疟疾的实时荧光PCR快速检测方法

目的:建立疟疾的实时荧光PCR检测方法并用于疟疾的快速检测。方法:设计了疟原虫通用型实时荧光PCR检测的引物和探针,建立了疟疾的实时荧光PCR检测方法。对40份疟疾镜检阳性样本和20份镜检阴性样本进行实时荧光PCR检测,并和巢式PCR方法检测结果进行了比较。结果:疟疾实时荧光PCR检测速度快,20分钟即可完成。40份镜检阳性样本的荧光PCR检测均为阳性,2份样本的巢式PCR结果为阴性;20份镜检阴性样本中有3份样本为实时荧光PCR阳性,其余17份样本为实时荧光PCR阴性,与疟疾巢式PCR检测结果相同。结论:该实时荧光PCR方法检测速度快,特异性强,准确性高,阳性率高,不易漏检,适用于疟疾的快速检验,这对防止该病在国境口岸的传入提供了技术依据。     

香港海鸥形菌荧光PCR快速检测方法的建立

目的:建立检测香港海鸥形菌的实时荧光PCR方法。方法:根据香港海鸥形菌以16SrDNA基因设计探针和引物,建立检测香港海鸥形菌荧光PCR方法,并对建立的方法进行特异性、灵敏性的评价。结果:检测体系灵敏度高,菌液的最低检出限可达40 CFU/反应体系;特异性好,对329株细菌的检测符合率达100%。结论:建立的实时PCR检测方法可应用于食源性致病菌中香港海鸥形菌的快速检测。     

实时荧光PCR法检测急性角结膜炎结膜拭子标本中的腺病毒

目的:利用实时荧光PCR技术,建立一套快速可靠的检测急性角结膜炎腺病毒的方法,并分析我院临床诊断为急性角结膜炎的疑似腺病毒感染患者的不同型别腺病毒的感染情况。方法:设计并利用ad1,ad3,ad4,ad8,ad19和ad37型的特异性引物,建立Real-time PCR方法检测角结膜炎标本中腺病毒DNA,并对我院168例急性角结膜炎腺病毒感染疑似患者结膜拭子样品进行实时荧光定量PCR检测。结果:验证5对引物,分别只扩增出相对应的Adv标准株,未见其他非特异性扩增,168份眼拭子标本中,共检出腺病毒49株,总阳性率为29.2%,其中ad1型2例,占腺病毒的4.1%,ad3型31例,占腺病毒的63.3%,ad8型9例,占腺病毒的18.4%,Ad4型4例,占8.2%,Ad19/37型6例,占12.2%,其余119例为阴性。结论:Real-time PCR技术具有敏感、特异等优点,适用于急性角膜结膜炎标本中腺病毒的检测及分型。2010年1月-2010年12月我院急性角膜结膜炎感染的腺病毒主要为Ad3型和Ad8型。     

荧光定量PCR在肺结核临床诊断及疗效评估中的应用

目的建立荧光定量PCR检测结核分枝杆菌方法,评估其在肺结核的t临床诊断和疗效评估中的应用价值。方法针对结核分枝杆菌保守序列IS6110基因设计引物和探针,建立荧光定量PCR方法。分别以荧光定量PCR、集菌培养法和抗酸染色法检测168份疑似肺结核和5例确诊病人随访的痰标本,比较各方法在结核病诊断以及治疗效果评估中的作用。结果该荧光定量PCR法能扩增巧6l1O基因片段（107bp）,序列分析显示为结核分枝杆菌核酸序列,方法的检测灵敏度为4copies／反应;而对偶发分枝杆菌、蟾分枝杆菌、草分枝杆菌、龟分枝杆菌、肺炎链球菌、大肠埃希菌、金黄色葡萄球菌、脑膜炎奈瑟茵的样本扩增结果均为阴性,荧光定量PCR法与集菌培养法和抗酸染色法对临床样本的检出率分别为64．29％（108／168）、35．12％（59／168）和12．50％（21／168）,前者高于后两者（P〈0．叭）。对抗酸染色法和集菌培养法阳性的样本,荧光定量PCR法的灵敏度分别为100．00％（21／21）和96．61％（57／59）。对20份非结核病人来源的痰标本．荧光定量PCR方法检测结果均为阴性,符合率为100．00％。用荧光定量PCR检测方法对5例活动性肺结核病例治疗期间进行了动态检测,结果显示病人晨痰中结核分枝杆菌的数量呈持续下降的趋势（即0值增加）。结论该荧光定量PER方法具有快速、敏感和特异性高的特点,可以快速、及时获取病人服菇詹的治府时票曲瞑辟的詹蛙督导妊井撂蚀饺摇臣者舌亚拍I性J妻音、;,     

荧光定量PCR快速检测沙门菌方法的建立及应用

目的:建立沙门菌实时荧光PCR的快速检测方法,探讨其可行性和应用价值。方法:根据沙门菌的特异性DNA作为靶序列,以沙门菌菌株提取核酸DNA作为模板进行荧光检测。结果:本研究在7种相关菌株的检测中,除沙门菌出现很好的阳性外,其余菌株均为阴性;在纯菌条件下,定量检测低限为30 cfu/ml;同一样品检测3次Ct值的变异系数均小于5%;对模拟标本与分离培养对比,二者符合率为100%。结论:该方法的建立,不仅为食源性沙门菌污染及食物中毒的快速检测提供依据,而且还可直接用于临床粪便标本的检测。     

Rapid identification of Babesia canis and Babesia gibsoni (Asian genotype) in canine blood samples using a customized portable real-time PCR analyzer and TaqMan-based assay

Canine babesiosis is a serious infectious disease in subtropical and tropical regions. Typically, clinical detection of canine babesiosis is performed by blood smear observation or the traditional polymerase chain reaction (PCR). Herein, we developed a new TaqMan-based real-time PCR assay combined with a customized portable real-time PCR platform for a rapid and accurate detection of canine babesiosis. Two new primer/probe pairs (B18S and BITS1) were designed based on 18S ribosomal RNA and an internal transcribed spacer 1 (ITS1) sequence to differentiate Babesia canis and B. gibsoni (Asian genotype) DNAs from canine blood samples. Additionally, a corresponding customized compact real-time PCR platform with low 6-carboxyfluorescein fluorescence detection (≤5 nM), including a fast and accurate thermal cycling ability with a user-friendly interface for thermal control and data analysis, was designed for the limited space use. Both assays (B18S and BITS1) demonstrated a sensitivity of 100 copies/reaction based on the 95 % confidence interval evaluation method. The self-developed customized portable real-time PCR analyzer presented high repeatability and reproducibility with the TaqMan-based assay. Moreover, 501 clinical specimens were collected for evaluating the performance of the proposed PCR. The positive and negative predictive values were 90 % (18 of 20) and 100 % (226 of 226), respectively, for samples suspected with B. canis infection and 98 % (55 of 56) and 100 % (199 of 199), respectively, for samples suspected with B. gibsoni infection.     

实时荧光定量PCR技术进展

实时荧光定量PCR(real_time quantitative PCR)技术是一种新型的核酸定量检测、分析技术,它通过在PCR扩增反应过程中加入荧光物质,使得对反应过程的实时监控成为可能。它具有实时监测、定量准确、灵敏度高、反应速度快、重复性好及PCR反应后不需电泳检测等优点,已逐步成为分子生物学研究中的重要工具。     

实时定量PCR检测人全血中金黄色葡萄球菌DNA方法的建立

目的建立实时定量PCR（RQ—PCR）快速检测人全血中金黄色葡萄球菌DNA的方法,以便早期定量评估肠屏障损伤所致肠道内细菌移位引起或加重的全身感染。方法对15份模拟全血标本及26份外科发热患者全血标本进行RQ—PCR检测。选择金黄色葡萄球菌高度保守的看家基因femA基因作为靶基因设计引物和Taqman探针,建立20ul的RQ—PCR反应体系,采用含靶基因扩增片段的重组质粒建立标准曲线,提取血标本中的细菌基因组DNA。结果引物和探针特异性好,检测限为10^0拷贝／ul（10^3CFU／ml）,灵敏度为99．7％,特异度为94．6％。标准曲线线性关系好,R。在0．9918—0．9997。不同浓度的金黄色葡萄球菌样本检测的平均准确性为（96．25±2．26）％,批内及批间重复性的平均变异系数分别为（8．06±0．07）％和（10．01±4．40）％。全血标本中金黄色葡萄球菌DNA平均回收率为（111．72±20．72）％。临床血标本RQ-PCR检测阳性率为15．4％（4／26）,血培养结果皆为金黄色葡萄球菌阴性。结论RQ—PCR可用于定量检测全血标本中的金黄色葡萄球菌DNA含量,具有快速、灵敏、特异性强、重复性好的优点。     

Risk factors for malaria in UK travellers

After observing an apparent increase in severe falciparum malaria among travellers returning from The Gambia to the United Kingdom (UK) in the last quarter of 2000, we conducted a case-control study to investigate risk factors for malaria. The study participants had visited The Gambia between 1 September and 31 December 2000, travelling with the largest UK tour operator serving this destination. The main outcome measures were risk factors associated with malaria. Forty-six cases and 557 controls were studied. Eighty-seven percent of all participants reported antimalarial use (41% chloroquine/proguanil, 31% mefloquine). On univariate analysis the strongest risk factors for disease were: early calendar period of visit, longer duration of stay, non-use of antimalarial prophylaxis, non-use of mefloquine, lack of room air-conditioning, less use of insect repellent, prior visit to another malarial area and accommodation in 'hotel X'. After adjustment in multivariate analysis, use of mefloquine remained strongly protective (odds ratios, OR 0.13 [95% confidence intervals, 95% CI 0.04-0.40]), and the strongest independent risk factors for malaria were early calendar period (OR 5.19 [2.35-11.45] for 1 September to 9 November 2000 versus 10 November to 31 December 2000), prior visit to another malarial area (OR 3.27 [1.41-7.56]), main accommodation in 'hotel X' (OR 3.24 [1.51-6.97]) and duration of stay (OR 2.05 per extra week [1.42-2.95]). Neither any use, nor > 90% adherence to chloroquine/proguanil were protective (adjusted OR for any use 0.57 [0.27-1.21], P = 0.14). We concluded mefloquine use was strongly protective against malaria (87% protective efficacy), whereas chloroquine/proguanil, which is no longer recommended but remains widely used, was less than half as effective (43% protective efficacy). Waning efficacy of chloroquine/proguanil may have contributed to the observed increase in malaria among travellers to The Gambia in 2000. Local factors may also influence the risk of malaria. Malaria could be prevented among travellers to West Africa if current national guidelines on antimalarial prophylaxis were better implemented.     

食品过敏原荞麦的实时荧光PCR检测

目的:荞麦是日本法规要求强制性标识的食品过敏原,我国需要建立出口日本食品中过敏原荞麦成分的标准方法。方法:参考现有文献,采用实时PCR方法建立食品过敏原荞麦成分的检测方法。结果:研究表明建立的检测方法具有特异性,灵敏度高,检测限为1 mg/kg。结论:本方法适用于食品中过敏原荞麦成分检测。     

快速检测试剂条、镜检和PCR在疟疾诊断中的应用研究

目的采用快速检测试剂条（rapiddiagnostictest,RDT）、镜检及聚合酶链反应（polymerasechainreac-tion,PCR）,3种检测方法进行疟原虫检测,比较3种方法在疟疾诊断中的敏感性、特异性,为科学分析检测结果和探究RDT能否在基层替代疟原虫镜检提供依据。方法收集安徽省2012年1月～2013年3月确诊以及疑似病例抗凝血148份,血片148张,采用RDT进行检测,并与镜检法和PCR的结果对比分析其敏感性与特异性。结果148份血样采用RDT、镜检和PCR3种方法检测结果阳性率分别为43．24％、36．49％、37．84％,经配对x2检验RDT阳性率高于镜检和PCR;镜检和PCR均检出2例卵形疟,而RDT结果为阴性;以镜检为标准,对间日疟和恶性疟进行分析得出,RDT的敏感度为100％,特异度87％,阳性预测值81％,阴性预测值100％;以PCR为标准,对间日疟和恶性疟进行分析得出,RDT的灵敏度为100％,特异度89％,阳性预测值84％,阴性预测值100％。3种方法均检出间日疟16例,而对恶性疟分别为RDT检出48例,镜检检出36例,PCR检出38例。结论RDT对间日疟和恶性疟敏感性较高,可以覆盖全部阳性病例,在今后疟疾低流行时期RDT有望取代基层镜检对疟疾进行诊断。     

实时荧光定量PCR快速检测嗜水气单胞菌方法的建立

目的利用实时荧光定量PCR技术,建立一种快速、特异、灵敏、定量检测嗜水气单胞菌的方法,用于病人腹泻物或水产品病变部位目的菌的快速检测。方法以GenBank中嗜水气单胞菌WP3的溶血素基因(hlyA)为靶序列,设计引物与TaqMan探针,对模板DNA制备方法,PCR反应时间和温度进行优化,以嗜水气单胞菌ATCC7966和20株相关细菌考核检测体系的特异性、灵敏性和重复性。结果本方法检测时间仅需30min,定量线性范围5.4×103～5.4×108cfu/ml,与20株常见肠道细菌均无反应,对4个浓度的嗜水气单胞菌测定的批内标准差在0.08～0.14之间。结论成功建立实时荧光定量PCR检测嗜水气单胞菌方法,该方法可在半小时左右直接定量检测病人腹泻物或水产品病变部位中的嗜水气单胞菌,具有推广应用价值。     

扎伊尔型埃博拉病毒实时荧光定量PCR方法的建立

目的 建立扎伊尔型埃博拉病毒实时荧光定量PCR快速检测技术。方法 人工合成扎伊尔型埃博拉病毒包膜糖蛋白GP基因上1 969～2 113 bp的保守特异片段,克隆到pUC57重组质粒中,构建阳性模板;以103～109拷贝数的重组质粒样品共7组作为标准品,制作标准曲线;设计上游引物5' - GAACCACATGATTGGACCAAGA - 3'、下游引物5' - TAACTCCAATACCTGCCGGTATC - 3'及TaqMan探针FAM 5' - TTGTTGATAAAACCCTTCCGGACCAGG - 3' BHQ1,采用普通PCR和实时荧光定量PCR,检测其特异性和灵敏度。结果 克隆出含GP 1 969～2 113 bp片段的阳性重组质粒pUC57 - GP,其浓度为97.75 ng/μl;以pUC57 - GP为标准品,制备标准曲线,拷贝数为103～109 copies/μL有较好线性关系,相关系数R2 = 0.999;能特异性检测扎伊尔型埃博拉病毒,而与I型登革病毒、寨卡病毒无交叉反应,其检测最低拷贝数达3.10×101copies/μl。 结论 建立了扎伊尔型埃博拉病毒荧光定量PCR快速检测技术,具有快速、灵敏、特异、定量检测等特点,可为埃博拉疫情的综合防控提供技术支撑。     

产气荚膜梭菌实时荧光定量PCR方法的应用

目的利用荧光定量PCR技术,建立快速敏感特异的产气荚膜梭菌检测方法。方法以产气荚膜梭菌基因作为靶序列,设计一对引物和探针,以产气荚膜梭菌菌株提取核酸DNA作为模板,优化引物和探针的浓度比,同时验证方法的灵敏度、特异性、重复性。结果反应体系在上、下游引物浓度均为1μmol/L,探针浓度为0.1μmol/L时,具有良好的特异性和灵敏度,与创伤弧菌等24种相关细菌均无交叉反应,对纯菌检测的灵敏度为9×102CFU/ml,重复性试验证实该体系重复性好,稳定性高;3例临床样本检测结果与细菌培养结果相符。结论建立的实时荧光PCR方法特异、灵敏、快速,能对临床气性坏疽做出快速准确的报告,实现对气性坏疽战时高发性疾病安全、快速和定量检测。     

Advantages and limits of real-time PCR assay and PCR-restriction fragment length polymorphism for the identification of cutaneous Leishmania species in Tunisia

Cutaneous leishmaniasis (CL), a public health problem in Tunisia, is associated to three species: Leishmania (L.) infantum, L. major and L. killicki. Accurate and sensitive procedures for the diagnostic of Leishmania infection and for species identification are required to enable adequate treatment and appropriate control measures. Several PCR-methods are applied for the diagnosis and the identification of Leishmania parasites such as PCR-restriction fragment length polymorphism (PCR-RFLP), DNA sequencing, hybridization probes and real-time PCR (RT-PCR). In this study, PCR-RFLP and RT-PCR were performed on skin scrapings from 27 patients with confirmed CL by microscopic examination, in order to compare their usefulness and efficiency for identification of Leishmania species in routine diagnostic laboratories. Identification of Leishmania species was successfully achieved in 96.3% and 81.5% respectively. Agreement between using internal transcribed spacer 1 (ITS1)-PCR-RFLP and kDNA-RT-PCR assays was 70% (19/27). Characterization problems using RT-PCR were mainly due to the difficulties in analyzing the melting temperatures.ITS1-PCR-RFLP and kDNA-RT-PCR presented an interesting alternative to conventional methods for the identification of Leishmania parasites from clinical samples. Both PCR assays can be used in a routine diagnostic, however, further prospective studies including largest sampling, are required to determine their performances in a routine use.     

Accuracy of the health information system on malaria surveillance in Vietnam

The health information system (HIS) is a key component of control programs and its accuracy is necessary for the assessment of disease risks, the formulation of priorities and the evaluation of the cost-effectiveness of different interventions. In order to assess the quality of the HIS in estimating malaria morbidity in Vietnam, we compared data obtained by a 2-year active (ACD) and passive case detection (PCD) study with those routinely collected at the local commune health centres (CHC) at three sites having different malaria epidemiology. The majority of malaria cases (80-95%) detected by ACD were missed by the HIS. Similarly, most malaria cases (50-90%) detected by PCD were also missed by the HIS, and this was proportional to the number of active private practitioners. Reasons for this low sensitivity are low CHC attendance, high attendance at private health facilities, widespread self-medication and attendance at central health facilities. In conclusion, although malaria has sharply decreased in Vietnam over the past 10 years, the current HIS greatly underestimates the malaria burden. Involvement of the private sector and the establishment of sentinel sites might improve the quality of data and the relevance of HIS in malaria control.     

实时荧光聚合酶链反应检测解脲脲支原体的临床意义

目的探讨实时荧光聚合酶链反应(PCR)在检测解脲脲支原体(Uu)中的临床意义。方法对278例未经治疗的门诊就诊者和35例确诊为Uu感染的患者经抗菌药物应用两周停药1周后分别采用实时荧光PCR和培养技术进行Uu的检测。结果 278例首次就诊者经实时荧光PCR检测Uu阳性125例,阳性率44.96%,培养阳性113例,阳性率40.65%,两者比较差异无统计学意义,35例确诊为Uu患者经抗菌药物治疗后停药1周,采用实时荧光PCR检测阳性12例,阳性率34.29%,但病原体含量要远远低于用药初期,35例患者经培养阳性5例,阳性率14.29%;两者比较差异有统计学意义(P<0.05)。结论实时荧光PCR在判断是否存在Uu感染时要较培养法灵敏,对经抗菌药物治疗停药后判断是否治愈时培养法要较荧光PCR更为准确。     

荧光定量PCR方法快速检测人博卡病毒的研究

目的建立特异、快速、灵敏的、用于人博卡病毒核酸检测的荧光定量PCR方法。方法对博卡病毒的ns1基因应用Clustal W软件进行序列同源性比对,在保守区设计特异性引物和TaqMan探针,并对荧光PCR反应条件进行优化,验证方法的特异性,并将该方法与国际上通用的针对人博卡病毒ns1基因和np基因的普通PCR方法进行灵敏度比对;同时将该方法应用于疑似患者临床标本检测。结果该方法针对ns1基因进行扩增,对人博卡病毒核酸检测有高度特异性,与副流感1、2、3型,甲型、乙型流感病毒,呼吸道合胞病毒,腺病毒,冠状病毒229E、OC43,偏肺病毒以及鼻病毒等均无交叉反应。检测的灵敏度均比国际上通用的扩增ns1基因及np基因的PCR方法高10倍。用该方法从144份急性上呼吸道感染患者咽拭标本中检出了2份博卡病毒阳性,从病毒核酸提取至检测完成仅需3 h左右;用普通PCR仅能检出1份阳性标本,且耗时需5 h左右。结论该研究建立的TaqMan荧光定量PCR是一种快速检测人博卡病毒特异、敏感的方法,适用于临床早期诊断。     

TaqMan-MGB探针实时荧光定量PCR联合检测难辨梭菌菌属基因及毒素基因方法学的建立

目的 建立一种简便、快速难辨梭菌菌属鉴定及其毒素基因筛查的荧光定量PCR检测方法。方法 采用TaqMan-MGB探针,通过real-time PCR分析系统,同时检测难辨梭菌菌属基因磷酸丙糖异构酶（Tpi）、A毒素（TcdA）、B毒素（TcdB）及缺失部分基因的A毒素（TcdAT）,从特异度、灵敏度及其抗干扰性等方面评价该方法,并联合全自动酶联荧光免疫系统（VIDAS）检测50 例临床不明原因腹泻病例粪便标本探讨其应用价值。结果 难辨梭菌非产毒株 Tpi 基因（tpi）的检测下限是6×10^-2 CFU/μl,产毒株 tpi、tcdA、tcdB、tcdAT 的检测下限为 6×10^-1 CFU/μl;难辨梭菌非产毒株tpi检测下限批内、批间变异率分别为2.1%和2.3%;产毒株tpi、tcdA、tcdB、tcdAT的检测下限批内、批间变异率依次为3.0%和3.4%、2.9%和3.2%、5.3%和5.7%、2.7%和2.8%。临床常见分离菌株及梭菌属其他细菌对检测无干扰。50 例不明原因腹泻病例粪便标本中,采用TaqMan-MGB 探针实时荧光 PCR 与 VIDAS 酶标免疫检测 39 例阴性标本其符合率为 100%;6 例两方法检测均为阳性;3例VIDAS酶标免疫检测为可疑及2例为阴性,经TaqMan-MGB探针实时荧光PCR检测为A-/B＋菌株。结论 建立的TaqMan-MGB探针实时荧光定量PCR具有高通量、高灵敏度和重复性好的特性,且可筛查携带缺失部分基因的TcdA难辨梭菌菌株。