PCR technique for detecting Toxoplasma gondii in animal amniotic fluid

The goal of diagnosing congenital toxoplasmosis is early detection of maternofetal transmission, for early treatment to prevent unwanted sequelae. Polymerase chain reaction (PCR) is a method used recently for detecting toxoplasmosis during pregnancy. Amniotic fluid is a the clinical specimen used, since it provides a rapid, simple and safe method to obtain accurate results. The advantages of the PCR technique are high sensitivity, specificity and positive predictive value compared with other laboratory methods. To determine the sensitivity, specificity and lower detection limits in our laboratory, amplification of the B1 gene by nested PCR was performed on Toxoplasma gondii tachyzoites added to animal amniotic fluid samples. From 48 samples, our technique detected T. gondii in 30 out of 41 positive samples, and gave negative results for all the negative samples. The sensitivity for this nested PCR was 73%, the specificity was 100%, and the efficiency of the test was 77.1%. The nested PCR technique is recommended as a diagnostic method for detecting T. gondii in suspected congenital toxoplasmosis animals.     

应用环介导等温扩增技术检测弓形虫

目的 用环介导等温扩增（LAMP）技术检测弓形虫。 方法　 用酚-氯仿法提取弓形虫速殖子基因组 DNA,设计两对扩增弓形虫 B1基因的 LAMP 引物。以间日疟原虫、恶性疟原虫、卡氏肺孢子虫、日本血吸虫及小鼠白细胞作对照,进行LAMP反应,产物经 SYBR Green I显色及电泳后观察结果,绿色判为阳性,棕色判为阴性。将弓形虫速殖子经倍比稀释为（2～3）×106个/ml至（2～3）×10-1个/ml 等8个浓度,进行LAMP反应,验证该方法的敏感性。 结果 LAMP反应结果显示,弓形虫速殖子检测管经显色后呈绿色,对照组均呈棕色。弓形虫的LAMP产物经电泳后呈LAMP特征性梯状条带,对照组均无扩增产物。LAMP技术可检测到的弓形虫速殖子最低浓度为2～3个/ml。 结论 LAMP技术在弓形虫检测中显示出较好的特异性与敏感性。     

Detection of Toxoplasma gondii in human placenta by PCR and placental histologic findings

Since isolation of Toxoplasma gondii from human placenta strongly correlates with fetal infection, the aims of the study were: to detect fragments of T. gondii B1 gene in human placentae by PCR and to evaluate their pathology. 36 placentae included in three groups were obtained: group I (n = 7) from pregnancies with prenatal diagnosis of fetal toxoplasmosis; II (n = 17) from women with serologic features of primary infection during pregnancy; III (n and 13) from pregnancies with fetal T. gondii infection based on clinical signs. T. gondii DNA was found in 2/4 samples from the I group and in 1/14 from the II group. Villitis was identified in 3/15 other placentae from the II group. In the III group we did not recognize neither T. gondii DNA nor villitis. We consider PCR and pathologic evaluations of placentae as the two complementary methods. PCR can be especially helpful in pregnancies not screened against T. gondii as positive result in placenta can confirm mother's primary infection.     

应用PCR技术检测孕早期绒毛组织弓形虫DNA的研究

应用ＰＣＲ技术检测２７０例孕６－１２周妇女外周血及相应绒毛组织中的弓形虫ＤＮＡ，结果弓形虫ＤＮＡ阳性２６例，相应的绒毛组织中检出８例，垂直传播率为３０．７７％，同时用ＥＬＩＳ法检测血清中弓形虫循环抗原、ＩｇＭ和ＩｇＧ抗体，结果阳性率分别为３．７０％、７．０３％和１５．５％。ＩｇＭ和／或ＣＡｇ阳性者，外周血皆检出弓形虫ＤＮＡ。提示应用ＰＣＲ技术检测绒毛组织弓形虫ＤＮＡ结合ＥＬＩＳＡ进行筛选，可早期诊     

用PCR技术检测大鼠弓形虫DNA

目的 探讨弓形虫病大鼠外周血弓形虫DNA检测的意义。方法 自行设计一对引物,用聚合酶链反应技术扩增弓形虫P30基因的一段保守序列。结果 设计的这对引物对健康人、大鼠、小鼠外周血白细胞以及阴道毛滴虫、溶组织内阿米巴均不能扩增,表明具有特异性。反应体系经35个循环扩增,可检测到2条弓形虫DNA,表明具有较高的敏感性。结论 PCR法对大鼠弓形虫感染可做出早期诊断。     

弓形体包囊的免疫荧光检查

应用直接和间接荧光抗体法可有效地检出病料中的弓形体包囊和速殖子。直接法在被检标本上用特异性荧光抗体染色,弓形体包囊呈圆形,发黄绿色荧光,其中慢殖子发强荧光;速殖子呈半月形,发黄绿色荧光。间接法采用双层染色法。包囊呈圆形,发黄绿色荧光,包囊周边部见一特亮的荧光圈,周围区绕有一淡色荧光层;速殖子发均匀的黄绿色荧光。     

Detection of Toxoplasma gondii DNA and specific antibodies in high-risk pregnant women

Primary maternal infection with toxoplasmosis during pregnancy is frequently associated with transplacental transmission to the fetus. This study was conducted to test the utility of a polymerase chain reaction (PCR) assay to detect recent infections with Toxoplasma in pregnant women. One hundred forty-eight women with high-risk pregnancies who had abnormal pregnancy outcomes (cases) and 100 with normal pregnancies (controls) were tested for the presence of Toxoplasma DNA in their blood by a nested PCR and specific antibodies to Toxoplasma by an enzyme-linked immunosorbent assay. The IgG results of the cases differed significantly from those of the controls (54% and 12%, respectively; P < 0.02). Four (2.7%) of the cases were IgM positive, but none of the controls were positive. Detection of Toxoplasma DNA in 20 (8.1%) of the IgG-positive cases suggests a recent infection. The risk factors associated with the infection were eating raw meat and contact with soil. The diagnostic serology of recent infection in early pregnancy could be confirmed by a positive Toxoplasma-specific PCR result in blood samples collected in the first half of pregnancy, even in the presence of serologic results difficult to interpret due to the lack of sequential follow-up during pregnancy.     

Detection of house-dust-mite allergen in amniotic fluid and umbilical-cord blood

Mononuclear cells in umbilical-cord blood display allergen-specific reactivity, but how allergen exposure occurs in utero is unknown. We investigated the presence of a common inhalant allergen (Der p 1), to which mothers are exposed throughout pregnancy, by ELISA in matched maternal blood and amniotic fluid samples at 16-17 weeks of gestation, and in matched maternal and umbilical-cord blood at term (> or =37 weeks of gestation). Der p 1 was detectable in 24 of 43 amniotic fluid samples where it was also present in maternal blood, and in 15 of 24 cord-plasma samples at significantly higher concentrations than in the maternal plasma (p=0.022). The detection of Der p 1 in the amniotic fluid and the fetal circulation provides direct evidence of transamniotic and transplacental allergen exposure.     

弓形虫感染家兔精液中虫体DNA检测初报

目的了解弓形虫感染家兔精液中是否有虫体存在，探明虫体在精液中动态变化情况。方法用RH株弓形虫速殖子不同剂量经腹腔感染家兔6只。在家兔感染前后分别采集精液，-40℃冻存备用，分别用普通PCR检测弓形虫感染家兔精液中弓形虫DNA。结果5只家兔在感染后的8～14d死亡，仅1只家兔在感染后的8d和72d，用PCR检测手段在精液中均可检测到弓形虫DNA。结论弓形虫感染雄兔精液中有虫体存在。     

弓形虫速殖子抗原的提取及蛋白分析

弓形虫病是一种严重危害人民健康的人兽共患病。全球约有 10亿人被弓形虫感染 ,我国弓形虫血清阳性率约 5 % [1 ] 。由于弓形虫的生物特性 ,人体感染后一般症状不明显 ,多呈隐性感染 ,无特异的临床症状与体征 ,且病原学检查难以查到病原体。因此 ,弓形虫感染的诊断多依靠免疫学方法。本研究主要对弓形虫 RH株速殖子抗原的提取、纯化和制备进行初探 ,对速殖子抗原的蛋白分子进行试验分析 ,以提高免疫诊断的敏感性和特异性 ,并试图了解其功能性抗原。材料与方法1　弓形虫速殖子的收集和抗原提取、制备从本实验室弓形虫 RH株感染、传代的昆明…     

Detection of circulating Toxoplasma gondii antigens by the dot-ELISA method

Dot-immunobinding technique was used to detect circulating Toxoplasma antigens in the sera of men and animals. Serum samples were collected from mice and rabbits infected experimentally with parasites of the RH strain of T. gondii while human sera were obtained from persons suspected of having active toxoplasmosis. Circulating Toxoplasma antigens were detected in the samples of mice sera that had been collected since the 2-nd day of infection and in the sera of rabbits during 2 and 3 week of infection. From amongst 146 human sera, 35 (24%) gave positive reaction in the test while 14 (9,5%) produced doubtful results. The experiments proved the method to be sensitive, reproducible and easy to perform. It obviates the need of special equipment while most of the necessary reagents are commercially available. High percentage of doubtful results is the main drawback of the method.     

The role of macrophages in protective and pathological responses to Toxoplasma gondii

The ability of Toxoplasma gondii to cause clinical disease in immune-competent and immune-deficient hosts coupled with its ease of use in vitro and availability of murine models has led to its use as a model organism to study how the immune system controls an intracellular infection. This article reviews the studies that established the role of the cytokine IFN-γ in the activation of macrophages to control T gondii and the events that lead to the mobilization and expansion of macrophage populations and their ability to limit parasite replication. Macrophages also have pro-inflammatory functions that promote protective NK and T-cell activities as well as regulatory properties that facilitate the resolution of inflammation. Nevertheless, while macrophages are important in determining the outcome of infection, T gondii has evolved mechanisms to subvert macrophage activation and can utilize their migratory activities to promote dissemination and these two properties underlie the ability of this parasite to persist and cause disease.     

刚地弓形虫感染致小鼠生殖细胞DNA损伤的作用

目的 探讨刚地弓形虫感染对小鼠睾丸细胞DNA的损伤作用.方法 选用9～10周龄雄性BALB/c小鼠20只,随机分为4组,每组5只,分为正常对照组(CG)和3个染虫组(G1,G2,G3).采用腹腔注射法建立小鼠刚地弓形虫感染的动物模型,CG组每只注射PBS 0.2 ml;染虫G1,G2,G3组注射纯化的刚地弓形虫速殖子悬液,每只注射刚地弓形虫速殖子的量依次为2.5×10 3、5×10 3、1×10 4,注射体积均为0.2 ml,应用单细胞凝胶电泳检测睾丸细胞DNA的损伤. 结果 正常对照组和染虫组的尾距分别为10.94±7.57、40.37±6.25、69.76±3.97、79.16±6.36,olive尾距分别为10.57±6.72、31.39±4.59、48.66±4.60、53.87±6.55,G1-G3组与CG组比较,差异具有统计学意义(P＜0.01).结论 弓形虫感染可导致小鼠睾丸生殖细胞DNA不同程度的损伤.     

刚地弓形虫感染致小鼠睾丸细胞DNA的损伤作用

目的探讨刚地弓形虫感染对小鼠睾丸细胞DNA的损伤作用。方法选用9～10周龄雄性BALB/c小鼠20只,随机分为对照组(CG)和3个染虫组(G1、G2和G3)。采用腹腔注射法建立小鼠刚地弓形虫感染的动物模型,CG组注射PBS 0.2 ml/只,染虫G1、G2和G3组各注射纯化刚地弓形虫速殖子悬液0.2 ml,剂量分别为2.5×10~3个、5×10~3个和1×10~4个。应用单细胞凝胶电泳(彗星实验)检测睾丸细胞DNA的损伤。结果对照组和3个染虫组的彗星实验尾矩分别为10.94±7.57、40.37±6.25、69.76±3.97和79.16±6.36;Olive尾矩分别为10.57±6.72、31.39±4.59、48.66±4.60和53.87±6.55,对照组和实验组彗星尾矩和Olive尾矩差异有统计学意义(P<0.01)。结论弓形虫感染可致小鼠睾丸细胞DNA不同程度的损伤。对小鼠有生殖遗传毒性。     

地高辛素标记弓形虫DNA探针的制备及在产前诊断的应用

本文建立了地高辛素标记的弓形虫ＤＮＡ探针并在２３例产前咨询孕妇中作产前诊断。通过聚合酶链反应扩增弓形虫ＲＨ株ＴＧＲ４核酸片段，扩增产物７７８ｂｐ片段经寡核苷酸层析柱纯化后，用地高辛素标记作为探针，与待检材料斑点杂交。结果显示该探针与待杂交的弓形虫ＲＨ、ＺＳ１、ＺＳ２、ＳＨ１株ＤＮＡ杂交；而与恶性疟原虫、卡氏肺孢子虫、杜氏利什曼原虫、大肠杆菌、巨细胞病毒ＤＮＡ无杂交。并能在待杂交液中检测出相当于１ｐｇ水平ＤＮＡ，表明该探针具有高度的特异性及敏感性。检测２３例孕期１０—１４周孕妇中，阳性３例，其中一孕妇外周血、绒毛组织及羊水均呈阳性。另一例的外周血及绒毛组织呈阳性。该探针稳定、敏感、操作简单，具有推广意义。     

Molecular diagnosis of cerebral toxoplasmosis: comparing markers that determine Toxoplasma gondii by PCR in peripheral blood from HIV-infected patients

As cerebral toxoplasmosis is the most common cerebral focal lesion in AIDS patients, this study evaluated three PCR markers for diagnosis, since some limitations remain present, such as low parasite levels in some clinical samples. The molecular markers were B22-B23 and Tg1-Tg2 (based on the B1 gene) and Tox4-Tox5 (non-coding fragment, repeated 200-300-fold). DNA samples from 102 AIDS patients with previously known diagnosis were analyzed. The cerebral toxoplasmosis group was constituted of DNA extracted from the blood of 66 AIDS patients, which was collected before or until the third day of the therapy for toxoplasmosis. DNA from the blood of 36 AIDS patients with other neurologic opportunistic infections was used as control group. Sensitivities of B22-B23, Tg1-Tg2, and Tox4-Tox5 markers were of 95.5%, 93.9%, and 89.3%, respectively. In the control group, the specificities were of 97.2% (B22-B23), 88.9% (Tg1-Tg2), and 91.7% (Tox4-Tox5). The association of at least two markers increased the PCR sensitivity and specificity. The concordance index between two markers varied from 83.3% to 93.1%. These data demonstrated that all markers evaluated here were highly sensitive for T. gondii determination, although B22-B23 has been shown to be the best. The association of two markers increases PCR sensitivity, but the procedure was more expensive and time-consuming.     

奶牛血清弓形虫抗体检测

弓形虫(Toxoplasma gondii)是猫科动物的肠道球虫,哺乳动物、鸟类以及冷血动物普遍易感,可引起人兽共患弓形虫病。世界上大部分地区都有牛弓形虫感染的报道,我国部分省牛弓形虫感染率也较高[1]。为了解国内奶牛弓形虫的感染状况,作者应用间接血凝试验(IHA)进行了奶牛血清弓形虫抗体检测,现报道如下。1　材料和方法1.1　待检血清　奶牛血清200份,采自光明乳业集团奶牛场。1.2　试剂　弓形虫病间接血凝(IHA)抗原(批号:20020822,浓度为1%,效价≥1∶1 024),阴、阳性对照血清,稀释液,均由中国农业科学院兰州兽医研究所惠赠。1.3　方法　在96孔…     

精神分裂症患者弓形虫感染的血清学调查

我国正常人群弓形虫血清抗体阳性率为 5 .89% [1 ] ,精神病患者感染率高于正常人 [2 ]。为了解本地区精神分裂症患者弓形虫感染情况 ,探讨弓形虫感染与精神分裂症之间的关系 ,作者于 1999年 10月～ 2 0 0 0年 7月 ,采用间接红细胞凝集试验(IHA)对 2 2 2例住院精神分裂症患者和 78例健康体检者进行了弓形虫抗体检测。1　材料和方法1.1　对象1.1.1　实验组　为 1999年 10月～ 2 0 0 0年 7月张家口沙岭子医院住院精神分裂症患者 ,共 2 2 2例。其中男性 14 0例 ,女性 82例 ,被选病例均符合《中国精神疾病分类方案和诊断标准》。1.1.2　对照组…