PCR技术检测猪肺炎支原体的研究进展

猪肺炎支原体(Mycopiasma hyopneumoniae)是引起猪支原体肺炎的重要病原,该病常引起继发感染和混合感染,严重威胁养猪业发展,造成巨大的经济损失.利用PCR技术对猪支原体肺炎早期正确诊断具有非常重要的意义.从猪肺炎支原体的特异性靶基因、临床样品采集方法与样品DNA处理方法、关键技术因素及普通PCR技术、多重PCR技术、套式PCR技术、荧光定量PCR技术、芯片检测和环介导等温扩增技术等在猪肺炎支原体检测中的研究进展、主要优缺点及应用进行综述.     

猴D型反转录病毒巢式PCR检测方法的建立

目的建立SRV-1巢式PCR检测方法并进行初步应用。方法针对SRV-1env基因的保守区序列,设计特异性引物,以感染SRV-1 Raji细胞提取出的含有前病毒DNA的基因组DNA为模板,进行巢式PCR反应。扩增产物测序后与GenBank报道的序列进行同源比对。将DNA样本进行10倍梯度稀释,以检测巢式PCR反应的灵敏度。使用该方法对正常Raji细胞以及感染SIV、STLV的外周血淋巴细胞DNA样本进行扩增,检测该方法的特异性。用建立的巢式PCR方法检测40份储存猴血标本。结果使用巢式PCR扩增出的特异片段经测序分析,结果证实与GenBank报道的序列一致。所建立的巢式PCR检测法检测限度可达1.5×10-3ng/μL,而且方法特异。用此方法检测40份猴血标本,未检测到阳性标本。结论初步建立SRV-1的巢式PCR检测方法,该方法灵敏、特异,为SRV-1的检测提供了一个快速、有效的手段。     

实时荧光定量PCR在猪肺炎支原体检测中的应用

实时荧光定量PCR技术是一种利用荧光检测方法来定量核酸的技术,具有高度的灵敏性、特异性和精确性.综述了荧光定量PCR技术的基本原理及其在猪肺炎支原体检测中的应用.     

Detection of Plasmodium vivax by Nested PCR and Real-Time PCR

Malaria is endemic in the Cukurova region while it is sporadic in other regions of Turkey. Therefore, the laboratory and clinical diagnosis of malaria is important for the treatment of malaria. In this study, 92 blood samples that were taken from the suspected malaria patients for routine diagnosis in a period of 10 years between 1999 and 2009 were analyzed. All of these blood samples were examined by microscopic examinations using Giemsa-stained thick blood films, nested PCR, and real-time PCR. The sensitivity-specificity and positive-negative predictive values for these diagnostic tests were then calculated. It was found that the positive predictive values of microscopic examination of thick blood films, nested PCR, and real-time PCR were 47.8%, 56.5%, and 60.9% for malaria, respectively. The real-time PCR was found to have a specificity of 75% and sensitivity of 100%, while specificity and sensitivity of nested PCR was found 81.2% and 97.7% according to the microscopic examination of thick blood films, respectively.     

绵羊肺腺瘤病特异性PCR及套式PCR诊断方法的建立

绵羊肺腺瘤是由外源性反转录病毒JSRV引起的接触性传染的肺肿瘤性疾病。感染羊没有针对JSRV的循环抗体,但在感染羊的肺肿瘤组织、淋巴网状系统及外周血单核细胞中可检测到外源性前病毒exJSRV及JSRV转录产物。健康羊基因组中存在15~20拷贝的内源性前病毒enJSRV,内外源性前病毒的结构基因高度相似,而在U3区存在差别,因而,设计了针对exJSRVU3区的特异性引物并在国内首次建立了一步法特异性PCR检测法及套式PCR检测法。以700ng健康羊基因组DNA为背景,梯度稀释阳性质粒pJSRV-LTR作为模板,比较两种方法的敏感性,结果表明套式法的敏感性是一步法的10倍以上,套式法也是目前可用于检测感染羊血液样品的唯一方法,可望在绵羊肺腺瘤病的流行病学调查及防控方面起重要作用。     

运用套式PCR检测传染性喉气管炎病毒核酸

选择鸡传染性喉气管炎病毒保守TK基因的蛋白编码区域,设计并合成了一对外引物和一对内引物,建立并优化了检测鸡传染性喉气管炎病毒DNA的套式PCR法.通过检测ILTV感染的鸡胚绒毛尿囊膜、实验室病料和临床病料,结果表明,套式PCR法能检测出ILTV感染后的非免疫鸡胚和SPF鸡胚绒毛尿囊膜研磨液中的被稀释了105倍的病毒(约1fg的ILTVDNA),攻毒后第10天还能从非免疫鸡和SPF鸡气管拭子中检出ILTV,第10天非免疫鸡气管拭子中ILTV的最大检出率为7/10,第10天SPF鸡气管拭子中ILTV的最大检出率为8/10.对非免疫鸡和SPF鸡的气管拭子中ILTV最佳检出时间均在攻毒后第5天.对临床样品中的ILTV的最大检出率为7/7.经过核酸杂交验证,套式PCR法具有很高的特异性和敏感性,为从分子水平探讨ILTV的发病机理、临床早期快速诊断提供了新的研究手段.     

Identification of Mycoplasma hyopneumoniae with a DNA probe

From a genomic library of Mycoplasma hyopneumoniae a 1.3 kb DNA fragment was cloned which showed specific Southern hybridization and dot hybridization with the type strain of several porcine and bovine Mycoplasma species. This probe selectively recognized M. hyopneumoniae sequences in purified DNA or in broth-grown organisms. The 35S-labelled probe could detect as little as 100 pg of DNA or 10(5) colour changing units. This is a possible alternative diagnostic procedure for enzootic pneumonia of pigs.     

In Situ Detection of the Clostridium botulinum Type C1 Toxin Gene in Wetland Sediments with a Nested PCR Assay

A nested PCR was developed for detection of the Clostridium botulinum type C₁ toxin gene in sediments collected from wetlands where avian botulism outbreaks had or had not occurred. The C₁ toxin gene was detected in 16 of 18 sites, demonstrating both the ubiquitous distribution of C. botulinum type C in wetland sediments and the sensitivity of the detection assay.     

大豆油中转基因成分的Nested PCR和Semi-nested PCR检测方法研究

对大豆油中DNA提取方法进行了研究,结果表明CTAB、SDS和Wizard试剂盒提取大豆油DNA均具有良好的效果。利用nested PCR和semi—nested PCR检测大豆（Roundup Ready）油中的转基因成分发现,该方法能够检测到大豆原油中的Lectin基因（112bp）、CaMV35S基因（147bP）和CP4-EPSPS基因（205bp）,检测灵敏度达到10^-6ng／μl;但该方法未能从人豆成品油（一级）中扩增到上述基因,当中的转基因成分DNA含量低于10^-12ng／μl。     

Detection of Sclerotinia sclerotiorum in Planta by a Real-time PCR Assay

Stem rot caused by Sclerotinia sclerotiorum is a very serious disease on oilseed rape worldwide. In this study, a pair of polymerase chain reaction (PCR) primers was designed based on the nucleotide sequence of a DNA region amplified by a microsatellite primer M13. The primer pair amplified a 252-bp fragment from all S. sclerotiorum isolates collected from oilseed rapes at different locations in different years, but not from any other fungus tested. Using this pair of primers, a real-time PCR assay was developed to rapidly detect early infection of S. sclerotiorum on petals of oilseed rape. The real-time PCR assay developed in this study could help growers make a timely decision on fungicide application.     

PCR技术在鼠肺支原体检测中应用的初步研究

目的　探讨PCR技术在鼠肺支原体检测中的应用,希望能建立一种可行、快速、敏感的检测方法。方法　使用支原体通用引物及鼠肺支原体特异性引物对14 份大鼠喉气管拭子洗液和拭子支原体培养液进行PCR扩增,2 % 琼脂糖电泳鉴定。另设M53 和ATCC19612 二株标准鼠肺支原体菌株作阳性对照。结果　通用引物对大鼠喉气管拭子洗液检出率8/14 ,拭子支原体培养液检出率14/14,鼠肺支原体特异引物PCR扩增对大鼠喉气管拭子洗液检出率0/14 ,拭子支原体培养液3/14。通用引物扩增M53 和ATCC19612 二株标准株均呈现阳性,而鼠肺支原体特异引物扩增M53 和ATCC19612,只有M53 呈现阳性。结论　PCR通用引物检测比普通分离培养省时省力,而我们采用国外某学者认为对鼠肺支原体有特异性的引物,是否可用于鼠肺支原体的特异性PCR 检查仍需进一步探讨。     

Rapid Detection of Ceratocystis platani Inoculum by Quantitative Real-Time PCR Assay

Ceratocystis platani is the causal agent of canker stain of plane trees, a lethal disease able to kill mature trees in one or two successive growing seasons. The pathogen is a quarantine organism and has a negative impact on anthropogenic and natural populations of plane trees. Contaminated sawdust produced during pruning and sanitation fellings can contribute to disease spread. The goal of this study was to design a rapid, real-time quantitative PCR assay to detect a C. platani airborne inoculum. Airborne inoculum traps (AITs) were placed in an urban setting in the city of Florence, Italy, where the disease was present. Primers and TaqMan minor groove binder (MGB) probes were designed to target cerato-platanin (CP) and internal transcribed spacer 2 (ITS2) genes. The detection limits of the assay were 0.05 pg/μl and 2 fg/μl of fungal DNA for CP and ITS, respectively. Pathogen detection directly from AITs demonstrated specificity and high sensitivity for C. platani, detecting DNA concentrations as low as 1.2 × 10⁻² to 1.4 × 10⁻² pg/μl, corresponding to ∼10 conidia per ml. Airborne inoculum traps were able to detect the C. platani inoculum within 200 m of the closest symptomatic infected plane tree. The combination of airborne trapping and real-time quantitative PCR assay provides a rapid and sensitive method for the specific detection of a C. platani inoculum. This technique may be used to identify the period of highest risk of pathogen spread in a site, thus helping disease management.     

巢式PCR检测食蟹猴和猕猴中SRV和SFV

目的利用巢式PCR方法检测圈养的食蟹猴（Macaca fascicularis）和猕猴（Macaca mulatta）中猴D型逆转录病毒（Simian Type D Retrovirus SRV）和猴泡沫病毒（Simian foamy virus SFV）。方法针对SRV-env和SFV-pol基因的保守区序列设计特异性外引物,然后再设计特异性内引物。将外引物扩增出的片段克隆到PJET1.2blunt载体中作为阳性对照,运用NCBI中BLAST软件比对测序结果。用巢式PCR方法分别检测食蟹猴和猕猴中SRV和SFV。结果发现食蟹猴中SFV感染率为65.2%,SRV感染率为9.5%,猕猴中SFV感染率为60.5%,SRV感染率为12.8%。结论圈养的食蟹猴和猕猴SFV的感染率均较高,SRV感染率很低。