胶体金标记单克隆抗体测定单增李斯特菌试剂盒制备

目的制备单增李斯特菌54001、54002型的单克隆抗体（McAb）,制备胶体金标记单增李斯特菌抗体免疫层析检测试剂板。方法以纯化单增李斯特菌为抗原,免疫Balb／c小鼠,制备单克隆抗体,鉴定其特性,建立并验证胶体金标记单增李斯特菌抗体免疫层析试剂板的检测方法。结果筛选出2株单抗杂交瘤细胞株F5、F9,免疫球蛋白亚类均为IgM。通过选用柠檬酸钠法制备胶体金,测定其最适结合蛋白浓度为28g/ml,制备胶体金标记单增李斯特菌抗体免疫层析检测板。结论为产单核李斯特菌提供了一个方便、快捷、切实有效的检测技术。     

Rapid detection of Listeria monocytogenes by a PCR assay specific for an aminopeptidase

Specific and rapid detection of Listeria monocytogenes is very important with regard to food safety since all other species of Listeria appear to be non-pathogenic to humans. Conventional microbiological detection methods are very time consuming. The polymerase chain reaction (PCR) is one of the most promising techniques for rapid detection of micro-organisms in food products. We have developed a PCR assay, specific for L. monocytogenes, based on the gene encoding an aminopeptidase, which previously has not been described for this species. The L. monocytogenes aminopeptidase shares strong sequence similarity with aminopeptidase C from Streptococcus thermophilous, Lactobacillus lactis, Lactobacillus helveticus, and with a cysteine proteinase from Saccharomyces cerevisiae. Polymerase chain reaction primers were synthesized based on the DNA sequence of the aminopeptidase gene. A 90 bp product was apparent with all L. monocytogenes strains tested but not with other species of Listeria or other bacterial genera. The PCR assay, which is performed directly from whole bacterial cells, does not involve DNA purification and can be conducted in 4 h. It provided positive identification of L. monocytogenes in mixed culture.     

Real-time PCRs assay for serogrouping Listeria monocytogenes and differentiation from other Listeria spp.

A 5′-exonuclease real-time triplex-PCR assay was developed for serogrouping Listeria monocytogenes, and differentiation from other Listeria spp. The assay was evaluated on 109 Listeria cultures, and results were compared with a previously validated gel-based multiplex-PCR procedure. All L. monocytogenes were correctly classified into four serogroups, including atypical serotype 4b strains, and differentiated from other Listeria species. The assay is a rapid method for categorisation of suspect L. monocytogenes.     

模拟粪便标本单增李斯特菌和伊氏李斯特菌TaqMan双重实时荧光定量-聚合酶链反应检测方法

目的建立一种TaqMan双重实时荧光定量-聚合酶链反应(real-time PCR),用于模拟粪便标本中单增李斯特菌和伊氏李斯特菌的快速检测。方法以单增李斯特菌特异基因hly和伊氏李斯特菌特异基因smcL作为靶基因,合成2对引物和其相应的荧光探针,制作标准曲线,建立从模拟粪便标本中直接检测单增李斯特菌和伊氏李斯特菌的TaqMan双重real-time PCR。利用李斯特菌属中其他种李斯特菌及常见致病菌验证引物、探针的特异性;通过制备模拟粪便标本并对其进行二次增菌后,分别提取DNA,采用TaqMan双重real-time PCR进行检测,以达到快速检测单增李斯特菌和伊氏李斯特菌的目的。结果采用本研究建立的TaqMan双重real-time PCR对模拟粪便标本的检测结果显示特异性良好,与其他种李斯特菌和其他病原菌均无交叉反应。模拟粪便标本中单增李斯特菌和伊氏李斯特菌的检测下限分别为2.45×103cfu/g和2.92×103cfu/g;模拟粪便标本在3 h内可得出检测结果,当模拟粪便标本中单增李斯特菌含量为6 cfu/g和伊氏李斯特菌含量为5 cfu/g时,经过增菌后使用双重real-time PCR可检测出阳性结果。结论本研究建立了以单增李斯特菌的特异基因hly和伊氏李斯特菌的特异基因smcL为靶基因的TaqMan双重realtime PCR检测方法,该方法具有特异性好、敏感性高、快速易操作等优点,可用于临床、食品及环境标本的快速诊断,以及我国人群中单增李斯特菌和伊氏李斯特菌的携带或感染状况的调查分析。     

Evaluation of an enzyme-linked immunosorbent assay for prostatic acid phosphatase

An enzyme-linked immunosorbent assay (ELISA) was developed to measure prostatic acid phosphatase in human sera. The value of this method was tested on four groups: patients with non-prostatic disease, with prostatic hypertrophy, and with untreated and treated prostatic carcinoma. The results of ELISA were also compared with those of enzyme immunoassay (EIA) and the conventional, enzymatic method. The specificity of ELISA as calculated from the hypertrophy group, was 76% against 68% for EIA and 71% for the conventional method. The sensitivity of ELISA, calculated from the untreated carcinoma group, was 57% against 60% for EIA and 70% for the conventional method. ELISA did not prove better than EIA or the conventional method in quantifying prostatic acid phosphatase in patients' sera.     

Detection of Listeria monocytogenes from food samples by PCR after IMS-plating

For rapid, accurate and sensitive detection of Listeria monocytogenes in food samples, colonies developed on the selective agar (Oxford agar) after immunomagnetic separation (IMS) were subjected to polymerase chain reaction (PCR) assay with the prf A1-2 primer pair. The proposed assay system was shown experimentally to be capable of specifically detecting the bacteria from food samples contaminated at more than 10(2) cfu/g. However, the enrichment culture after a short period of 16 h with the appropriate selective broth was needed before IMS-plating, because the bacterial contents in most actual food were as low as less than 10(2) cfu/g. However, even if the enrichment cultivation was employed before IMS, L. monocytogenes was detected within 3 days.     

Evaluation of an enzyme-linked immunosorbent assay kit (GTI PakPlus) for the detection of antibodies against human platelet antigens

Twenty-six serum samples from 24 patients were investigated for the presence of platelet-specific antibodies in a partly retrospective (n = 15) and partly prospective (n = 9) study. The sera contained either alloantibodies to human platelet antigens (HPA) (n = 23) or were from clinically suspected cases of fetomaternal alloimmune thrombocytopenia (FMAITP) in which platelet-specific antibodies had not been detected (n = 3). Three techniques were used to detect platelet antibodies: the platelet immunofluorescence test, the monoclonal antibody immobilization of platelet antigens (MAIPA) assay and a commercially available enzyme-linked immunosorbent assay--GTI PakPlus (GTI kit). Two alkaline phosphatase-conjugated antiglobulin reagents provided by the manufacturer were used in the GTI kit: an antihuman IgG/IgA/IgM (IgGAM) conjugate and an antihuman IgG conjugate. The GTI kit with the anti-IgGAM conjugate failed to detect eight antibody specificities in seven sera (anti-HPA-1a [n = 3], anti-HPA-3a [n = 1], anti-HPA-3b [n = 1] and anti-HPA-5b [n = 3]). Greater signal-to-background ratios were achieved in the GTI kit with the anti-IgG conjugate but five antibody specificities (anti-HPA-1a [n = 1], anti-HPA-3a [n = 1], anti-HPA-3b [n = 1], anti-HPA-5b [n = 2]) remained undetectable. All the sera were detected by MAIPA assay and, furthermore, the MAIPA assay achieved the greatest signal-to-background ratio in the majority of sera tested. These findings re-emphasize the value of the MAIPA assay in reference laboratories and illustrate that the GTI kit may either fail to detect or incorrectly identify clinically significant HPA antibodies.     

抗-HCV酶联免疫吸附试验试剂的评价

目的评价国内外11种抗-HCV酶联免疫吸附试验(ELISA)试剂的质量。方法采用艾滋病参比实验室基础血清盘、BBI阳转血清盘、美国CAP特性血清盘,对国产和进口的试剂进行质量测评。评价指标包括敏感性、特异性、阳性预测值、阴性预测值等。结果11种抗-HCV ELISA检测试剂的敏感性均为100%,多数试剂特异性>90%。阳转血清盘评价显示5种试剂平均延长天数<2d,大多数试剂未检出抗-NS3。所有试剂均检测能出抗核心区抗体。美国CAP特性血清盘评价显示所有试剂检测结果与标准结果一致率均为100%。结论11种抗-HCV酶联免疫吸附试验试剂有较高的敏感性,特异性有差异。由于ELISA抗-HCV试剂敏感性高,实用性强,适合于我国一般人群的筛查检测。     

空肠弯曲菌、单增李斯特菌和大肠杆菌O157多重PCR分子检测研究

目的建立同时检测空肠弯曲菌、单核细胞增生性李斯特菌(简称单增李斯特菌)和大肠杆菌O157的多重聚合酶链反应(PCR)检测技术。方法根椐空肠弯曲菌mapA基因序列、单增李斯特菌的编码溶血素(hly)基因序列和大肠杆菌的O157抗原(rfbE)基因序列,分别设计了3对特异引物,PCR扩增的目的基因片段为589 bp、360 bp和194bp;并对反应条件进行了优化。结果对3种致病菌检测,多重PCR检测DNA的敏感度分别为空肠弯曲菌2.264 ng、单增李斯特菌37.92 ng、大肠杆菌2.100 ng,样品检测时间6~8 h。结论该方法操作简便,检测周期短,特异性和敏感度高,为同时检测污染样品中空肠弯曲菌、单增李斯特菌和大肠杆菌O157奠定了基础。     

空肠弯曲菌、单增李斯特菌和大肠杆菌O157多重PCR分子检测研究

目的建立同时检测空肠弯曲菌、单核细胞增生性李斯特菌（简称单增李斯特菌）和大肠杆菌O157的多重聚合酶链反应（PCR）检测技术。方法根椐空肠弯曲菌mapA基因序列、单增李斯特菌的编码溶血素（hly）基因序列和大肠杆菌的O157抗原（rfbE）基因序列,分别设计了3对特异引物,PCR扩增的目的基因片段为589 bp、360 bp和194bp;并对反应条件进行了优化。结果对3种致病菌检测,多重PCR检测DNA的敏感度分别为空肠弯曲菌2.264 ng、单增李斯特菌37.92 ng、大肠杆菌2.100 ng,样品检测时间6-8 h。结论该方法操作简便,检测周期短,特异性和敏感度高,为同时检测污染样品中空肠弯曲菌、单增李斯特菌和大肠杆菌O157奠定了基础。     

Development of an enzyme-linked immunosorbent assay and counterimmunoelectrophoresis for the detection of Cryptosporidium parvum copro-antigens

Diagnosis of intestinal parasitic infections is still mainly dependent upon the cumbersome, insensitive technique of stool microscopy. Alternatively, detection of parasite antigens that are shed in stool denotes infection regardless of symptomatology, physical integrity or excretion pattern of the parasite. Cryptosporidium copro-antigen (CCA) was detected in human and calf stool using enzyme-linked immunosorbent assay (ELISA) and counterimmunoelectrophoresis (CIE). Fifty-nine stool samples (25 samples positive for Cryptosporidium parvum by microscopy, 17 uninfected and 17 positive for other parasites) were tested against monospecific antibody raised against 20 kDa CCA. CCA was detected by ELISA in all Cryptosporidium microscopy positive samples, while CIE demonstrated CCA in 23 out of 25 positive samples. No crossreactions were observed when faecal samples harbouring other parasites were screened by the two techniques. CCA remained detectable after freezing, boiling or upon polyvinyl alcohol preservation. ELISA and CIE together with the developed monospecific antibody may represent new, reliable and confirmative immunodiagnostic tools for cryptosporidiosis.     

A facile fluorescent sensor based on nitrogen-doped carbon dots derived from Listeria monocytogenes for highly selective and visual detection of iodide and pH

Sensitive and visual analysis of iodide (I⁻) and pH is significant in environmental and food applications. Herein, we present a facile fluorescent sensor for highly selective and visual detection of I⁻ and pH based on nitrogen-doped carbon dots derived from Listeria monocytogenes (NCDs-LM). The NCDs-LM-based fluorescent sensor showed a good linear relationship to I⁻ concentrations, and the detection limit was calculated as 20 nmol L⁻¹. The developed sensor was successfully applied to the detection of I⁻ in drinking water and milk samples. Meanwhile, the as-synthesized NCDs-LM sensor can be used to detect pH, achieving a wide linear pH range. Furthermore, fluorescent test papers based on NCDs-LM were designed for semi-quantitative detection of I⁻ and pH via the naked-eye colorimetric assay. The present work indicates that the NCDs-LM-based fluorescent sensor has high potential for use in environmental monitoring and food analysis.

Listeria monocytogenes-derived nitrogen-doped carbon dots served as a facile fluorescent sensor with excellent sensing performances for iodide with low detection limit of 20 nmol L⁻¹ and wide pH range from 1.81 to 11.82.     

Tetracycline-resistant L-forms isolated from an antibiotic-susceptible strain of Listeria monocytogenes.

A tetracycline-susceptible strain of Listeria monocytogenes type 4b was converted to stable L-forms by penicillin. L-form variants resistant to tetracycline were then selected from a predominantly tetracycline-susceptible L-form population on plates containing penicillin and increasing concentrations of tetracycline. The origin of tetracycline-resistant L-forms from the parent Listeria strain was confirmed biochemically, by immunofluorescence, and by polyacrylamide gel electrophoresis. Scanning and transmission electron microscopy confirmed the typical L-form structure and the complete lack of cell wall in both L-form strains. The level of [3H]tetracycline uptake was lower in tetracycline-resistant than in susceptible cells.     

Evaluation of a new enzyme-linked immunosorbent assay for detection of Chagas antibody in US blood donors

BACKGROUND: Chagas disease, caused by the parasite Trypanosoma cruzi, represents a serious blood safety problem due to increasing immigration from Latin America. The Food and Drug Administration recently recommended implementation of Chagas antibody screening for US donors as soon as a suitable assay is licensed. An anonymized preclinical study of a prototype T. cruzi lysate-based enzyme-linked immunosorbent assay (ELISA) developed by Ortho-Clinical Diagnostics was conducted. STUDY DESIGN AND METHODS: Two populations of specimens were evaluated: 1) 10,192 sequential donations from blood donors residing in the El Paso, Texas, area and 2) 178 specimens from South America which were presumptively positive for antibodies to T. cruzi and purchased from commercial vendors. RESULTS: A total of 10,189 (99.97%) of the 10,192 screened donor specimens did not react, whereas 3 (0.03%) tested initially reactive. The 3 initially reactive specimens tested repeat reactive and were confirmed by radioimmunoprecipitation analysis (RIPA). Based on antibody profile analysis, 2 of the 3 Chagas-positive specimens were from the same donor. Observed specificity of the test was therefore 100 percent. Of the specimens from South America, 173 of 178 were reactive by the prototype ELISA. Of the 5 nonreactive specimens, all did not react by indirect fluorescence assay, but 4 were positive by RIPA. Therefore, calculated sensitivity of the ELISA was 97.7 percent (173/177). CONCLUSIONS: These studies indicate that the prototype ELISA has excellent sensitivity and specificity for detection of antibodies to T. cruzi in donors. Moreover, among donations from a geographically selected collection region of the United States, observed seroprevalence was 0.03 percent.     

生物素-链霉亲和素酶免疫法PSA测定的评价

目的评价生物素-链霉亲和素酶免疫分析法(BSA-ELISA)测定前列腺特异性抗原(PSA)并与化学发光法(CLIA)进行比较.方法根据EP9-A文件要求采集数据,并以相应软件分析这两种方法的相关性;同时对BSA-ELISA 的测定范围,变异系数(CV),最小检测限进行测定.结果两种方法测定的结果具有较好的相关性,回归方程y=0.985X-0.131,相关系数(r)=0.997.PSA的批内平均CV为2.21%,批间平均CV为3.08%.测定范围为1.50～85.μg/L.最小检测限为0.30μg/L.结论采用全自动酶免疫分析仪的BSA-ELISA与CLIA自动分析仪测定PSA的相关性好,结果稳定,偏差小,可以在临床上常规应用.     

不同酶联免疫吸附试验检测系统评价方法探讨

目的：探讨不同酶联免疫吸附试验（ELISA ）检测系统的评价方法,以保证同一采供血机构实验室内多套检测系统检测结果的稳定性、准确性和一致性。方法稳定性评价,采用A、B两套ELISA检测系统同时检测同一个弱阳性标本,连续20次,比较两套系统变异系数（CV值）大小。准确性和一致性评价,A、B两套系统同时检测室间质评阳性样本,根据本试剂组反馈结果的吸光度／临界值比值（S／CO值）均值（x）、标准差（SD）,以 x作为参考结果分别计算每个样本A、B系统结果的S／CO值标准差指数 SDI1、SDI2;分析两套系统检测结果之间的差异。结果 A、B检测系统 CV值分别为6．5％和5．3％;A系统结果中有3个标本︱ SDI1︱大于2,其他结果均小于2,且无趋势性偏移;B系统结果︱ SDI2︱均小于2,且无趋势性偏移;两套系统检测结果之间的差异无统计学意义（P＞0．05）。结论两套检测系统稳定性较好,检测结果均处于可受控状态,且具有较好的一致性。     

生物素-链霉亲和素酶免疫法PSA测定的评价

目的 评价生物素-链霉亲和素酶免疫分析法(BSA-ELISA)测定前列腺特异性抗原(PSA)并与化学，发光法(CLIA)进行比较。方法根据EP9-A文件要求采集数据，并以相应软件分析这两种方法的相关性；同时对BSA-ELISA的测定范围，变异系数(CV)，最小检测限进行测定。结果两种方法测定的结果具有较好的相关性。回归方程Y=0．985X-0．131，相关系数(r)=0．997。PSA的批内平均CV为2．21％，批间平均CV为3．08％。测定范围为1．50～85．00μg／L。最小检测限为0．30μg／L。结论采用全自动酶免疫分析仪的BSA-ELISA与CLIA自动分析仪测定PSA的相关性好，结果稳定，偏差小，可以在临床上常规应用。