用PCR扩增tim基因检测蓝氏贾第鞭毛虫

采用聚合酶链反应 (PCR)对蓝氏贾第鞭毛虫 (Giardialamblia)的磷酸丙糖异构酶 (triosephosphateisomerase ,缩写为tim)基因进行特异性扩增 ,结果扩增出 1条 6 83bp的DNA片段。此方法的特异性可高达10 0 % ,而其它DNA样本 ,如日本血吸虫 (Schistosomajaponicum)、刚地弓形虫 (Toxoplasmagondii)、微小隐孢子虫 (Cryptosporidiumparvum)、溶组织内阿米巴 (Entamoebahistolytica)、旋毛虫 (Trichinellaspiralis)和阴道毛滴虫 (Trichomonasvaginalis) ,以及人体血细胞等均未出现扩增反应。本法的敏感性也很高 ,可检测到0 4pg贾第虫包囊的DNA。 13株来自不同地理位置和 或宿主的贾第虫DNA样本在PCR中均各产生 1条长为 6 83bp的目的片段。上述结果表明本实验建立的检测贾第虫的PCR方法有效     

用PCR扩增tim基因检测蓝氏贾第鞭毛虫

采用聚合酶链反应(PCR)对蓝氏贾第鞭毛虫(Giardia lamblia)的磷酸丙糖异构酶(triose phosphate isomerase,缩写为tim)基因进行特异性扩增,结果扩增出1条683bp的DNA片段.此方法的特异性可高达100%,而其它DNA样本,如日本血吸虫(Schistosoma japonicum)、刚地弓形虫(Toxoplasma gondii)、微小隐孢子虫(Cryptosporidium parvum)、溶组织内阿米巴(Entamoeba histolytica)、旋毛虫(Trichinella spiralis)和阴道毛滴虫(Trichomonas vaginalis),以及人体血细胞等均未出现扩增反应.本法的敏感性也很高,可检测到0.4pg贾第虫包囊的DNA.13株来自不同地理位置和/或宿主的贾第虫DNA样本在PCR中均各产生1条长为683bp的目的片段.上述结果表明本实验建立的检测贾第虫的PCR方法有效.     

用PCR扩增tim基因检测蓝氏贾第鞭毛虫

采用聚合酶链反应(PCR)对蓝氏贾第鞭毛虫(Giardia lamblia)的磷酸丙糖异构酶(triose phosphate isomerase,缩写为tim)基因进行特异性扩增,结果扩增出1条683bp的DNA片段.此方法的特异性可高达100%,而其它DNA样本,如日本血吸虫(Schistosoma japonicum)、刚地弓形虫(Toxoplasma gondii)、微小隐孢子虫(Cryptosporidium parvum)、溶组织内阿米巴(Entamoeba histolytica)、旋毛虫(Trichinella spiralis)和阴道毛滴虫(Trichomonas vaginalis),以及人体血细胞等均未出现扩增反应.本法的敏感性也很高,可检测到0.4pg贾第虫包囊的DNA.13株来自不同地理位置和/或宿主的贾第虫DNA样本在PCR中均各产生1条长为683bp的目的片段.上述结果表明本实验建立的检测贾第虫的PCR方法有效.     

实验感染C57 BL/6N小鼠粪内蓝氏贾第鞭毛虫tim基因检测

建立蓝氏贾第鞭毛虫感染动物模型 ,用PCR技术对感染动物粪内该虫tim基因进行检测 ,探讨蓝氏贾第鞭毛虫基因检测方法。用人源蓝氏贾第鞭毛虫河北株 (CD)和江苏株 (XZ)的包囊分别接种两组C5 7BL 6N小鼠 ,于接种后第 6天、1 0天和 1 4天收集粪便标本 ,采用酚 氯仿法提取总DNA。将提取液分别按 1 0 0 、 1 0 - 1 、 1 0 - 2 和 1 0 - 3稀释并纯化。根据该虫磷酸丙糖异构酶 (Triosephosphateisomerase,tim)基因合成 1对特异性引物 ,采用PCR技术分别扩增各个稀释度的样本。结果贾第虫阳性样本均被扩增出相应基因的 1条 683bp长的片段 ,纯化后的各个稀释度样本的PCR检测阳性率随稀释倍数的增加而增高。本研究结果为贾第虫感染基因检测方法的进一步研究提供了实验资料。     

贾第鞭毛虫和隐孢子虫的流行病学研究进展

蓝氏贾第鞭毛虫和隐孢子虫在我国的流行状况还不是很清楚,本文简单介绍了贾第鞭毛虫和隐孢子虫的特性及分类,综述了国外贾第鞭毛虫和隐孢子虫的流行状况及其分子流行病学方面的内容。     

新孢子虫核酸检测方法的建立及应用评价

以新孢子虫Nc-5基因为目的基因,建立检测新孢子虫的PCR方法并初步应用。根据Gen Bank发布的新孢子虫Nc-5特异性基因序列设计引物,以新孢子虫基因组DNA为模板,利用梯度PCR法优化反应条件,建立PCR检测方法。以弓形虫、疟原虫、牛环形泰勒虫、包拉米虫和马尔太虫为模板进行扩增以验证建立方法的特异性。采用紫外分光光度计测定新孢子虫基因组DNA浓度和纯度,倍比稀释后的DNA进行PCR扩增,产物电泳分析确定建立方法的敏感性;选择高浓度和低浓度的新孢子虫基因组DNA重复检测3次,分析建立方法的重复性和稳定性。利用该方法对实验室建立的小鼠感染模型进行初步应用,评价建立方法的检测效果。结果成功建立新孢子虫的核酸扩增检测方法,扩增序列与Gen Bank(LN714476.1)中Nc-5基因序列一致性为100%,建立的新孢子虫核酸扩增检测方法与弓形虫、疟原虫、牛环形泰勒虫、包拉米虫和马尔太虫均无交叉反应,最低能检测新孢子虫DNA浓度为0.5788 pg/μL,应用该方法检测感染新孢子虫的BALB/c小鼠模型,可在肺和脑组织中检测到新孢子虫的感染。结果表明,建立的新孢子虫核酸扩增检测方法有效,并为后续研究奠定基础。     

尿液中GSTP1基因甲基化数字PCR方法的建立和优化

目的 基于微滴式数字PCR(droplet digital PCR,ddPCR)建立一种检测前列腺癌(prostatic cancer, PCa)尿液谷胱甘肽硫转移酶P1(glutathione S-transferase pi-1,GSTP1)基因甲基化的方法,并对其进行优化。方法 本研究收集20例前列腺癌尿液样本,设计并筛选引物和探针,通过调整亚硫酸氢盐转化时间及ddPCR反应体系中引物浓度、探针浓度、退火温度、循环次数、变性时间和退火延伸时间对ddPCR检测方法进行优化。结果 尿液DNA亚硫酸氢盐转化的最佳时间是2h 10min,最佳引物浓度是700nmol/L,最佳扩增温度为58.3℃,最佳循环次数是50次,最佳变性时间是30s,最佳退火延伸时间是60s,本研究范围内探针浓度对ddPCR影响不大,可根据实验自行调整探针浓度。结论 本研究建立并优化了ddPCR检测前列腺癌尿液样本GSTP1基因甲基化方法,提高了ddPCR检测尿液中痕量DNA的性能。     

郑州市某医院儿童肠道寄生虫感染情况调查

目的了解郑州地区儿童肠道寄生虫感染情况,为制定肠道寄生虫病防治措施提供参考依据。方法采用卢戈氏碘液染色法、饱和蔗糖溶液漂浮法和改良抗酸染色法对儿童新鲜粪便样品进行检查。结果共调查1996份粪便样品,肠道寄生虫总感染率为1.5%,发现蓝氏贾第鞭毛虫、环孢子虫、隐孢子虫、阿米巴原虫和粪类圆线虫5种肠道寄生虫,其感染率分别为0.6%、0.5%、0.1%、0.3%和0.1%。不同性别、不同季节儿童肠道寄生虫感染情况差异无统计学意义（P〉0.05）。蓝氏贾第鞭毛虫和阿米巴原虫主要发现于春季和冬季,隐孢子虫和环孢子虫仅发现于夏季和秋季。结论郑州地区儿童土源性肠道寄生虫感染率显著下降,以机会性原虫感染为主,应进一步加强健康卫生教育。     

人源性蓝氏贾第鞭毛虫Elp3基因的克隆及序列分析

目的克隆蓝氏贾第鞭毛虫Elp3基因,并应用生物信息学方法进行分析。方法根据蓝氏贾第鞭毛虫Elp3已知基因序列的特点设计引物,利用PCR技术扩增获得Elp3的核苷酸序列,连接到pET28a载体并测定序列。应用生物信息学方法分析Elp3基因的序列同源性与结构特征。结果扩增片段长度为1767bp,测序结果与蓝氏贾第鞭毛虫WB株（GL50830）同源性100%。可构建生物进化树,进行同源结构建模发现,71-362aa具有一个保守的S-腺甙基蛋氨酸区域,458-584aa具有组蛋白乙酰基转移酶区域。结论成功克隆了蓝氏贾第鞭毛虫Elp3基因,生物信息学分析发现,其在进化上与其它物种不属同一起源,具有SAM和HAT的结构和功能,这些发现为进一步研究其生物学功能提供了依据和线索。     

实时荧光定量PCR检测沙眼衣原体的研究

目的采用TaqMan探针建立检测沙眼衣原体的实时荧光定量PCR（real-time PCR）方法。方法根据沙眼衣原体外膜蛋白A的基因（ompA）序列设计引物和探针,以克隆的ompA部分基因片段作DNA模板,建立实时荧光定量检测方法。结果建立的荧光定量PCR检测方法的最低检出限为5 copies/反应,检测线性范围100～107线性关系良好（r2=0.997）,比巢式PCR敏感100倍;且与鹦鹉热衣原体、淋球菌、解脲脲原体、大肠杆菌等病原菌DNA以及人基因组DNA均无交叉反应,表明该方法具有良好的特异性。以巢式PCR作参比,建立的荧光定量PCR法检测沙眼衣原体的阳性符合率为100.00%,阴性符合率为95.09%,总符合率为96.81%。结论建立的检测沙眼衣原体实时荧光定量PCR具有特异性强和敏感性高的特点,可快速检测样本中微量沙眼衣原体DNA,适用于对沙眼衣原体进行大规模筛选。     

Evaluation of nine immunoassay kits (enzyme immunoassay and direct fluorescence) for detection of Giardia lamblia and Cryptosporidium parvum in human fecal specimens.

It is well known that Giardia lamblia and Cryptosporidium parvum can cause severe symptoms in humans, particularly those who are immunologically compromised. Immunoassay procedures offer both increased sensitivity and specificity compared to conventional staining methods. These reagents are also helpful when screening large numbers of patients, particularly in an outbreak situation or when screening patients with minimal symptoms. The data obtained by using 9 diagnostic kits were compared: direct fluorescent-antibody assay (DFA) kits (TechLab Giardia/Crypto IF kit, TechLab Crypto IF kit, and Meridian Merifluor Cryptosporidium/Giardia) and enzyme immunoassay (EIA) kits (Alexon ProSpecT Giardia EZ Microplate Assay, Alexon ProSpecT Cryptosporidium Microplate Assay, Cambridge Giardia lamblia Antigen Microwell ELISA, Meridian Premier Giardia lamblia, Meridian Premier Cryptosporidium, TechLab Giardia CELISA, Trend Giardia lamblia EIA). The test with the Meridian Merifluor Cryptosporidium/Giardia kit was used as the reference method. In various combinations, 60 specimens positive for Giardia, 60 specimens positive for Cryptosporidium, 40 specimens positive for a Giardia-Cryptosporidium mix, and 50 negative fecal specimens were tested. Different species (nine protozoa, three coccidia, one microsporidium, five nematodes, three cestodes, and one trematode) were included in the negative specimens. The sensitivity of EIA for Giardia ranged from 94% (Alexon) to 99% (Trend and Cambridge); the specificity was 100% with all EIA kits tested. The sensitivity of EIA for Cryptosporidium ranged from 98% (Alexon) to 99% (Meridian Premier); specificities were 100%. All DFA results were in agreement, with 100% sensitivity and specificity; however, the TechLab reagents resulted in fluorescence intensity that was generally one level below that seen with the reagents used in the reference method. In addition to sensitivity and specificity, factors such as cost, simplicity, ease of interpretation of results (color, intensity of fluorescence), equipment, available personnel, and number of tests ordered are also important considerations prior to kit selection.     

Comparison of techniques for detecting antigens of Giardia lamblia and Cryptosporidium parvum in faeces.

AIM--To compare the use of commercial monoclonal antibody test systems--the Giardia CEL IF test and the Crypto CEL IF test--for the detection of Giardia lamblia and Cryptosporidium parvum antigens in faeces with conventional techniques. METHODS--Sensitivity and specificity were evaluated using preparations of cysts of G lamblia and purified oocysts of C parvum. Evaluation of 59 random faecal samples passing through the Department of Clinical Parasitology, Hospital for Tropical Diseases, London, was carried out for both organisms. RESULTS--The fluorescence staining techniques proved more sensitive than other tests routinely used for diagnosis.     

Detection and genotyping of Entamoeba histolytica, Entamoeba dispar, Giardia lamblia, and Cryptosporidium parvum by oligonucleotide microarray

Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum are the most frequently identified protozoan parasites causing waterborne disease outbreaks. The morbidity and mortality associated with these intestinal parasitic infections warrant the development of rapid and accurate detection and genotyping methods to aid public health efforts aimed at preventing and controlling outbreaks. In this study, we describe the development of an oligonucleotide microarray capable of detecting and discriminating between E. histolytica, Entamoeba dispar, G. lamblia assemblages A and B, and C. parvum types 1 and 2 in a single assay. Unique hybridization patterns for each selected protozoan were generated by amplifying six to eight diagnostic sequences/organism by multiplex PCR; fluorescent labeling of the amplicons via primer extension; and subsequent hybridization to a set of genus-, species-, and subtype-specific covalently immobilized oligonucleotide probes. The profile-based specificity of this methodology not only permitted for the unequivocal identification of the six targeted species and subtypes, but also demonstrated its potential in identifying related species such as Cryptosporidium meleagridis and Cryptosporidium muris. In addition, sensitivity assays demonstrated lower detection limits of five trophozoites of G. lamblia. Taken together, the specificity and sensitivity of the microarray-based approach suggest that this methodology may provide a promising tool to detect and genotype protozoa from clinical and environmental samples.     

Prospective comparison of direct immunofluorescence and conventional staining methods for detection of Giardia and Cryptosporidium spp. in human fecal specimens.

In a prospective comparative study, 2,696 consecutive fresh stool specimens over the course of 1 year were examined for Giardia lamblia and Cryptosporidium parvum by using a direct immunofluorescent-monoclonal antibody stain (for unspun specimens) and conventional staining methods (chlorazol black E for Giardia cysts and modified Kinyoun acid-fast for Cryptosporidium oocysts). The direct immunofluorescent-monoclonal antibody method resulted in a significantly increased detection rate for both giardia (118 versus 79 specimens, 49.4%; P = 0.006) and cryptosporidia (39 versus 23 specimens, 69.6%; P = 0.055).     

Evaluation of an enzyme-linked immunosorbent assay for detection of Cryptosporidium spp. in stool specimens.

We evaluated a commercially produced enzyme-linked immunosorbent assay (ELISA; LMD Laboratories, Inc.) for the detection of Cryptosporidium spp. in 296 stool specimens submitted to the Mayo Clinic parasitology laboratory for routine examination. The specimens examined were fresh (4 specimens), were stored frozen at -65 degrees C (49 specimens), or were preserved in 10% formalin (243 specimens). Results were compared with those obtained by indirect immunofluorescent antibody detection (Merifluor Cryptosporidium/Giardia; Meridian Diagnostics, Inc.). One hundred of the specimens were positive by indirect immunofluorescent antibody and ELISA, while 187 were negative by both methods; 91 of these negative stool samples contained 121 parasites of 17 different species. Eight ELISA false negatives and one false positive were observed. The ELISA sensitivity was 93%, specificity was 99%, and the positive predictive value was 99%. Storage of specimens preserved in 10% formalin or frozen fresh at -65 degrees C for up to 18 months did not appear to affect the results. There was no cross-reactivity with Giardia lamblia (54 negative specimens) or with the 16 other parasites present in the ELISA-negative stool samples. The ELISA is a fast, easy-to-read, and accurate method for the detection of Cryptosporidium spp. in stool specimens.     

Parasitological diagnosis combining an internally controlled real-time PCR assay for the detection of four protozoa in stool samples with a testing algorithm for microscopy

Molecular detection of gastrointestinal protozoa is more sensitive and more specific than microscopy but, to date, has not routinely replaced time-consuming microscopic analysis. Two internally controlled real-time PCR assays for the combined detection of Entamoeba histolytica , Giardia lamblia , Cryptosporidium spp. and Dientamoeba fragilis in single faecal samples were compared with Triple Faeces Test (TFT) microscopy results from 397 patient samples. Additionally, an algorithm for complete parasitological diagnosis was created. Real-time PCR revealed 152 (38.3%) positive cases, 18 of which were double infections: one (0.3%) sample was positive for E. histolytica , 44 (11.1%) samples were positive for G. lamblia , 122 (30.7%) samples were positive for D. fragilis , and three (0.8%) samples were positive for Cryptosporidium . TFT microscopy yielded 96 (24.2%) positive cases, including five double infections: one sample was positive for E. histolytica / Entamoeba dispar , 29 (7.3%) samples were positive for G. lamblia , 69 (17.4%) samples were positive for D. fragilis , and two (0.5%) samples were positive for Cryptosporidium hominis / Cryptosporidium parvum . Retrospective analysis of the clinical patient information of 2887 TFT sets showed that eosinophilia, elevated IgE levels, adoption and travelling to (sub)tropical areas are predisposing factors for infection with non-protozoal gastrointestinal parasites. The proposed diagnostic algorithm includes application of real-time PCR to all samples, with the addition of microscopy on an unpreserved faecal sample in cases of a predisposing factor, or a repeat request for parasitological examination. Application of real-time PCR improved the diagnostic yield by 18%. A single stool sample is sufficient for complete parasitological diagnosis when an algorithm based on clinical information is applied.     

Detection of diarrhoea-causing protozoa in general practice patients in The Netherlands by multiplex real-time PCR

The diagnostic value of a multiplex real-time PCR for the detection of Entamoeba histolytica, Giardia lamblia and Cryptosporidium parvum/Cryptosporidium hominis was evaluated by comparing the PCR results obtained with those of routinely performed microscopy of faecal samples from patients consulting their general practitioner (GP) because of gastrointestinal complaints. Analysis of 722 faecal DNA samples revealed that the prevalence of G. lamblia was 9.3% according to PCR, as compared to 5.7% by microscopy. The number of infections detected was more than double in children of school age. Furthermore, G. lamblia infection was detected in 15 (6.6%) of 228 faecal samples submitted to the laboratory for bacterial culture only. C. parvum/C. hominis infections were not diagnosed by routine procedures, but DNA from these organisms was detected in 4.3% of 950 DNA samples. A strong association with age was noted, with Cryptosporidium being detected in 21.8% of 110 children aged <5 years. C. hominis was the most prevalent species. E. histolytica was not detected in this study population. Analysis of microscopy data revealed that the number of additional parasites missed by PCR was small. Overall, the study demonstrated that a multiplex real-time PCR approach is a feasible diagnostic alternative in the clinical laboratory for the detection of parasitic infections in patients consulting GPs because of gastrointestinal symptoms.     

Comparison of the sensitivity of microscopy and culture in the laboratory diagnosis of intestinal protozoal infection.

Concentration of protozoal cysts from faeces by the formol-ether method and faecal culture on Robinson's medium were studied to determine their diagnostic value among patients attending a sexually transmitted diseases clinic in Edinburgh. Of 450 men studied Entamoeba histolytica and Giardia lamblia were identified in the faeces of 10.7 and 5.1% respectively. Thirty (81.1%) of 37 men with E histolytica and 11 (47.8%) of 23 men with G lamblia infections would not have been identified if formol-ether concentration had not been undertaken. Culture of faeces in Robinson's medium led to the detection of a further 11 men with amoebiasis. Oocysts of Cryptosporidium spp were not identified in faecal samples from 130 men.     

Evaluation of three commercial assays for detection of Giardia and Cryptosporidium organisms in fecal specimens

There is an increasing demand for diagnostic testing for Giardia intestinalis (G. lamblia) and Cryptosporidium parvum, with a priority being placed on obtaining diagnostic results in an efficient and timely manner. Several commercial companies have developed rapid diagnostic tests that are simple to perform and can be completed in less time than traditional methods for detecting Giardia and Cryptosporidium: We compared one of these rapid tests, the ImmunoCard STAT! (Meridian Bioscience, Inc.) lateral-flow immunoassay, with the MERIFLUOR direct fluorescent-antibody (DFA) test, the ProSpecT EZ microplate assay for Giardia and the ProSpecT microplate assay for Cryptosporidium, and modified Kinyoun's acid-fast stained smears for the detection of Cryptosporidium using 246 specimens. The MERIFLUOR DFA (Meridian Bioscience, Inc.) test detected the largest number of cases (32 Giardia and 37 Cryptosporidium) infections and was used to calculate the sensitivity and specificity of the other tests. For Giardia, the sensitivities of the ImmunoCard STAT! and the ProSpecT Giardia EZ microplate assay (Alexon-Trend, Inc.) were 81 and 91%, respectively. For detection of Cryptosporidium, the sensitivities of the ImmunoCard STAT!, the ProSpecT Cryptosporidium microplate assay (Alexon-Trend, Inc.), and modified Kinyoun's acid-fast stained smears were 68, 70, and 78%, respectively. Test specificities were equal to or greater than 99%. Specimens with very small numbers of organisms were not detected by the ImmunoCard STAT!, the ProSpecT microplate assay or modified Kinyoun's acid-fast stained smears.