用4对引物聚合酶链式反应检测鼠疫耶尔森氏菌的实验研究

目的用4对引物聚合酶链式反应(以下简称4-Prim er-PCR)来检验鼠疫耶尔森氏菌,对于探索鼠疫的快速诊断方法将起到推动作用。方法选择不同疫源地的40株鼠疫耶尔森氏菌;目的基因选择于与鼠疫耶尔森氏菌毒力因子有关的FI抗原质粒基因cafI鼠疫杆菌素Ⅰ基因、与色素沉着有关的质粒基因hm s以及染色体基因inv。结果该法所有的鼠疫耶尔森氏菌全部出现4对引物的预期扩增带;该试验在4 h内完成;结论四对引物鼠疫PCR法具有快速、特异、敏感等优点,可以用于鼠疫的快速诊断和流行病学调查。     

荧光定量PCR快速检测鼠疫耶尔森氏菌的实验研究

目的　利用LightCycler建立一种简便、特异的荧光定量PCR检测方法 ,用于鼠疫耶尔森氏菌的快速检测。 方法　采用SYBRGreenI随机掺入法 ,以Roche试剂盒为标准 ,比较两公司的DNA聚合酶 ,用鼠疫菌 310 0 4建立荧光PCR反应体系 ;针对鼠疫耶尔森氏菌特异的染色体标识序列和质粒上F1抗原基因设计引物 ,检测其灵敏度和特异性 ,以盲测试验进行验证 ;在此基础上鉴定 2 75株鼠疫耶尔森氏菌 ,并测试该法检测脏器污染模拟标本的灵敏度。结果　建立以一个公司试剂为主较低成本的PCR反应体系 ;EV76的检测灵敏度可达每反应体系 1.6个菌 ;检测我国 18个生态型共计 2 75株鼠疫耶尔森氏菌及 2 8株非鼠疫菌株的PCR扩增结果表明 ,鼠疫菌株均出现特异的扩增结果 ,2 8株对照菌株均为阴性 ;肝、脾、肺模拟标本检测灵敏度可达每反应体系 4 0 0 0个菌。结论　该方法对于检测鼠疫耶尔森氏菌具有简便、快捷、高敏感性和特异性的特点 ,适用于鼠疫紧急疫情时的快速诊断和疫源地监测     

多重PCR快速鉴定鼠疫耶尔森氏菌

目的　建立鼠疫耶尔森氏菌多重PCR鉴定系统 ,用于鼠疫耶尔森氏菌的快速鉴定。方法　针对分别存在于pFra、pRst质粒上毒力基因caf1和pla ,以及一段 2 76bp染色体序列 3a分别设计引物。采用多重PCR技术 ,同时检测caf1、pla、3a三个靶序列。结果　应用该多重PCR反应体系 ,对我国鼠疫耶尔森氏菌 17个生态型及新发现的青海田鼠鼠疫疫源地菌株的多重PCR扩增结果表明 ,实验菌株中除 2株分别缺失pFra、pPst质粒而扩增出 2条产物带外 ,其余 5 2株均扩增出预期的 3条产物带 ,相关菌株均阴性 ,其检测灵敏度为 1× 10 -4ngDNA。 结论　该方法用于鼠疫耶尔森氏菌的鉴定简便、快捷 ,具有很高的特异性和敏感性 ,为鼠疫耶尔森氏菌的快速鉴定提供了有力手段     

三种致病耶尔森氏菌Hsp60基因序列的比较研究

目的　研究 3种致病耶尔森氏菌Hsp6 0基因序列的差异。方法　根据文献和基因库中的Hsp6 0基因序列设计了两对PCR引物 ,获得Hsp6 0基因的部分序列。将扩增产物纯化回收 ,克隆至 pGEM -T载体后进行测序。 结果　用CLUSTALX软件进行序列分析 ,发现 3种耶尔森氏致病菌的Hsp6 0基因序列较为保守 ,其中鼠疫耶尔森氏菌和假结核耶尔森氏菌的该基因序列在所研究范围内仅相差一个碱基。结论　 3种菌的Hsp6 0基因保守 ,不足以在此区间设计种特异性探针。小肠结肠炎耶尔森氏菌与鼠疫耶尔森氏菌和假结核耶尔森氏菌的Hsp6 0基因序列差异较大 ,易与这两个种区分开     

随机扩增多态性DNA(RAPD)技术用于青海田鼠鼠疫菌株亲缘性的研究

目的　对青海省称多县境内和四川省石渠县境内青海田鼠体内分离的 32株鼠疫耶尔森氏菌的基因进行分析 ,明确两地鼠疫耶尔森氏菌间的亲缘关系。方法　用随机扩增多态性DNA(RAPD)技术。结果　扩增产物在凝胶电泳上显示的条带 ,除 7株菌略有不同外 ,其余 2 5株均相同。TreeView[Win32 ]统计软件分析的结果也显示两地鼠疫耶尔森氏菌之间具有很近的亲缘关系。结论　青海省称多县和四川省石渠县青海田鼠体内分离到的鼠疫耶尔森氏菌在遗传学上属同一来源。     

随机扩增多态性DNA技术检测不同地区田鼠型鼠疫耶尔森氏菌基因结构

目的　了解不同地区田鼠型鼠疫耶尔森氏菌 (以下简称鼠疫菌 )之间的亲缘关系和基因类型。方法　随机扩增多态性DNA(RAPD)技术。结果　扩增的 13株田鼠型鼠疫耶尔森氏菌在凝胶电泳所显示的条带 ,除 3株菌缺少部分条带外 ,其余菌株基本相同。结论　青海田鼠型鼠疫耶尔森氏菌和布氏田鼠型鼠疫耶尔森氏菌具有相似性 ,在遗传学上属于同源。     

贵州省鼠疫耶尔森菌生物学特征研究

目的研究贵州省鼠疫耶尔森菌的生物学特征,探讨疫源地性质。方法对37株鼠疫菌进行生化试验、营养需求试验、毒力基因检测、随机扩增多态性DNA分析和脉冲场电泳分析。结果37株鼠疫菌均不发酵鼠李糖和甘油,发酵麦芽糖和阿拉伯糖,脱氮阳性。选择20株代表菌株检测均为苯丙氨酸依赖(Phe )、谷氨酸半依赖(Glu±);用Pla、Cafl、inv、hms4对引物分别进行PCR扩增,均得到456、249、1000和700bp的目标基因条带;随机引物(RAPD)分析均获得505、790、1140和1680bp的条带;脉冲场电泳图谱显示338、242.5、168和77kbp的条带。结论贵州省鼠疫菌株的生物学特征与云南省滇闽居民生态型鼠疫菌相同。     

基于核酸预混室温储运技术的荧光定量PCR快速检测鼠疫耶尔森氏菌

目的 实现荧光定量PCR技术在鼠疫现场和基层的应用。方法 本试验将已建立的基于核酸预混室温储运技术的荧光定量PCR方法应用于我国各鼠疫疫源地野生菌株的检测,进行特异性评价。选取分布于我国境内不同鼠疫疫源地野生鼠疫耶尔森氏菌株、鼠疫减毒菌株、耶尔森菌属近缘菌株假结核菌及小肠结肠炎菌进行荧光定量PCR检测。结果 55株鼠疫耶尔森氏菌及鼠疫减毒菌株扩增阳性,假结核耶尔森氏菌、小肠结肠炎耶尔森氏菌无阳性扩增,冻干试剂在室温25 ℃和37 ℃条件下可保存6个月,敏感性与冷冻保存无差异,核酸扩增诊断可在1 h内完成。结论 应用基于核酸预混室温储运技术的荧光定量PCR方法对我国各鼠疫疫源地55株鼠疫耶尔森氏菌检测呈现高度特异性,本试验冻干试剂具有可室温保存、便于运输、检测结果精准、快速等特点,具有较好的鼠疫现场应用前景。     

鼠疫耶尔森菌F1抗原蛋白大肠杆菌分泌表达载体的构建

目的构建鼠疫耶尔森菌F1抗原蛋白的大肠杆菌分泌表达载体。方法 PCR法扩增鼠疫耶尔森菌的F1抗原基因,并插入大肠杆菌分泌表达载体p BAD/gⅢC中构建分泌表达载体。结果扩增出了长约500 bp的鼠疫耶尔森菌F1抗原基因并构建了该抗原的大肠杆菌分泌表达载体。结论本研究中我们用载体p BAD/gⅢC构建鼠疫耶尔森菌F1抗原的大肠杆菌分泌表达质粒,为后期制备针对鼠疫F1抗原的单克隆抗体或多克隆抗体,进一步建立便捷高效的鼠疫诊断方法奠定基础。     

鼠疫耶尔森氏菌荧光标记扩增片段长度多态性分型方法

目的 建立荧光标记扩增片段长度多态性(FAFLP)技术平台，探讨鼠疫菌基因分型。方法 用5种核酸限制性内切酶消化鼠疫菌基因组DNA，选择最佳内切酶组合，酶切片段经接头连接后，用荧光素标记的5条EcoRⅠ引物和9条MseⅠ引物组成的引物配对扩增，选择最佳引物组合，进行系列PCR条件的优化。扩增产物经ABI PRISM 3100 Genetic Analyzer(遗传分析仪)检测，GeneScan等软件进行分析。结果 成功建立FAFLP技术平台。结论 FAFLP具有快速、简便、廉价、分辨率高、重复性好和污染少的优点，可用于鼠疫菌的基因分型分析。     

快速检测牛羊猪种布鲁杆菌多重PCR的建立

目的 建立一种能同时快速检测并能鉴别牛、羊、猪种布鲁杆菌的多重PCR方法.方法 根据IS711插入序列设计1条公共引物和3条牛、羊、猪种布鲁杆菌(544A、16M、1330S)特有序列引物,进行多重PCR反应;选择耶尔森菌O:9、大肠埃希菌O157:H7、鼠伤寒沙门菌47729进行多重PCR反应的特异性检测;倍比稀释定量法观察牛种布鲁杆菌多重PCR反应的敏感性.结果 牛、羊、猪种布鲁杆菌多重PCR反应扩增片段产物长度分别为485、731、248 bp;耶尔森菌O:9、大肠埃希菌O157:H7、鼠伤寒沙门菌47729加入布鲁杆菌中进行多重PCR反应.扩增结果呈阴性;牛种布鲁杆菌多重PCR反应敏感性为0.0967 Pg.结论 成功建立快速检测牛、羊、猪种布鲁杆菌多重PCR扩增反应方法,且其特异性、敏感性较好.     

Linear-After-The-Exponential (LATE)-PCR: primer design criteria for high yields of specific single-stranded DNA and improved real-time detection

Traditional asymmetric PCR uses conventional PCR primers at unequal concentrations to generate single-stranded DNA. This method, however, is difficult to optimize, often inefficient, and tends to promote nonspecific amplification. An alternative approach, Linear-After-The-Exponential (LATE)-PCR, solves these problems by using primer pairs deliberately designed for use at unequal concentrations. The present report systematically examines the primer design parameters that affect the exponential and linear phases of LATE-PCR amplification. In particular, we investigated how altering the concentration-adjusted melting temperature (Tm) of the limiting primer (TmL) relative to that of the excess primer (TmX) affects both amplification efficiency and specificity during the exponential phase of LATE-PCR. The highest reaction efficiency and specificity were observed when TmL - TmX 5 degrees C. We also investigated how altering TmX relative to the higher Tm of the double-stranded amplicon (TmA) affects the rate and extent of linear amplification. Excess primers with TmX closer to TmA yielded higher rates of linear amplification and stronger signals from a hybridization probe. These design criteria maximize the yield of specific single-stranded DNA products and make LATE-PCR more robust and easier to implement. The conclusions were validated by using primer pairs that amplify sequences within the cystic fibrosis transmembrane regulator (CFTR) gene, mutations of which are responsible for cystic fibrosis.     

PCR-HRM方法快速鉴定布鲁氏菌的初步应用

目的 本研究旨在建立准确、快速地鉴定布鲁氏菌菌种及生物分型的PCR-HRM(高分辨率熔解曲线)方法。方法 根据目的基因序列,参考文献合成6对基因扩增引物(1对布鲁氏菌属特异引物,5对种间特异引物),应用PCR-HRM方法鉴定布鲁氏菌属6个种19个生物型的标准菌株,并初步应用到临床分离的35株布鲁氏菌中。结果 采用布鲁氏菌属特异引物(Bspp),6个种19个生物型的标准菌株均扩增出同样形状的溶解曲线,与其它对照菌株的曲线不同;采用5对种特异引物,6个种的标准菌株均有特征性PCR-HRM曲线;临床分离的35株布鲁氏菌的PCR-HRM曲线与羊种布鲁氏菌标准菌株的一致。结论 该研究采用的PCR-HRM分析方法,获得了布鲁氏菌属及6个种标准菌株的不同曲线图,可准确鉴定临床分离的羊种布鲁氏菌,可用于临床微生物实验室疑似布鲁氏菌感染的初步鉴定。     

Evaluation of different primers for detecting mecA gene by PCR in comparison with phenotypic methods for discrimination of methicillin-resistant Staphylococcus aureus

Detection of the mecA gene by polymerase chain reaction (PCR) is the gold standard for identifying methicillin-resistant Staphylococcus aureus (MRSA). PCR assays, employing MR1-MR2 primers (primer set 1) and MR3-MR4 primers (primer set 2) to generate 154 and 533 bp fragment, respectively, are most widely used for amplification of mecA gene. The purpose of this study was to evaluate the presence of mecA gene in 100 clinical isolates of S. aureus using PCR with the two pairs of primers. The results were compared to the broth dilution MIC method, oxacillin salt screening method (OSS) and oxacillin disk agar diffusion method (ODD). Fifteen of the 100 isolates showed a discrepancy between the mecA primer sets 1 and 2. Three isolates (3%) without the mecA gene showed discrepancies with phenotypic methods. The sensitivity, specificity and positive and negative predictive values for the 154 and 533 bp products of mecA were 79, 85, 83, 81 and 94, 100, 100, 94%, respectively. The results indicated that primer set 2 was more appropriate than primer set 1 for the detection of mecA gene in MRSA. There was a good correlation among the mecA gene detection, ODD and OSS methods. The discrepancy of three isolates between PCR and phenotypic methods should be clarified for other resistant mechanisms.     

Newer developments in the identification of beta-thalassemia

One of the easiest and most sensitive methods of detecting mutations in the beta-globin gene leading to beta-thalassemia is by the use of oligonucleotide probes. The current method involves digestion of 5-10 micrograms of genomic DNA followed by gel electrophoresis, and blotting onto nitrocellulose. The membrane is then hybridized with a 32P-radiolabeled oligonucleotide probe containing the specific point mutation of interest. Finally, the membrane is subjected to X-ray film for 3-10 days. We wish to report a method for detecting these mutations which involves 1 microgram of genome DNA or less. The method involves the use of a gene amplification technique. A series of primers are synthesized which span the beta-globin gene. In each primer set, one primer is complementary to the beta-gene and the other primer is complementary to the non-coding strand. The suspected mutation point is located between these two primers. With the use of this primer set, the beta-globin gene region is amplified by denaturing, annealing, and DNA synthesis. The amplification cycle is repeated 25 to 30 times. The amplification is conducted using the Klenow fragment of DNA polymerase I or Taq polymerase in the presence of all four deoxynucleotide triphosphates. The resulting amplified DNA is applied to a nylon membrane with the aid of a dot-blot apparatus and directly hybridized with normal and mutant deoxynucleotide probes. The entire process requires one to two days. More than 300 beta-thalassemia homozygotes have been identified in our laboratories; over 20 different mutations have been observed.     

陕西省鼠疫耶尔森菌间区规律短回文重复序列基因分型研究及流行病学分析北大核心CSCD

目的通过CRISPR(间区规律短回文重复序列)对陕西省鼠疫菌进行基因分型,分析流行病学特征,为陕西省鼠疫防治工作提供科学依据。方法提取分离自陕西省鼠疫疫源地的66株鼠疫菌核酸,利用针对鼠疫菌的3对CRISPR引物进行PCR扩增,对扩增产物进行测序及分析,确定CRISPR基因分型。结果 66株鼠疫菌在3个位点上共有12种spacer,其中YPa 3种、YPb 5种、YPc 4种,具体为al′、a2′、a3′、b1′、b2′、b29′、b1″、b2″、c1、c2、c3、c3′,基因簇为Cb2′,基因型分为1′和4′两种。结论疫情发生年份不同,鼠疫菌CRISPR基因型不同,鼠疫疫情处置进行溯源应将生态分型与基因分型结合。     

福氏志贺菌4av和Yv血清型PCR鉴定方法的建立及应用

目的 建立福氏志贺菌4av和Yv血清型PCR鉴定方法。方法 根据福氏志贺菌4av和Yv血清型O抗结构,针对O抗合成基因wzx、IV型抗原决定基因gtrIV和MASF IV-1抗原决定基因opt,建立血清型4av和Yv 的PCR鉴定方法。并应用该方法对126株福氏志贺菌临床分离株进行血清型检测。结果 建立了一种福氏志贺菌4av和Yv血清型PCR鉴定方法,在一个反应中包括4对引物,Yv血清型PCR扩增为wzx及opt阳性;4av血清型PCR扩增为wzx、opt及gtrIV阳性。该方法可将4av和Yv血清型与目前已知的其他福氏志贺菌血清型完全区分。对126株不同血清型福氏志贺菌临床分离株的鉴定结果显示,该方法具有很好的特异性。结论 本研究建立的福氏志贺菌4av和Yv血清型PCR鉴定方法,可以用于志贺菌检测和监测。     

Quantitation of mRNA by the polymerase chain reaction.

A method for the quantitation of specific mRNA species by the polymerase chain reaction (PCR) has been developed by using a synthetic RNA as an internal standard. The specific target mRNA and the internal standard are coamplified in one reaction in which the same primers are used. The amount of mRNA is then quantitated by extrapolating against the standard curve generated with the internal standard. The synthetic internal standard RNA consists of a linear array of the sequences of upstream primers of multiple target genes followed by the complementary sequences to their downstream primers in the same order. This quantitative PCR method provides a rapid and reliable way to quantify the amount of a specific mRNA in a sample of less than 0.1 ng of total RNA. In addition, the same internal standard RNA is used, with appropriate primer pairs, to quantitate multiple different mRNA species.     

Newer Developments in the Identification of β-Thalasseuia

One of the easiest and most sensitive methods of detecting mutations in the β-globin gene leading to β-thalassemia is by the use of oligonucleotide probes. The current method involves digestion of 5–10 μg of genomic DNA followed by gel electrophoresis, and blotting onto nitrocellulose. The membrane is then hybridized with a 32P-radiolabeled oligonucleotide probe containing the specific point mutation of interest. Finally, the membrane is subjected to X-ray film for 3–10 days. We wish to report a method for detecting these mutations which involves 1 μg of genome DNA or less. The method involves the use of a gene amplification technique. A series of primers are synthesized which span the β-globin gene. In each primer set, one primer is complementary to the β-gene and the other primer is complementary to the non-coding strand. The suspected mutation point is located between these two primers. With the use of this primer set, the β-globin gene region is amplified by denaturing, annealing, and DNA synthesis. The amplification cycle is repeated 25 to 30 times. The amplification is conducted using the Klenow fragment of DNA polymerase I or Taq polymerase in the presence of all four deoxynucleotide triphosphates. The resulting amplified DNA is applied to a nylon membrane with the aid of a dot-blot apparatus and directly hybridized with normal and mutant deoxynucleotide probes. The entire process requires one to two days. More than 300 β-thalassemia homozygotes have been identified in our laboratories; over 20 different mutations have been observed.