大鼠仙台病毒ELISA、间接免疫荧光和免疫印迹三种检测方法比较

目的比较ELISA（enzyme-linked immunosorbent assay）、IFA（immuno-fluorescence assay）和WB（Western blot）三种方法在大鼠仙台病毒血清学检测中的差异。方法仙台病毒蛋白抗原经凝胶电泳分离转移后用于血清学检测的WB方法;使用IFA、ELISA方法对20份无菌大鼠、227份SPF大鼠以及63份清洁级大鼠送检血清样品进行检测,阳性及可疑样品用WB方法进行了验证。结果 20份无菌大鼠血清样品被3种方法检测为仙台病毒抗体阴性;SPF级大鼠样品被IFA方法判定为阴性,1.32%（3/227）被ELISA方法判定为阳性,其中有2/3被WB确认为阳性;ELISA、IFA和WB在清洁级大鼠样品中检出仙台病毒的阳性率分别为为18.12%、11.34%和15.87%。结论三种检测方法灵敏度从高到低依次为ELISA、WB和IFA。WB方法可作为IFA和ELISA难以确定结果的替代方法。     

C57BL/6小鼠幽门螺杆菌感染动物模型的建立

目的：建立幽门螺杆菌（Helicobacter pylori,Hp）小鼠感染模型。方法：建立Hp经口感染SPF级小鼠的动物模型,取小鼠胃粘膜组织,利用PCR技术、尿素酶实验、细菌培养等方法检测接种小鼠,对结果进行判定。结果：Hp可感染C57BL／6小鼠并在小鼠胃部定植。     

小鼠脑脊髓炎病毒标准血清制备及间接ELISA检测方法的应用

目的制备小鼠脑脊髓炎病毒（TMEV）标准血清,建立ELISA检测方法,实现一种病毒多种检测方法的比对研究。方法用TMEV感染3周龄SPF级BALB／c小鼠,制备免疫用抗原。分别在0、7、14d以腹腔注射的方式免疫SPF级6-8周龄BALB／c小鼠,免疫血清用IFA、IEA法进行鉴定;TMEV感染BHK-21细胞,制备EHSA抗原,确定酶结合物、抗原和标准阳性血清的最佳工作浓度,建立TMEVEHSA检测方法,进行敏感性、特异性、重复性和稳定性实验。结果制备TMEV免疫血清,以IFA、IEA测定血清效价分别为1：640和1：320,与痘苗病毒、小鼠肺炎病毒、小鼠肝炎病毒、仙台病毒和呼肠孤病毒-Ⅲ型均无交叉反应;建立了ELISA检测方法,确立了各种试剂的最佳工作浓度,其中酶结合物最佳工作浓度为1：10000,抗原为2.5μg／mL,阳性参考血清为1：200;EHSA检测灵敏度为1：3200。板内特异抗原、正常抗原平均变异系数为4.67％和5.7％,板间为4.39％和7.61％。稳定性实验相对偏差均小于20％。结论本研究制备的TMEV免疫血清可作为血清学检测方法中的标准质控血清;建立的ELISA方法重复性、稳定性好,敏感性和特异性强,可用于小鼠脑脊髓炎病毒血清抗体检测。     

电化学发光法在检测孕产妇乙肝中的应用

背景与目的：探讨电化学发光法（electrochemiluminescence ECL）在检测孕产妇乙肝病毒中的应用价值,从而正确指导乙肝孕产妇产后的喂养方式。方法：用ELISA法和ECL法平行测定孕产妇血清乙肝病毒血清标志物（HBV—M）,并对ELISA法检测表面抗原为阴性而ECL法为低浓度的标本,用荧光定量PCR测定病毒DNA。结果：ECL法表面抗原的检出率（9．66％）明显高于ELISA法（6.54％）,p〈0.05,ELISA法检测为阴性而ECL法为低浓度的标本,病毒DNA均为阳性。结论：ECL法测定HBV—M的敏感性比ELISA法高,可以更灵敏的检出低浓度的表面抗原,从而可以更好的指导孕产妇产后的喂养方式,但HBcAb阳性率过高。     

小鼠仙台病毒标准化血清的制备及鉴定

目的制备标准化抗小鼠仙台病毒（Sendai Virus,SV）免疫血清,为清洁级及SPF级实验小鼠质量检测提供批量、稳定、敏感的质控血清。方法使用动物来源的仙台病毒（sv）感染BALB／c小鼠,获得高效价免疫血清,经IFA、IEA、ELISA等方法检测血清效价。结果制备多量抗SV血清,经多种方法检定,IFA效价达1：640,IEA效价达1：320,ELISA效价达1：102400～1：204800。结论本研究中制备的sV抗血清达到同批次大量高滴度的水平,可作为检测实验小鼠中sv病原的标准化质控血清。     

三江地区妊娠妇女风疹病毒感染的血清学分析

目的调查2005年三江地区妊娠妇女风疹病毒感染情况。方法收集400例妊娠8～24周孕妇血清,采用酶联免疫吸附法（ELISA）检测风疹特异性IgM抗体,对IgM阳性患者进一步用PCR检测风疹病毒RNA,对该人群的风疹病毒感染率进行调查。结果检测400例孕妇风疹病毒IgM,阳性20例,阳性率为5．00％;检测IgM阳性者的风疹病毒RNA,结果阳性8例,占IgM阳性者的40．00％。结论用ELISA和PCR法进行妊娠早期风疹病毒感染的检测,对降低先天性风疹综合征患儿的出现具有重要意义。     

长爪沙鼠小鼠肝炎病毒（MHV）RT-PCR检测方法的建立及初步应用

目的建立长爪沙鼠小鼠肝炎病毒（MHV）RT-PCR检测方法,应用于长爪沙鼠、小鼠等实验动物MHV的检测。方法根据已发表的小鼠肝炎病毒（MHV）S基因序列,设计合成引物。提取MHV细胞毒RNA,以其为模板,进行PCR扩增。优化反应条件,进行特异性、敏感性、稳定性、重复性试验。并对65只长爪沙鼠及12只小鼠进行检测。结果建立的MHVRT-PCR检测方法特异、敏感、稳定。以MHVRNA逆转录产物为模板,所能检测RNA最小模板浓度为3．1pg／μL,可检测病毒最小滴度为10^-3／mL。65只沙鼠经RT-PCR检测,均为阴性,12只小鼠经RT．PCR检测,有3只MHV阳性,测序结果与Genbank中MHV核酸序列同源性均为97％。结论建立的长爪沙鼠小鼠肝炎病毒（MHV）RT-PCR检测方法可用于长爪沙鼠、小鼠等实验动物MHV的检测。     

SPF级小鼠超数排卵效果

目的探求小鼠周龄、品系和季节对SPF级小鼠超数排卵效果的影响。方法在春、夏、冬季对4～10周龄的SPF级BALB／c小鼠和KM小鼠进行超数排卵处理,通过见栓率、产胚率、3．5d胚胎获得量及胚胎类型等方面进行统计学分析。结果小鼠周龄、品系和季节对SPF级BALB／c小鼠和KM小鼠见栓率没有影响;对产胚率、3．5d胚胎获得量和胚胎类型等方面有一定影响。结论SPF级BALB／c小鼠宜用9、10周龄的进行超数排卵处理以供应3．5d胚胎为需要所用,而SPF级KM小鼠使用4～10周龄的都可以;另外,SPF级小鼠超数排卵处理仍宜在春、夏季进行,可以保证获得最佳的效果。     

儿童抗幽门螺杆菌IgG和IgA水平的酶联免疫吸附试验检测

目的：了解北京地区儿童幽门螺杆菌（Hp)的感染状况,探讨儿童幽门螺杆菌感染与年龄、性别的关系,比较分析HP感染后血清中抗体(IgG、IgA)水平。方法：采用ELISA方法对我院消化道门诊227例患儿同时检测血清抗Hp抗体IgG、IgA,任一项阳性者即诊断为Hp感染。结果：(1)227例门诊患儿Hp平均感染率53．7％,男孩感染率57．1％,女孩48．1％。(2)3岁以下就诊儿童Hp感染率57．1％,4—7岁组39．1％,8—12岁组60．5％,13岁以上47．1％;其中66．9％(81／120)的Hp阳性患儿在8—12岁之间;(3)HP感染总阳性率53．7％;若单独检测IgG,阳性率42．7％,假阴性率11％;单独检测IgA,阳性率22％,假阴性率31．7％;两项诊断符合率达57．3％。结论：北京地区儿童幽门螺杆菌感染率较高。有消化道症状伴HP感染的患儿以8—12岁居多,其感染率明显高于全国无症状儿童平均染率。儿童Hp感染性别差异无显著性,感染后血清抗体水平IgG显著高于IgA,同时检测IgG、IgA有助于提高ELISA方法Hp的感染检出率。     

一期梅毒实验室诊断差异性研究

目的通过梅毒螺旋体初筛试验、确认试验和鉴别诊断试验,探讨一期梅毒实验室诊断差异性,最大限度减少漏诊与误诊,为深入研发新型早期梅毒诊断试剂奠定基础。方法依据2000年中国卫生部防疫司颁布的性病诊断标准,临床筛选一期梅毒患者86例（研究组）和非梅毒患者100例（对照组）,对患者血清进行甲苯胺红不加热血清试验（TRUST）初筛和梅毒螺旋体明胶颗粒凝集试验（TPPA）确认。筛选临床体征、TRUST法和TPPA法三者结果有差异的患者进一步鉴别诊断,鉴别诊断主要应用荧光定量PCR（FQ-PCR）法、免疫PCR法与自身抗体检测等试验。结果初筛TRUST法灵敏度和特异性分别为62．8％、93．0％;确认TPPA法灵敏度与特异性分别为66．3％、100％。TRUST法和TPPA法两者结果差异占12．8％;临床体征诊断、TRUST法和TPPA法三者结果差异占41．9％。TPPA法与TRUST法两者均阴性的一期梅毒患者中,FQ-PCR阳性率达88．0％,免疫PCR阳性率占40．0％。TPPA法阳性、TRUST法阴性的一期梅毒患者免疫PCR法与TPPA法结果一致;TPPA法阴性、TRUST法阳性11例患者中结核抗体阳性2例,类风湿因子阳性3例与抗Sm抗体结果阳性6例。结论一期梅毒患者实验室诊断结果差异性较大,漏诊与误诊的比例较高,有待研发新型的诊断试剂和提高诊断水平。     

上海地区小鼠诺瓦克病毒的检测分析及病毒分离

目的了解上海地区实验小鼠自然感染小鼠诺瓦克病毒(murine norovirus,MNV)的状况,并分离毒株。方法抽取委托检测单位送检的SPF小鼠319只,分别采集盲肠内容物及血液样本,应用逆转录-聚合酶链反应(RT-PCR)方法扩增小鼠盲肠内容物样本中MNV的特异性基因片段来检测MNV的感染情况,同时采用酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)与核酸检测方法进行对比。将RT-PCR扩增结果为阳性的盲肠内容物样本稀释并经0.22μm滤膜过滤,接种到小鼠巨噬细胞系RAW 264.7细胞,盲传后采用RT-PCR方法鉴定。结果 RT-PCR检测的319份小鼠盲肠内容物样本中,阳性样本95份,阳性率为29.78%。对180份经RTPCR检测的小鼠血清进行ELISA检测,阳性样本70份,阳性率为38.89%。RAW 264.7细胞盲传5代后在72 h内出现细胞病变,经RT-PCR鉴定,显示187 bp的目的条带。结论通过核酸检测方法和血清学方法证实上海地区实验小鼠存在MNV自然感染,且感染率较高,应加强实验小鼠的饲养管理。     

Detection of rodent Helicobacter spp. by use of fluorogenic nuclease polymerase chain reaction assays

Polymerase chain reaction (PCR) analysis is the standard method for detection of Helicobacter spp. infections in laboratory rodents, with H. hepaticus, H. bilis, and H. typhlonius considered primary pathogens. Fluorogenic nuclease PCR assays that detect all known rodent Helicobacter spp., or that specifically detect H. hepaticus, H. bilis, or H. typhlonius were developed to eliminate post-PCR processing, enhance specificity, and provide quantitative data on starting template concentration. Each fluorogenic PCR assay detected a minimum of 10 copies of target template, had comparable or greater sensitivity when compared directly with corollary gel detection PCR assays, and detected only targeted species when numerous Helicobacter spp. and other enteric bacteria were analyzed. Fluorogenic nuclease PCR analysis of fecal DNA samples obtained from numerous laboratory mice sources detected all samples with positive results by use of Helicobacter spp., H. hepaticus, H. bilis, and/or H. typhlonius gel detection PCR analysis, except for one sample that had positive results by H. typhlonius gel detection PCR but negative results by H. typhlonius fluorogenic nuclease PCR analysis. Among fecal DNA samples that were Helicobacter spp. negative by use of all gel detection PCR assays, the fluorogenic nuclease PCR assays detected target template in only one sample that was positive by use of the Helicobacter spp. and the H. bilis fluorogenic nuclease PCR assays. In conclusion, fluorogenic nuclease PCR assays provide sensitive, specific, and high-throughput diagnostic assays for detection of Helicobacter spp., H. hepaticus, H. bilis, and H. typhlonius in laboratory rodents, and the quantitative data generated by these assays make them potentially useful for bacterial load determination.     

Eradication of Helicobacter bilis and H. hepaticus from infected mice by using a medicated diet

Infection of laboratory mice with Helicobacter spp. is a serious problem for many laboratory animal facilities worldwide. Rederivation and antibiotic treatment are two of the most common methods used to eliminate the bacterial infection from rodent colonies. Forty-seven newly imported mice were suspected to be positive for Helicobacter infection based on PCR analysis of pooled fecal samples from sentinel animals. We treated the mice with a medicated feed containing four antibiotic compounds (amoxicillin, clarithromycin, metronidazole, omeprazole). After eight weeks of continuous administration the animals were negative for H. bilis and H. hepaticus. Frequent retesting of the animals for up to one year proved that the mouse colony remained negative for Helicobacter spp.     

抗胆汁螺杆菌单克隆抗体的制备

目的制备抗胆汁螺杆菌单克隆抗体（McAbs）。方法用胆汁螺杆菌B2m株皮下免疫BALB/c小鼠,采用杂交瘤技术进行融合。以酶联免疫吸附实验（ELISA）筛选抗胆汁螺杆菌单克隆杂交瘤细胞株并初步鉴定其特异性;免疫印迹试验测定单抗所结合的抗原表位;免疫双向扩散试验确定IgG亚类;腹腔接种法、辛酸-硫酸铵盐析法大量制备、纯化单克隆抗体。结果经过ELISA筛选获得11个阳性杂交瘤细胞株,其效价最高达1：4×10^5以上,并与实验动物常见的15种病原菌呈阴性反应;IgG亚类为IgG2a和IgG2b;免疫印迹试验显示,6株（A-F）与胆汁螺杆菌大约相对分子质量（172、0、21、30、52、66）×10^3的抗原特异结合,5株（G-K）皆与胆汁螺杆菌、幽门螺杆菌等三种螺杆菌大约相对分子质量（52、82）×10^3的抗原呈阳性反应,表明A-F株针对的是胆汁螺杆菌特异性抗原,G-K株可能具有属特异性。结论筛选的单克隆抗体具有较高的特异性和敏感性,所结合的抗原为胆汁螺杆菌或螺杆菌的免疫优势抗原,为进一步的种、属生物学特性研究、菌株分型及血清学检测方法建立等奠定了基础。     

Prevalence of viral, bacterial and parasitological diseases in rats and mice used in research environments in Australasia over a 5-y period

Viral, bacterial and parasitological infections in rats and mice used in biomedical research continue to occur despite improved housing and biosurveillance. The presence of disease in laboratory animals can lead to spurious results for research undertaken in universities, research institutes and the pharmaceutical industry. Here the authors report the results of serological, microbiological, parasitological and molecular tests done on mice and rats from Australasia submitted to a rodent health monitoring laboratory (Cerberus Sciences) from 2004 to 2009. In tested mice, norovirus was the most prevalent virus and ectromelia virus was the least prevalent virus. In tested rats, pneumonia virus of mice was the most prevalent virus and adenoviruses 1 and 2 were the least prevalent viruses. In mice, Helicobacter hepaticus was the most prevalent bacterium, and in rats, Proteus spp. were the most prevalent bacteria. The most common positive helminthological finding in mice and rats was the presence of all pinworms (including Aspicularis spp. and Syphacia spp.). The most common positive protozoan findings in mice and rats were Chilomastix spp. and Trichomonads.     

2003-2008年我国实验猴BV抗体检测结果及抗体水平变化规律的分析

目的对近年来我国实验猴BV（猴疱疹病毒Ⅰ型）抗体检测结果进行比较分析,以了解我国实验猴BV感染情况及其抗体水平变化规律,为我国实验猴质量控制及标准化提供依据。方法根据国标中ELISA方法 ,对2003～2008年我国11个单位送检的2个品种猴血清进行BV抗体检测,并对检测结果进行统计分析。结果检测的4612份猴血清中,有1843份BV抗体呈阳性,阳性率为39.96%;6年中检测猴群BV抗体阳性率基本在30%～50%。幼年（≤2岁）、青年（2.1～4.0岁）、成年（4.1～6.5岁）、老年（≥6.5岁）4个不同年龄段猴BV感染率分别为26.28%、31.53%、53.74%、87.27%。不同年龄感染率差异显著（P〈0.01）。雌猴BV感染率（35.91%）高于雄猴（34.93%）,但两者差异不显著（P〉0.05）。结论不同年龄猴BV感染率不同,随着年龄的增长BV感染率升高。     

Natural colonization with Helicobacter species and the development of inflammatory bowel disease in interleukin-10-deficient mice

BACKGROUND: The interleukin-10-deficient (IL-10-/-) mice maintained in specific-pathogen-free (SPF) conditions develop typhlocolitis when experimentally infected with Helicobacter species. However, there is limited information regarding the role of Helicobacter species that naturally colonize IL-10-/- mice in typhlocolitis development. The aim of this study was to examine in SPF IL-10-/- mice the association between natural colonization specific Helicobacter species and typhlocolitis development. MATERIAL AND METHODS: Cecum and proximal colon from 72 C57BL/6 x 129/Ola IL-10-/- mice (8-20 weeks old) were removed for DNA extraction and histologic evaluation. Genus-specific polymerase chain reaction- denaturing gradient gel electrophoresis (PCR-DGGE) and species-specific PCR were used to detect Helicobacter species. Mice were grouped by age, sex, and Helicobacter colonization status, and their histologic scores were compared. The development of clinical typhlocolitis was observed in a further 12 mice. RESULTS: Species-specific PCR showed that mice were colonized with Helicobacter ganmani and/or Helicobacter hepaticus. The PCR-DGGE detected H. ganmani, H. hepaticus and an H. ganmani-like organism. The histologic scores in mice colonized with H. hepaticus were significantly higher than that in mice colonized with H. ganmani. Male mice showed significantly higher histologic scores than female mice. Four of the 12 mice developed clinical typhlocolitis in 38 weeks. CONCLUSION: Natural colonization with different Helicobacter species was found in IL-10-/- mice within the same breeding colony. The severity of typhlocolitis differed according to the colonizing Helicobacter species. Furthermore, the rate of typhlocolitis development in IL-10-/- mice naturally colonized with Helicobacter species was significantly slower than that reported in experimentally infected mice.     

Employment of broad range 16S rDNA PCR and sequencing in the detection of aetiological agents of meningitis

To employ partial 16S rDNA PCR and automated sequencing technique to identify non-culturable causal agents of bacterial meningitis, 73 peripheral blood samples and 413 culture-negative and eight culture-positive CSF clinical specimens from patients with suspected acute meningitis were examined for the presence of bacterial genomic DNA employing broad range 16S rDNA PCR followed by sequencing of the amplicons. In blood samples, 63/73 specimens were PCR positive (86.3%) and after direct sequencing of the PCR amplicons, only 12.7% (8/63) gave clear sequencing results and 55/63 (87.3%) were mixed with more than one organism detected. The mixed PCR amplicons were separated by using PAGE and mixed amplicons from 29/55 (52.7%) specimens were successfully identified through sequencing. Of the CSF samples, 8/8 culture-positive samples were also PCR positive and 45/413 (10.9%) of culture-negative gave a strong PCR signal and 88/413 (21.3%) specimens yielded a weak PCR signal. The remaining 280 culture-negative specimens were also PCR negative. Nested PCR was set up for the 88 weak positive samples and yielded 72/88 (81.8%) strong positives, with the remainder failing to amplify 133/413 (32.2%) culture-negative samples were PCR positive. In this study, the most common bacteria identified from blood specimens were Neisseria meningitidis, 13/63 (20.6%); Streptococcus spp, 5/63 (7.9%); Acinetobacter spp and Pseudomonas spp 4/63 (6.3%). From culture-negative CSF, the pattern was different in that Staphylococcus spp (13/58, 22.4%), Neisseria meningitidis (9/58, 15.52%) and Pseudomonas spp (8/58, 14.79%), were the most frequent. Overall, 16S rRNA broad-range PCR combined with direct DNA sequencing is a valuable molecular tool to aid with the detection as well as identification of non-culturable aetiological agents of acute bacterial meningitis and can augment cultural methods in the diagnosis of central nervous system infections in patients who have been treated with antibiotics. However, this study demonstrates that contamination is an important complication of the molecular assay, which should be attempted to be eliminated through careful laboratory controls. Hence there should be careful interpretation of any molecular finding, in tandem with other laboratory findings, such as culture, immunological and biochemical markers, and the clinical scenario of the patient.     

实时荧光定量PCR技术在SPF鸡四种垂直传播疾病监测中的应用

目的探讨荧光定量PCR检测技术对SPF鸡四种垂直传播病毒的检测应用。方法采集60份SPF鸡及70份普通鸡群蛋清、泄殖腔试子样品,提取样品核酸,分别进行ARV、REV、CAV、ALV四种病毒实时荧光定量PCR检测,根据标准曲线及溶解曲线分析判读样品病毒拷贝数。结果 SPF鸡ALV 2份阳性,检出率3.3%,其余病毒检测均为阴性;普通鸡样品REV检测2份阳性,检出率2.9%,ALV 10份阳性,检出率14.3%。结论荧光定量PCR检测方法最低可检测到100个拷贝核酸,检测灵敏度较高,有望应用于SPF鸡临床样品的病原检测。