PCR-ELISA检测弓形虫实验研究

目的建立快速、敏感、特异、稳定的PCR—ELISA方法，并用其检测感染动物体内的弓形虫。方法将生物素标记的PCR产物与地高辛标记的特异性探针杂交，再通过酶免显色反应测出OD值。以判断弓形虫感染情况。测定该方法的敏感性、特异性及稳定性。再分别以10^4、10^3弓形虫RH株速殖子腹腔接种小鼠。取全血、肝组织用PCR—ELISA检测小鼠感染情况。结果本实验中，PCR-ELISA方法的检测闻值为20fg弓形虫DNA，其灵敏度是电泳法的10倍，并且与人、小鼠、疟原虫、旋毛虫等DNA均无交叉反应。同一样本重复测试5次，结果经统计学检验，一致性良好（Alpha=0．72）。检测感染动物肝组织及全血标本，10^4、10^3组分别在感染后第二d、第三d即可测出阳性，两种标本的阳性检出效率无统计学差异（P〉0．05）。结论PCR—ELISA是一种快速、敏感、特异、稳定的检测方法，可试用于临床弓形虫病的诊断及流行病学调查。     

乙肝病毒DNA定量检测的临床分析

PCR技术检测血清或肝组织HBV DNA是诊断乙型肝炎病毒感染的指标之一 ,直接说明病毒的复制和传染性 ,是临床抗病毒治疗的指征。PCR ELISA定量检测对检测乙肝病毒含量及考核抗病毒治疗疗效 ,具有很好的使用价值。为了探讨HBV DNA在乙肝病毒感染者血清中的水平 ,我们对 92例ELISA检测为阳性的几种模式的乙肝感染者 ,作了HBV DNA定量检测和分析。为临床正确诊断和及时有效的治疗提供了可靠的依据。1　对象和方法1 1　对象　 92例乙肝病毒感染者 :其中大三阳 38例 ,小三阳 34例 ,其他乙肝指标阳性 2 0例。所有病例的HAV、HCV、H…     

比较PCR酶免法与套式PCR法在不孕症中沙眼衣原体的临床检测

目的 比较PCR酶免法(PCR—ELISA)与套式PCR法(nPCR)法检测沙眼衣原体(CT)在不孕症宫颈分泌物中的临床意义。方法 应用PCR-ELISA法对185份宫颈分泌物进行CT检测，nPCR法对l12份宫颈分泌物进行CT检测，两种方法同时对38份宫颈分泌物进行检测，两种方法的检测结果进行对比分析。结果 PCR-ELISA法，CT阳性率为36．22％，(67／185)。nPCR法检到法CT阳性率为19.64％(22／112)两种方法同时检测的CT阳性率分别为34.21％及13．16％。PCR-ELISA与nPCR法比较差异有显著性(P＜0.05)。结论 PCR—ELISA法检测CT比nPCR法更敏感，特异，简单，并且没有EB污染等优点，结合临床治疗结果分析更适用于对长期慢性CT感染不孕症的诊断。     

端粒酶活性与肿瘤相关性研究

本研究采用TRAP－银染色法和（或）PR－ELISA技术进行端粒酶活性研究。1997－1999年对肝癌、胃癌、急性淋巴细胞白血病、子宫颈癌、鼻窦部浆细胞肉瘤、恶性甲状腺瘤、精原细胞癌、阴茎癌等22种肿瘤共115份肿瘤组织标本检测，端粒酶阳性率为80％（92／115），同时检测这些肿瘤的临近正常组织共96份，端粒酶阳性率为5．2％（5／96）；34份子宫平滑肌瘤和8份非肿瘤病变组织端粒酶阳性率为7．1％（3／42），它们的临近正常组织阳性率为2．4％（1／42）；肿瘤细胞克隆株3例均为阳性。结果表明，恶性肿瘤组织细胞中端粒酶活性明显高于良性肿瘤和正常细胞（P＜0．01）；1999－2000年对胃癌组织进行研究，得出同样结果，因此端粒酶活性的检测有望成为一种新的肿瘤标志物用于临床诊断研究。端粒酶活性的增高与恶性肿瘤高度相关，对试图通过调控端粒酶活性来达到对恶性肿瘤的治疗研究提供了一个新的靶点和可靠依据。     

A new PCR-ELISA for diagnosis of visceral leishmaniasis in blood of HIV-negative subjects

The PCR-ELISA represents a promising advance for diagnosis of visceral leishmaniasis (VL) in blood samples. However, the method has been validated mostly with HIV-positive patients who are known to have high levels of parasitaemia. We developed a new PCR-ELISA assay for specific detection of Leishmania in patients' blood and validated it in Nepalese subjects with clinically suspected VL, almost all of whom were HIV-negative. For blood samples, PCR-ELISA was more sensitive (83.9%) than conventional PCR (73.2%), and demonstrated 100% and 87.2% specificity when using healthy controls who had never travelled to a VL-endemic area and controls from a VL-endemic area as references, respectively. We have demonstrated the ability of PCR-ELISA to detect parasites in blood of HIV-negative patients. The method could be used for epidemiological as well as clinical purposes, as it reduces the need for traumatic bone marrow sampling and risky spleen aspiration.     

端粒酶在子宫腺肌病中的表达及其临床意义

目的:通过检测端粒酶在子宫腺肌病患者异位内膜灶中的表达情况,探讨端粒酶在子宫腺肌病发生发展中的作用和临床应用价值。方法:采用PCR-ELISA技术检测30例子宫腺肌病患者异位内膜、在位内膜中端粒酶的表达情况,并与20例子宫肌瘤患者进行比较。结果:(1)研究组端粒酶阳性表达率与对照组比较差异显著(P<0.0001)。(2)研究组重度弥漫性子宫腺肌病患者的端粒酶阳性表达率明显高于轻中度患者,差异显著(P<0.05)。(3)研究组异位内膜端粒酶表达情况与在位内膜比较无显著差异(P>0.05)(4)轻中度腺肌病异位内膜灶端粒酶表达情况与周围正常组织比较无显著差异(P>0.05)。结论:端粒酶的活性与子宫腺肌病发生发展的关系密切。     

PCR-ELISA检测端粒酶活性的方法及其在人体肿瘤中的应用

目的：介绍ＰＣＲ－ＥＬＩＳＡ端粒酶活性测定方法。并以示同组织来源肿瘤端粒酶活性检测结果进行分析。方法：采用ＰＣＲ－ＥＬＩＳＡ端粒酶活性检测方法对２９９例孙同组织来瘤端粒酶活性进行检测。结果：本组结果显示ＰＣＲ－ＥＬＩＳＡ端粒酶性测定在敏感性与划性方面与国外报道的同位素方法结果一致。在多种人类恶性肿瘤组织中可检出端粒酶活性，而与各种肿瘤对应的正常组织及良性病变中则多为阴性，结论：本组结果提示ＰＣＲ＝     

卵巢上皮性肿瘤端粒酶活性定量检测

目的:探讨端粒酶在上皮性卵巢肿瘤发生发展中的作用,评价端粒酶作为卵巢癌诊断及预后指标的价值。方法:采用端粒酶PCR-ELISA法对25例卵巢上皮性肿瘤(8例良性、3例交界性和14例恶性)和6例正常卵巢表面上皮进行端粒酶活性定量检测。结果:2例良性、2例交界性和12例恶性卵巢上皮性肿瘤及1例正常卵巢上皮存在端粒酶活性增强,8例良胜、3例交界性、14例恶性和6例正常卵巢上皮端粒酶活性吸光值的x±s分别为0.080±0.070、0.408±0．208、1.659±0．930和0.086±0.060。统计学分析表明恶性卵巢癌中端粒酶活性明显高于良性、交界性肿瘤和正常卵巢组织;端粒酶活性和卵巢上皮性肿瘤组织学类型、临床分期无关;而与肿瘤的分化程度有关。结论:研究结果初步证明端粒酶活性增强在卵巢癌的无限增殖中是一重要环节,并提示端粒酶活性增强有可能成为卵巢癌的标志物之一。     

Direct identification of Pseudomonas aeruginosa from blood culture bottles by PCR-enzyme linked immunosorbent assay using oprI gene specific primers

A PCR-enzyme-linked immunosorbent assay (PCR-ELISA) was developed for direct identification of Pseudomonas aeruginosa from positive BACTEC blood culture bottles. PCR primers were designed to target a 249 bp sequence of the oprI gene in P. aeruginosa. Biotin-labeled probe (PA3) targeted to the species-specific motif were hybridized to the digoxigenin-labeled amplified products from P. aeruginosa and captured on streptavidin-coated microtiter plates. The specificity of the assay using the PA3 probe was investigated with a range of microorganisms, which are commonly isolated from blood culture bottles serving as negative controls. The PCR-ELISA assay was shown to be highly specific for the identification of P. aeruginosa and was 10-fold more sensitive than an agarose gel-based detection method using the same pair of primers, with a detection limit at 10 fg of template. The PCR-ELISA assay developed in this study is 100% sensitive and 100% specific as it correctly identified all 73 positive and 42 negative controls as well as 25 double blind clinical samples. It significantly reduces the time needed for the identification of P. aeruginosa from positive BACTEC blood cultures bottles from 2-3 days to 6-8h.     

Detection of hepatopancreatic parvovirus (HPV) infection in Penaeus monodon using PCR-ELISA

A rapid and sensitive PCR-ELISA has been developed for detection of hepatopancreatic parvovirus (HPV) in Penaeus monodon. The specific primer set amplified 156 bp fragment and could detect as a little as 0.01 fg of purified HPV DNA which equivalent to three viral particles. No cross-reactivity was observed when nucleic acid templates from white spot syndrome virus, yellow-head virus, monodon baculovirus and shrimp were tested. The crude DNA simple prepared from hepatopancreas can be used as DNA template and provide a favorable result. Using this technique for detection of HPV infection in 87 carrier shrimps revealed the higher sensitivity and efficiency of detection when compared to histological examination and conventional PCR. Sixty-two percent infection was detected by PCR-ELISA from samples with HPV negative diagnosed by histological examination. Therefore, this sensitive and specific method is promisingly useful for early detection of HPV infection in broodstock, carriers and for ex situ application where large numbers of samples can be analyzed simultaneously.