具有很高的特异性;粒DNA和卵囊的检测阈值分别达到5个拷贝和10个卵囊,奶牛粪便阳性率为21.15%(11/52).建立的安氏隐孢子虫TaqMan荧光定量PCR检测方法简便、快速,特异性强,敏感度高,可用于安氏隐孢子虫的快速定量检测.     

实时荧光定量PCR检测猪细小病毒

根据已报道的猪细小病毒基因组序列,设计并合成了引物和探针,从猪细小病毒(PPV)感染的细胞中提取DNA,经PCR扩增,产物纯化后与pGEM-T-easy连接,转化大肠杆菌JM109,筛选后得到重组标准品质粒,对重组标准品质粒进行PCR和测序鉴定,表明目的片段已经成功克隆.将104～108拷贝反应的重组标准品质粒进行荧光定量PCR,系统自动分析软件显示Ct值与标准品浓度的对数之间存在良好的线性关系.动力学曲线分析表明,在该反应体系和反应条件下,标准曲线的灵敏度为102拷贝.本方法的建立为猪细小病毒感染的早期诊断奠定了基础.     

Levels of detection of Cryptosporidium oocysts in mussels (Mytilus galloprovincialis) by IFA and PCR methods

Cryptosporidium spp. are monoxenous protozoan parasites that cause gastrointestinal diseases in humans and animals. Shellfish harvesting areas can become contaminated by the infectious stage of the parasite and humans are therefore at risk of infection either by consumption of shellfish, or by taking part in recreational activities in these areas. In the present study we determined the levels of detection, by IFA and PCR techniques, of Cryptosporidium oocysts in mussels experimentally contaminated with a theoretical number of oocysts. There was a significant correlation between the results obtained by both techniques (P<0.05). IFA and PCR were also applied to a total of 222 samples of mussels (Mytilus galloprovincialis) destined for human consumption. In the naturally contaminated samples, we detected a 31.1% of contamination and only Cryptosporidium parvum (previously denominated C. parvum genotype II) was identified.     

Detection of Cryptosporidium oocysts in soil samples by enzyme-linked immunoassay

An ELISA protocol was adapted for detection of Cryptosporidium parvum oocysts in soil samples and the limit of detection of the test was determined. A modified indirect antigen capture ELISA protocol was developed using monoclonal antibodies against the oocyst outer wall. The accuracy of the ELISA was compared to spiked soil samples and measured in terms of sensitivity and specificity of the test. The performance of the ELISA was evaluated in field soil samples and measured using the kappa-statistics. Similarly, the performance of the ELISA was compared to the concentration flotation method, to a modified concentration flotation method and to a commercial ELISA (ProSpecT) in field fecal and soil samples.The limit of detection of the test was selected to be 10,000 oocysts/g. At this limit of detection, the ELISA had a sensitivity of 95% and specificity of 100%. The agreement between the ELISA and the modified flotation-concentration method in detecting Cryptosporidium oocysts in soil samples was 32% (kappa=0.32). The ELISA had the same relative sensitivity (82%) in comparison to both the flotation and ProSpecT in determining Cryptosporidium-infection status of an animal. The kappa-statistics was 0.26 for both tests. The developed ELISA proved to be a valuable diagnostic test for detecting oocysts in soil samples and has a potential application in determining the infection status of animals.     

猪细小病毒与猪伪狂犬病毒二重SYBR GreenⅠ实时荧光PCR方法的建立与检测

本研究旨在建立一种能快速、灵敏、同时检测出猪细小病毒与猪伪狂犬病毒的基于SYBR GreenⅠ实时荧光定量PCR方法。参照GenBank中登录的相关基因序列,设计了2对引物分别用于扩增PRV gH基因与PPV NS1基因的部分片段。将测序正确的PRV gH基因与PPV NS1基因片段克隆入pGEM-T Easy载体,转化大肠杆菌DH5α,经测序鉴定后得到阳性重组质粒,作为标准品模板建立SYBR GreenⅠ荧光定量PCR标准曲线和熔解曲线,并对其灵敏性、特异性和重复性进行验证。结果表明,猪细小病毒与猪伪狂犬病毒荧光定量PCR的标准曲线的Tm值分别为80.9℃和86.5℃,熔解曲线特异,灵敏度分别可达248拷贝/μL和160拷贝/μL,是普通PCR检测方法的100倍。本次建立的猪细小病毒与猪伪狂犬病毒荧光定量PCR检测方法实现了2种病毒的同时检测,能够对PRV、PPV混合感染的临床病料进行快速诊断。     

Quantification of Mycobacterium avium subspecies in pig tissues by real-time quantitative PCR

Background

Mycobacterioses in animals cause economical losses and certain Mycobacterium avium subspecies are regarded as potential zoonotic agents. The evaluation of the zoonotic risk caused by M. avium subspecies requires information about the quantities of Mycobacterium strains in infected animals. Because M. avium subspecies in pig tissues are difficult or even impossible to quantify by culturing, we tested the suitability of a culture-independent real-time quantitative PCR (qPCR) assay for this purpose.

Methods

Mycobacterial DNA was extracted from porcine tissues by a novel method and quantified by Mycobacterium genus specific qPCR assay targeting the 16S rRNA gene.

Results

The response of the qPCR assay to the amount of M. avium subspecies avium mixed with porcine liver was linear in the range of approximately log10⁵ to log10⁷Mycobacterium cells per 1 g of liver. The assay was validated with three other M. avium subspecies strains. When the assay was applied to porcine lymph nodes with or without visible lesions related to Mycobacterium avium subspecies infections, around 10⁴–10⁷ mycobacterial genomes per gram of lymph nodes were detected.

Conclusions

The qPCR assay was found to be suitable for the quantification of Mycobacterium avium subspecies in porcine lymph nodes and liver.     

荧光定量PCR检测硬蜱体内莱姆病螺旋体的研究

为建立一个荧光定量PCR检测硬蜱体内莱姆病螺旋体的方法,根据GenBank登录的莱姆病螺旋体鞭毛蛋白FlaB序列,应用生物学软件进行序列比对,在保守的C段区设计与筛选特异引物和TaqMan探针。对荧光定量PCR反应体系与条件进行优化,验证方法的特异性、敏感性,并通过对感染螺旋体的蜱样本的检测,评价该方法的实用价值。结果显示,自然感染莱姆病螺旋体的35份蜱标本检测阳性符合率100%,正常蜱20份标本的检测结果均为阴性。该方法对牛巴贝斯原虫、泰勒原虫、边缘无浆体、金龟子绿僵菌、大肠杆菌等蜱体常见病原微生物所抽提的DNA的检测均呈阴性。荧光定量PCR方法检测质粒的灵敏度可达1×102拷贝/μL。TaqMan荧光定量PCR方法检测硬蜱体内莱姆病螺旋体具有较好的敏感性和特异性,可适于莱姆病的流行病学调查和监控。     

实时荧光定量PCR技术在生物饲料中的应用

生物饲料技术主要指通过微生物发酵,有效降解转化饲料中的抗营养因子,促进养分的消化吸收,分泌小肽、有机酸等功能物质,菌群演变是其重要内容。目前,发酵菌群的定量数据大多通过传统的依赖于培养的微生物学方法获得,由于其局限性,发酵饲料的微生物生态学尚未被很好地了解。因此,本文主要介绍了实时荧光定量PCR技术的原理和分类,综述了近年来实时荧光定量PCR技术在生物饲料研究领域中的应用及注意事项,为该领域的深入发展提供理论依据。     

猪伪狂犬病毒与猪圆环病毒2型二重SYBR GreenⅠ实时荧光PCR检测方法的建立

参照GenBank登录的相关基因序列,设计了2对引物分别用于扩增伪狂犬病毒(PRV)gH基因与猪圆环病毒2型(PCV2)ORF2基因的部分片段。将测序正确的PRV gH基因与PCV2ORF2基因片段克隆入pGEM-T Easy载体,转化大肠杆菌DH5α,经测序鉴定后得到阳性重组质粒,作为标准品模板建立SYBR GreenⅠ荧光定量PCR标准曲线和熔解曲线,并对其灵敏性、特异性和重复性进行验证。结果显示,PCV2与PRV荧光定量PCR的标准曲线的Tm值分别为80.8℃和86.7℃,熔解曲线特异,灵敏度分别可达215拷贝/μL和180拷贝/μL,是普通PCR检测方法的100倍。结果表明,建立的PCV2与PRV荧光定量PCR检测方法实现了2种病毒的同时检测,能够对PRV、PCV2混合感染的临床病料进行快速诊断。     

猪伪狂犬病毒与猪圆环病毒2型二重SYBR Green I实时荧光PCR检测方法的建立

参照GenBank登录的相关基因序列,设计了2对引物分别用于扩增伪狂犬病毒（PRV）gH基N与猪圆环病毒2型（PCV2）ORF2基因的部分片段。将测序正确的PRVgH基因与PCV2ORF2基因片段克隆入pGEMTEasy载体,转化大肠杆菌DH5a,经测序鉴定后得到阳性重组质粒,作为标准品模板建立SYBRGreenI荧光定量PCR标准曲线和熔解曲线,并对其灵敏性、特异性和重复性进行验证。结果显示,PCV2与PRv荧光定量PCR的标准曲线的Tm值分别为80．8℃和86．7℃,熔解曲线特异,灵敏度分别可达215拷贝／μL和180拷贝μL,是普通PCR检测方法的100倍。结果表明,建立的PCV2与PRV荧光定量PCR检测方法实现了2种病毒的同时检测,能够对PRV、PCV2混合感染的临床病料进行快速诊断。     

应用荧光PCR诊断鸡腺胃型传染性支气管炎

应用荧光PCR试剂盒对临床表现为消瘦和腺胃肿大为特征的患鸡病料进行鸡传染性支气管炎病毒(IBV)、禽网状内皮组织增殖病病毒(REV)和鸡马立克氏病病毒(MDV)核酸检测。结果显示:鸡传染性支气管炎病毒荧光RT-PCR检测为阳性,禽网状内皮组织增殖病病毒荧光RT-PCR检测为阴性,鸡马立克氏病病毒荧光PCR检测为阴性。结合患鸡精神沉郁,极度消瘦,腺胃肿大如球状等的临床表现,综合分析表明该患鸡发病主要病因为感染鸡腺胃型传染性支气管炎病毒。     

Rapidly quantitative detection of Nosema ceranae in honeybees using ultra-rapid real-time quantitative PCR

BackgroundThe microsporidian parasite Nosema ceranae is a global problem in honeybee populations and is known to cause winter mortality. A sensitive and rapid tool for stable quantitative detection is necessary to establish further research related to the diagnosis, prevention, and treatment of this pathogen.ObjectivesThe present study aimed to develop a quantitative method that incorporates ultra-rapid real-time quantitative polymerase chain reaction (UR-qPCR) for the rapid enumeration of N. ceranae in infected bees.MethodsA procedure for UR-qPCR detection of N. ceranae was developed, and the advantages of molecular detection were evaluated in comparison with microscopic enumeration.ResultsUR-qPCR was more sensitive than microscopic enumeration for detecting two copies of N. ceranae DNA and 24 spores per bee. Meanwhile, the limit of detection by microscopy was 2.40 × 10⁴ spores/bee, and the stable detection level was ≥ 2.40 × 10⁵ spores/bee. The results of N. ceranae calculations from the infected honeybees and purified spores by UR-qPCR showed that the DNA copy number was approximately 8-fold higher than the spore count. Additionally, honeybees infected with N. ceranae with 2.74 × 10⁴ copies of N. ceranae DNA were incapable of detection by microscopy. The results of quantitative analysis using UR-qPCR were accomplished within 20 min.ConclusionsUR-qPCR is expected to be the most rapid molecular method for Nosema detection and has been developed for diagnosing nosemosis at low levels of infection.     

Asymptomatic shedding of Cryptosporidium oocysts by red deer hinds and calves

Levels of Cryptosporidium infection in a group of red deer were monitored over a period of 1 year. Faecal samples were examined on an approximate monthly basis from adult hinds and calves for the presence of Cryptosporidium oocysts. The water-ether sedimentation method followed by sucrose flotation and a monoclonal antibody identification procedure were used. It was found that apparently healthy adult deer were shedding low numbers of oocysts in their faeces throughout the year and that there appeared to be a periparturient increase in the numbers of oocysts shed. Samples taken from 6-month-old deer calves, both in-house and on pasture, had low numbers of Cryptosporidium oocysts, indicating that the calves were also asymptomatically shedding oocysts.     

Detection of equine herpesvirus-1 in nasal swabs of horses by quantitative real-time PCR

Background: Early identification of inhalation-transmitted equine herpesvirus type 1 (EHV-1) infections has been facilitated by the availability of a number of real-time quantitative PCR (qPCR) tests. A direct comparison between nasal swab qPCR and traditional virus isolation (VI) requires a method for normalizing the qPCR samples and controlling for PCR inhibitors present in some clinical samples.
Objectives: To quantify EHV-1 shedding in viral swabs using an internal control and to compare fast qPCR to VI for the detection of EHV-1 in nasal swabs from horses.
Animals: Fifteen horses experimentally infected with EHV-1.
Methods: Experimental study : Nasal swab samples were collected daily after experimental infection for up to 21 days. VI was performed by conventional methods. The DNA was prepared for qPCR with the addition of a known quantity DNA of Marek's disease virus as an internal control. qPCR was performed.
Results: The qPCR method detected virus up to day 21 after challenge, whereas VI detected virus only to day 5. The median Kaplan-Meier estimates for EHV-1 detection were 12 days for qPCR and 2 days for VI ( P < .0001). When compared with VI, the sensitivity and specificity of qPCR were 97 (95% CI: 86–100) and 27% (95% CI: 20–35).
Conclusions and Clinical Importance: We conclude that fast qPCR of nasal swab samples should be chosen for diagnosis and monitoring of herpesvirus-induced disease in horses. Recommended reference ranges of C _T values are provided as well as justification of a minimum 10-day quarantine period.     

猪源隐孢子虫卵囊的分离及基因型鉴定

应用RT-PCR方法对南京、上海和合肥猪源隐孢子虫卵囊SSU rRNA部分序列进行扩增,产物测序后提交GenBank,收录号为DQ855266、DQ855267;用BLAST和DNAStar软件与GenBank参考序列进行比较,分析其同源性,绘制系统发育进化树,结合卵囊形态学观察和对小鼠、大鼠、兔、山羊和鸡的传染性试验确定隐孢子虫种类或基因型。结果表明,3地区猪源隐孢子虫分离株与微小隐孢子虫(C.parvum)同源性达94%~100%,与C.parvummouse型有99.8%~100%的同源性,并处于进化树的同一分支。因此,3地区猪源隐孢子虫是C.parvummouse型,提示猪和鼠之间存在交叉传播的可能。     

Detection of Actinobacillus pleuropneumoniae in pigs by real-time quantitative PCR for the apxIVA gene

A real-time quantitative PCR (qPCR) for detection of the apxIVA gene of Actinobacillus pleuropneumoniae was validated using pure cultures of A. pleuropneumoniae and tonsillar and nasal swabs from experimentally inoculated Caesarean-derived/colostrum-deprived piglets and naturally infected conventional pigs. The analytical sensitivity was 5colony forming units/reaction. In comparison with selective bacterial examination using tonsillar samples from inoculated animals, the diagnostic sensitivity of the qPCR was 0.98 and the diagnostic specificity was 1.0. The qPCR showed consistent results in repeatedly sampled conventional pigs. Tonsillar brush samples and apxIVA qPCR analysis may be useful for further epidemiological studies and monitoring for A. pleuropneumoniae.     

基于TaqMan探针的Real-time PCR定量检测空肠弯曲杆菌

空肠弯曲杆菌 (Campylobacterjejuni ,C .jijuni)被认为是人类主要食源性病原菌之一。由于空肠弯曲杆菌特殊的生长条件和容易进入不可培养但存活的状态 (Viablebutnonculturable ,VNC) ,所以传统的生化鉴定结果并不一定可靠 ,并且是一项费时而繁琐的工作。核酸检测方法的出现为空肠弯曲杆菌的检测带来了方便。在本文基于LightCycler为平台 ,建立一种基于TaqMan探针的Real_timePCR方法来定量检测空肠弯曲杆菌。用该方法检测时 ,发现所有空肠弯曲杆菌 (11株 )都呈阳性 ,所有其它弯曲菌 (3株 )和其它菌株 (5株 )都是阴性。整个检测过程 6 0min内可以完成 ,检测限度为5CFU ,标准曲线的相关系数为 0 .988。结果表明荧光定量PCR方法既为空肠弯曲杆菌提供了一种特异、敏感、快速和简洁的定量检测方法 ,又为研究空肠弯曲杆菌致病机理提供了一种重要方法。     

应用巢式PCR检测隐孢子虫

应用巢式聚合酶链反应(Nested PCR)建立了一种检测隐孢子虫(Cryptosporidium)的方法。试验中隐孢子虫卵囊纯化采用庶糖密度梯度离心法,以液氮-热水浴反复冻融及酚-氯仿抽提冷乙醇沉淀法制备模板DNA,根据隐孢子虫18S rRNA序列高度保守区设计2对引物,建立Nested PCR诊断方法。该方法特异性强,可检出牛源微小隐孢子虫(C.parvum)、羊源微小隐孢子虫(C.parvum)、牛源安氏隐孢子虫(C.andersoni)、鸡源贝氏隐孢子虫(C.baileyi)及猪源隐孢子虫(C.suis);敏感性高,该方法最低核酸DNA检测量达到10fg。初步应用结果表明,所建立的Nested PCR方法适合于隐孢子虫病的诊断和分子流行病学调查。     

副猪嗜血杆菌及巴氏杆菌双重荧光定量PCR检测方法的建立

为建立副猪嗜血杆菌(Hps)、多杀性巴氏杆菌(Pm)的双重荧光定量PCR检测方法,本研究基于Hps的Omp P2基因,Pm的PlpE基因设计两对特异性引物及探针,通过对反应条件优化,建立了一种同时检测Hps及Pm的双重荧光定量PCR方法。该方法能够特异性地检测Hps和Pm,其对重组质粒标准品的最低检测浓度分别为5.60×10^2拷贝/μL、7.58×10^2拷贝/μL。双重与单一荧光定量PCR最低检测限相同,且均是常规PCR的100倍。重复性试验结果显示,该方法的组内和组间变异系数均小于2.5%。临床应用结果显示:该方法对阳性样品的检出率为53.57%,明显优于常规PCR和细菌分离鉴定。该方法能够用于两种疾病的同时检测和快速排查疾病。为两种疾病的防治提供有效检测工具。     

实时定量PCR对猪圆环病毒2型感染猪体内病毒分布规律的研究

根据发表的猪圆环病毒2型（PCV2）ORF2基因序列设计了一对特异引物,并以克隆重组质粒作为阳性标准品,采用SYBR-GreenⅠ嵌合荧光染料建立了一种用于检测PCV2的实时定量PCR方法。该方法在10^7～10^1范围内具有良好的线性关系,相关系数为R2=0．997,扩增效率为E=0．920;对质粒标准品检测下限值为8．2拷贝／μL,检测变异系数低于2．0％。该方法与常规PCR及过氧化物酶单层细胞试验（IPMA）比较,其敏感性提高10^3倍以上。采用该法对PCV2人工感染猪的心、肝、脾、肺、肾、腹股沟淋巴结等组织中病毒核酸载量检测结果表明,在多种脏器中均可检9n,4到病毒,其中腹股沟淋巴结、扁桃体和脾脏中病毒含量较高（为3．4&#215;10^9拷贝儋～1．7&#215;10^10拷贝／g）,这表明病毒主要在免疫器官增殖,导致淋巴细胞耗损。对来自国内不同地区的28份临床发病猪病料进行病毒DNA定量检测,有半数以上病料的病毒载量达到10^9-10拷贝／g。实验表明,实时定量PCR可用于PCV2核酸定量检测,为该病毒体内外定量检测提供了一种技术手段。