猴副流感病毒SV5 PCR检测方法的建立与初步应用

目的建立检测SV5的PCR方法并加以初步应用。方法根据GenBank中报道的SV5序列,针对其中的SH基因设计引物进行PCR反应,扩增产物进行测序并用BLAST软件进行同源性比对,同时利用限制性内切酶的酶切反应以证实此PCR反应的特异性。在此基础上设计巢式PCR提高此方法的灵敏度。利用此方法对20份猴肾源细胞培养物和40份血清标本进行检测。结果利用设计的引物扩增出的序列测序结果证实与报道的SV5SH基因相对位置的序列一致。AccⅢ限制性内切酶可对PCR产物进行特异性酶切。巢式PCR比一次PCR的敏感度有所提高。用此方法检测的20份猴肾源细胞培养物和40份血清标本结果为阴性。结论初步建立了检测SV5病毒的PCR方法,排除实验室用20份猴肾源细胞培养物和40份血清标本SV5的污染。     

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。     

应用逆转录酶链反应对我国流行性出血热病毒分型

针对我国流行性出血热病毒的S片段核蛋白 (NP)编码基因保守核苷酸序列 ,合成了一对引物 ,扩增出编码核蛋白的 5 90bp碱基。扩增的II型产物存在限制性内切酶PstI的酶切位点 ,而I型产物不存在。因此 ,用酶切扩增产物的方法达到病毒分型的目的。试验提取 8份鼠脑传代毒种 ,1份细胞培养物和 2 5份病人血清中的出血热病毒RNA ,同时做 6份正常人血清对照 ,经逆转录PCR酶切分型。试验结果同间接免疫荧光技术 (IFA)分型结果相一致 ,可特异地对我国流行性出血热病毒进行分型。     

SIV模型猴石蜡包埋淋巴结组织DNA的提取及病毒基因检查

从取材于３只ＳＩＶ感染治疗猴和４只ＳＩＶ艾滋病模型猴治疗前后的１１份石蜡包埋淋巴结活检组织中提成基因组ＤＮＡ,分别用ＰＣＲ和巢式ＰＣＲ方法检测ＳＩＶ病毒基因,ＰＣＲ共检出１０份阳性,巢式ＰＣＲ检测１１价样品均阳性,而同期采样的猴外周血标本病毒分离仅３份阳性,ＰＣＲ扩增产物的特异性用限制性内切酶酶切反应得到证实。实验说明,从淋巴结组织中检测ＳＩＶ的病毒基因较外周血病毒分离更能真实地反映病毒感染状况,本研究将对全面和正确评价艾滋病药吻和疫苗治疗效果提供帮助。     

猕猴巨细胞病毒(RhCMV)nested PCR检测方法的建立与初步应用

目的建立猴巨细胞(RhCMV)病毒的nested PCR检测方法,并初步应用。方法根据GenBank中报道的RhCMV全基因序列,针对其中的保守区域Rh85设计两对引物进行nested PCR反应,利用此方法对20份猕猴全血标本进行检测,将检测到的猕猴阳性标本扩增片段进行克隆测序。结果利用保守区域Rh85设计的引物可对人HCMV阳性对照进行扩增。用此方法检测的20份猕猴全血标本,出现2例阳性。其中一例扩增片段经纯化、回收克隆测序后用BLAST软件进行同源性对比,与GenBank中报道的RhCMV序列基本相同。结论建立了从猴全血中直接检测猴RhCMV病毒DNA的敏感、特异的nested PCR方法。     

犬腺病毒、犬细小病毒联合PCR方法的建立与应用

根据GenBank报道的犬腺病毒(CAV)和犬细小病毒(CPV)序列,设计两对联合PCR引物,其中一对为CAV-1和CAV-2通用引物,另一对为CPV引物。在建立单项PCR基础上,通过优化Mg^2 离子浓度和循环参数等反应条件建立联合PCR,确定联合PCR条件为：96℃180s,96℃230s,56℃30s,72℃280s,30个循环。联合PCR结果显示：CAV—l扩增片段大小为497bp,CAV-2为l019hp,CPV为719hp;细胞和其它相关病毒对照均无扩增带。上述PCR产物经用限制性内切酶酶切和克隆测序,结果均与相应病毒的应有条带和序列相同。敏感性比较试验结果表明。联合PCR比用细胞培养分离病毒敏感。将联合PCR应用于15份CAV和CPV细胞培养物,5份CAV-1、1份CAV-2和3份CPV人工感染犬病料以及30份临床病料检测,并与电镜负染、HA／HI及病毒分离等方法的结果进行比较,结果显示,联合PCR的检出率和病毒分离结果一致,高于电镜负染和HA／HI试验。以上结果说明：CAV-1／12AV-2和CPV联合PCR不仅具有很好的特异性和敏感性,而且可以在短时间内(2．5h～3h)同时鉴定出上述三种病毒。因此具有良好的实验室诊断和临床应用价值。     

HCV E2区基因的分子克隆及序列分析

用RT－PCR KIT从西安血站大样抗HCV阳性血清中筛选出HCV RNA阳性血清,提取HCV的RNA,利用随机引物反转录合成其cDNA并进行半巢式PCR反应。将纯化的PCR产物酶切后与表达载体PET－22b^＋连接,经过双脱氧末端终止法双向测序,得到852bp长的核苷酸序列,通过将该序列与已知不同型的HCV E2序列比较得知,此序列正是HCVⅡ型目的基因。     

肺炎链球菌自溶素和溶血素基因的PCR法鉴定

肺炎链球菌是一种致病率和致死率很高的病原菌 ,若无丰富临床检测经验从临床标准中分离鉴定此菌较困难 ,本实验以寡核苷酸引物YH1 YH2、YH7 YH8分别扩增肺炎链球菌自溶素和溶血素基因的 35 4bp和 30 7bp的DNA片段 ,通过改变各种反应条件 ,建立了这两种病原因子基因的PCR检测方法。用此方法对 2 0株肺炎链球菌标准菌株及 7株对照菌株进行了鉴定 ;其扩增产物分别经限制性内切酶TthHB81和和AccI进行酶切以确认扩增产物是否正确 ;用酚 氯仿抽提纯化的全细胞DNA对PCR方法的检测灵敏度进行了测定 ;并利用此方法对 2 8份临床标本分离物进行了鉴定。结果　所有 2 0株肺炎链球菌均可分别用引物YH1 YH2、YH7 YH8扩增出 35 4bp和 30 7bp的DNA片段 ,而对照菌株均呈阴性 ;自溶素及溶血素基因的扩增产物分别经限制性内切酶TthHB81和AccI消化后产生的片段和预期的完全一致 ;两对产物均可从 10fg的全细胞DNA中扩增出目的DNA片段。所建立的两套PCR系统对 2 8份临床标本分离物进行鉴定 ,其中PCR阳性的 15份分离物经生化学特性检查被鉴定为肺炎链球菌。本试验所建立的两套PCR检测系统具有特异性强 ,灵敏度高及操作简单等优点 ,均可用于肺炎链球菌的鉴定。     

人白细胞介素-36受体拮抗剂基因的克隆及序列分析

[目的]构建人白细胞介素-36受体拮抗剂(interleukin-36 receptor antagonist,IL-36Ra)的真核表达载体,并对其基因全长编码区进行分析。[方法]提取总RNA,逆转录出IL-36Ra c DNA序列。设计人IL-36Ra基因的上游引物和下游引物。利用热启动PCR扩增目的基因,将扩增出的IL-36Ra基因编码区通过限制性内切酶酶切后,把酶切的目的片段与载体pc DNA3.1连接,转化到感受态细胞大肠杆菌DH5α中。通过氨苄青霉素抗性筛选、双酶切及菌落PCR鉴定,最后选取阳性克隆进行测序。[结果]重组载体pc DNA3.1-h IL-36Ra经菌落PCR和酶切鉴定,最后通过序列分析证实,其序列与Gen Bank中数据一致。[结论]人IL-36Ra基因的重组真核表达载体pc DNA3.1-h IL-36Ra的成功构建,为进一步研究IL-36Ra在免疫性疾病中的作用奠定了实验基础。     

猴血SV40抗体和SV40 DNA携带关系分析

目的　检测 5 7份恒河猴血清及对应的猴血中抗SV4 0抗体、SV4 0DNA的携带情况 ,找出抗体滴度与DNA携带的相关关系。方法　用间接免疫荧光法检测猴血中SV4 0中和抗体 ,聚合酶链反应 (PCR)法检测SV4 0st抗原DNA的携带情况。结果　 5 7份猴血清中 ,SV4 0抗体阳性率为 93% (5 3 5 7) ,抗体滴度最高为 1∶12 80 ,最低为 1∶10 ,GMT为 15 9.5 6 ,PCR检测猴血淋巴细胞SV4 0DNA阳性率为 2 4 .5 6 % (14 5 7) ,当抗体为 1 80以下时 ,病毒整合于血细胞 ,1∶80以上时 ,对st抗原基因的整合有抑制作用。结论　恒河猴SV4 0DNA的携带情况与抗体阳性和滴度呈反相关     

弓形虫（Toxoplasma gondii）的PCR检测方法在笼养猕猴群中的应用

目的建立快速、灵敏、特异及检测结果易判断的PCR方法,并应用于大规模猕猴种群的弓形虫常规检测中。同时比较巢式PCR和单一PCR的一致性。方法根据弓形虫保守基因p30（SAG1）设计了内、外两对进行巢式PCR扩增以及B1基因设计一对引物进行单一PCR扩增,将DNA样本进行10倍倍比稀释,以检测巢式PCR反应的灵敏度;并对医学生物学研究所自繁猕猴共150只进行了弓形虫检测。结果巢式PCR检测法检测限度可达10^-3ng/uL,而且方法特异。两种PCR法检测结果基本一致,其中巢式PCR检测阳性率（10％）稍高于单一PCR检测阳性率（8．67％）。结论巢式PCR和一次PCR方法都可应用于猕猴弓形虫的常规检测中,并提示巢式PCR比单一PCR更敏感、检出率更高。     

Detection and typing of BKV,JCV, and SV40 by multiplex nested polymerase chain reaction

A multiplex nested polymerase chain reaction (PCR) method was developed for detecting and differentiating simultaneously the DNA of polyomaviruses JC, BK, and SV40 in a single tube. In the first amplification step the same set of primers was used to amplify a conserved DNA region of the large T antigen gene of JCV, BKV, and SV40. The second round was carried out using a set of primers designed to obtain products of different size for each related virus. Subsequently, the sensitivity of the multiplex nested PCR was maximized by optimizing parameters such as primer, magnesium, and dNTP concentrations. The sensitivity of the method ranged between 1 and 10 copies of the polyomavirus genome. The assay was then used for detecting polyomavirus DNA in urine, serum, and biopsy specimens from renal transplant recipients. Based on the results obtained, the multiplex nested PCR developed in our study represents a useful tool for supporting the diagnosis of polyomavirus infection and could be used for epidemiological purposes and to better define the role of polyomaviruses in human pathology.     

猪流行性腹泻病毒嵌套式RT—PCR检测方法的建立

Two pairs of primers were designed according to the N gene sequence of PEDV-CV777 strain in Genebank (No. AF353511). PCR amplification product of outer-primers was 1328bp, and the inter-primers amplification product was 538bp, With the primers, a nested PCR assay was established to detect PEDV. This method was sensitive and specific and could be used in PEDV diagnosis and epidemiological investigation.     

TAIL-PCR方法快速分离Xcc致病相关基因序列

以mini-Tn5 gfp-km转座子中nptⅡ片段作为探针,对已获得的五株野油菜黄单胞菌野油菜黑腐病致病型（Xcc)非致病突变体进行了Southern blot分析,结果表明,这五株突变体确由mini-Tn5 gfp-km转座子插入致病相关基因所致,且为单拷贝不同位点的插入。提取这五株突变体总DNA作为模板,采用改进的热不对称交错PCR（TAIL－PCR）方法从其中克隆到了各自转座子插入区侧翼序列,对这些侧翼序列进行了序列测定并将分析结果与GenBank database及Xcc全基因组序列做了比较,结果表明,五个侧翼序列所在的基因确与Xcc致病性有关。这种改进后的TAIL－PCR方法为突变体特别是转座子插入突变体中目的基因的克隆提供了一种简要高效的新方法。     

Rapid Detection of Phytophthora nicotianae in Infected Tobacco Tissues and Soil Samples Based on Its Ypt1 Gene

This paper describes the development of a polymerase chain reaction (PCR) assay for the detection of Phytophthora nicotianae , the causal agent of Phytophthora blight of tobacco and other plants. The PCR primers were designed based on a Ras-related protein ( Ypt 1) gene, and 115 isolates representing 26 species of Phytophthora and 29 fungal species of plant pathogens were used to test the specificity of the primers. PCR amplification with species-specific (Pn) primers resulted in a product of 389 bp only from isolates of P. nicotianae . The detection sensitivity with Pn primers was 1 ng of genomic DNA. Using Ypt 1F/ Ypt 1R as first-round amplification primers, followed by a second round using the primer pair Pn1/Pn2, a nested PCR procedure was developed, which increased the detection sensitivity 100-fold to 10 pg. PCR with the Pn primers could also be used to detect P. nicotianae from naturally infected tobacco tissues and soil. The PCR-based methods developed here could simplify both plant disease diagnosis and pathogen monitoring as well as guide plant disease management.     

Pseudomonas syringae pv. actinidiae detection in kiwifruit plant tissue and bleeding sap

The rapid spreading of the disease during last few years highlighted the need of a quick, sensitive and reliable method for Pseudomonas syringae pv. actinidiae (Psa) detection, to find possible inoculum sources and limit the pathogen spreading. A PCR method, using new primers designed on the gene encoding a putative outer membrane protein P1, was developed to detect Psa in symptomatic and asymptomatic tissue; a nested‐PCR was also applied. Bleeding sap samples, collected in early spring from orchards with symptomatic and asymptomatic trees, were used both for PCR assays and for pathogen isolation and identification. The PCR and nested PCR methods were able to detect Psa presence at very low concentration from plant and pollen extracts; RFLP analyses with BclI on PCR and nested PCR amplicons confirmed the assay specificity, while the digestion with BfmI and AluI allowed to discriminate Psa strains isolated before 2008 from those isolated after 2008. Furthermore, the PCR and nested PCR on crude bleeding sap samples detected the presence of the pathogen in 3 and 5 of the 15 assayed samples, respectively. Direct isolation from the same samples and bacterial identification confirmed the results of molecular analysis.     

Rapid detection of viable bacteria by nested polymerase chain reaction via long DNA amplification after ethidium monoazide treatment

In assays to determine whether viable cells of Enterobacteriaceae are present in pasteurized milk, the typical ethidium monoazide (EMA) polymerase chain reaction (PCR) targets a short stretch of DNA. This process often triggers false-positive results owing to the high level of dead cells of Enterobacteriaceae that had initially contaminated the sample. We have developed a novel, direct, real-time PCR that does not require DNA isolation (DQ-PCR) to detect low levels of cells of Enterobacteriaceae regardless of live and dead cells first. We confirmed that the DQ-PCR targeting a long DNA (the 16S ribosomal RNA [rRNA] gene, amplified length of 1514 bp) following EMA treatment is a promising tool to detect live bacteria of all genera owing to the complete suppression of background signal from high levels of dead bacteria in pasteurized milk. However, when identifying viable bacteria in pasteurized milk, commercial PCR primers designed for detecting long stretches of DNA are generally not available. Thus, we treated samples with EMA and then carried out an initial round of PCR of a long stretch of DNA (16S gene, 1514 bp). We then performed another round of PCR, a novel nested PCR to generate short products using commercial primers. This procedure resulted in the rapid detection of low levels of viable cells of Enterobacteriaceae.     

Specific PCR assays for the detection of <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> causing green mold disease during mushroom cultivation

We have developed a polymerase chain reaction (PCR)-based detection method for Trichoderma harzianum, which causes green mold disease in mushroom cultivation fields and facilities. Based on the sequence data of the internal transcribed spacer (ITS) region of T. harzianum strains and several other species, six primers consisting of three forward and three reverse primers were designed. Among the nine possible combinations of these primers, PCR with the pair THITS-F2 and THITS-R3 distinguished most T. harzianum strains from other Trichoderma species. The optimal annealing temperature for detection of T. harzianum strains was from 62° to 63°C with this primer combination. We designed new primers derived from THITS-F2 and THITS-R3. Annealing temperatures to detect T. harzianum ranged from 64° to 67°C using the new primers. The detection limit of T. harzianum DNA was 50 fg by nested PCR with THITS-F1 and LR1-1 for the first PCR and the new primers for the second PCR. T. harzianum was readily detectable in contaminated cultures of Lentinula edodes by this method.