直接酶联免疫吸附法和聚合酶链反应联合检测沙门菌的应用

目的　通过比较试验 ,得到准确、快速的沙门菌检测方法。方法　 2 2 8株沙门菌经过前增菌、选择性增菌后 ,采用在聚合酶链反应 (PCR)和酶联免疫吸附法 (ELISA)同时检测 ,并将 2种快速检测方法进行比较研究。结果　PCR法的敏感性优于直接ELISA法 ,2种方法的检测符合率达 99%以上。直接ELISA结合PCR法对饮食行业工作人员健康检查的 15 4 6份人粪便样品进行检测 ,同时以国家标准方法为参照 ,直接ELISA法的敏感性和特异性达 10 0 %和 97 14 % ,PCR法的敏感性和特异性均为 10 0 %。结论　优化的沙门菌检测程序是对大量样品采用直接ELISA筛检 ,除去大量阴性样品 ,阳性样品用PCR法作进一步鉴定 ;血清型的确定用国家标准方法。     

疱疹病毒6型感染与儿童风湿热关系的研究

目的:探讨人类疱疹病毒6型(HHV-6)与儿童风湿热之间的关系。方法:采用巢式PCR方法检测38例儿童风湿热患者外周血单个核细胞和血清中的HHV-6 DNA。结果:风湿热患者HHV-6 DNA阳性率为55.26%,而对照组为52.00%,两组比较无显著性差异(P>0.05)。部分患者血清中存在HHV-6 DNA,提示这些患者可能存在HHV-6活动性感染。结论:HHV-6感染与风湿性关节炎相关。     

荧光定量PCR法评价核黄素光化学法灭活全血病毒的效果

目的了解荧光定量PCR法(qPCR)评价核黄素光化学法灭活含疱疹病毒8型(HHV-8)全血的效果。方法在常规检测合格的全血中,分别加入含105、106、107 copy/mL HHV-8的淋巴细胞BC-3和核黄素,以40~160J/mLRBC的紫外光辐照进行病毒灭活。以HHV-8ORF26为中心设计4对PCR套式扩增引物(产物长度为2 998、1 252、561、206bp),构建和优化PCR预扩增条件,以qPCR检测灭活前、后4个片段在不同辐照条件下的扩增产物经梯度稀释的Ct值,计算相应的病毒滴度ΔLog值,以评估对病毒核酸的损伤情况。结果核黄素光化学法对全血中HHV-8核酸有损伤作用,PCR预扩增HV-1(2 998bp)片段可用于评估辐照后HHV-8的病毒核酸损伤情况。病毒滴度ΔLog值随着病毒滴度、辐照强度和扩增片段长度的增加,最高达1.9Log值。结论在HHV-8高流行区缺乏常规血液筛查HHV-8方法情况下,可采用核黄素光化学法进行体外灭活,降低其传播的可能性。     

水产品中霍乱弧菌的监测方法研究

目的:研究水产品中霍乱弧菌的监测方法,及时发现被霍乱弧菌污染的水产品,采取有效防控措施,防范霍乱疫情的发生。方法:用实时荧光PCR法、胶体金法、分离培养法三种方法分别对180份水产品进行霍乱弧菌检测,阳性样品再进行霍乱肠毒素检测。结果:三种方法检出了44份核酸阳性标本,18份霍乱肠毒素阳性标本,分离出8株霍乱弧菌。结论:在进行水产品霍乱监测时,可以先用实时荧光PCR进行初筛,筛出核酸阳性标本,进行霍乱肠毒素检测,及时发现被霍乱产毒株污染的水产品,再对筛出的核酸阳性标本结合胶体金法进行分离培养和分型鉴定,进一步完成霍乱疫情的确证和分析。     

家禽中艾伯特埃希菌的分离培养和鉴定

目的分析聊城市家禽中艾伯特埃希菌感染状况,为预防艾伯特埃希菌感染引起的食源性疾病提供依据。方法于2017年10月选取聊城市3个规模较大的食品公司采集鸡肠、鸭肠样品进行EC增菌肉汤培养后,采用PCR检测eae基因,阳性增菌液接种到麦康凯琼脂平板上分离纯化,随后进行生化鉴定,采用clpX、LysP和mdh管家基因对疑似菌株进一步进行三重PCR和多位点序列分型(MLST)鉴定。结果共采集样品250份,检出51株eae阳性且不发酵乳糖和木糖的艾伯特埃希菌,检出率为20.40%,全部来自于乙公司采集的新鲜鸡肠。三重PCR结果显示51株菌株的clpX、lysP和mdh均为阳性,且与艾伯特埃希菌参考菌株KF1和LMG20976高度聚集,形成独立的分支,51株菌均为艾伯特埃希菌。结论聊城市家禽中存在艾伯特埃希菌,三重PCR和MLST聚类分析对艾伯特希菌鉴定可行有效。     

辽宁省首次在分割禽肉中分离出2株空肠弯曲菌

目的 2018年辽宁省食品污染和有害因素风险监测项目中首次在分割禽肉中分离出2株空肠弯曲菌。方法对6份分割禽肉进行选择性增菌后,采用直接滤膜法进行分离培养。对滤过的可疑菌落进行纯培养后,采用VITEK全自动生化鉴定系统及弯曲菌特异性微量生化补充试验联合多重PCR技术进行鉴定。结果从6份分割禽肉中(4份鸡肉、2份鸭肉)分离出2株空肠弯曲菌,并通过弯曲菌多重PCR技术进行鉴定,2株菌均证实为空肠弯曲菌。结论通过选择性增菌后直接滤膜法联合多重PCR技术,可以精准快速检测食源性弯曲菌污染,有助于从根本上预防控制弯曲菌引发的疾病。     

两种方法检测冻肉中的小肠结肠炎耶尔森氏菌

目的:建立适用于本实验室快速、准确检测致病性小肠结肠炎耶尔森氏菌的方法。方法:采用常规分离培养鉴定法与多重PCR方法对49份生肉进行检测,分析比较两种方法的检测结果。结果:两种方法的检测结果相同,符合率为100%。49份样品中均检出8份检出小肠结肠炎耶尔森氏菌,3株阳性对照以及6株阴性对照也均符合检测结果。但多重PCR方法所需的检测时间远少于常规分离培养鉴定法。结论:多重PCR较传统鉴定方法省时省力,对于监测小肠结肠炎耶尔森氏菌有重要意义。     

食品中副溶血性弧菌检测能力验证分析

目的通过参加CNAS(中国合格评定国家认可委员会)食品中副溶血性弧菌检测能力验证计划,对实验室检测检测能力及质量进行确认。方法采用国标法及实时荧光PCR法对标识为VP-162考核样品及超市随机抽取的50份动物性海水产品进行副溶血性弧菌定性检测,通过检测结果比较进行方法评价。结果 VP-162考核样品中检出副溶血性弧菌,考核结果为满意。随机抽取的50份动物性海水产品,干制生化试剂及API20E均检出6份(检出率12.0%),全自动微生物生化鉴定系统检出5份(检出率10.0%),实时荧光PCR法检出8份(检出率16.0%)。结论实时荧光PCR检测结果准确、灵敏、快速,在运用国标法进行检测过程中,采用生化鉴定试剂与全自动微生物生化鉴定系统分别进行鉴定时,其鉴定结果存在一定差异。     

上海奉贤区公共场所嗜肺军团菌污染状况调查

目的 应用PCR技术对奉贤地区部分公共场所空调冷却塔水及其他环境(淋浴设施等)进行军团菌污染状况调查.方法 于2010-05和2010-10,分别采集中央空调冷却塔水样品及环境样品用聚合酶链(PCR)对其进行嗜肺军团菌检测,同时用常规法进行分型鉴定.结果 采集的125份样品中,PCR法检出嗜肺军团菌45份,阳性检出率为36％,水样检出率远高于环境(分别为67.19％和3.28％).在检出的59株军团菌中共鉴定出6种不同菌型,其中LP1为重点菌型,占50.85％.且在同一样品中检出不同菌型有2株以上的占35.29％.结论 奉贤地区中央空调冷却塔及环境中存在军团菌污染.     

铜绿假单胞菌质粒介导的AmpCβ-内酰胺酶耐药性的分子生物学研究

目的研究铜绿假单胞菌（PAE）质粒介导的AmpCβ-内酰胺酶的耐药性，检测AmpC酶并鉴定其基因表型，为临床抗菌药物的合理使用提供实验室依据。方法收集108株临床分离鉴定的PAE，采用K—B药敏纸片法和头孢西丁三维试验方法检测产AmpC酶阳性菌株，对AmpC酶阳性菌株采用SDS碱裂解法抽提质粒，以纯化的质粒DNA为模板，应用PCR技术扩增AmpC酶基因产物，对PCR产物测序并对测序结果进行同源性比较，鉴定AmpC酶基因类型。结果在临床分离的108株PAE中，产AmpC酶28株，产AmpC酶株对多种抗菌药物耐药，新发现1株产质粒介导的CMY-7型AmpC酶铜绿假单胞菌株。结论PAE对多种抗菌药物耐药、存在产质粒介导AmpC酶；产AmpC酶是PAE对多种抗菌药物产生耐药的重要机制；通过序列比较，国内首次在PAE中发现1株产质粒介导的CMY-7型AmpC酶菌株。     

斑点杂交技术用于蛙钩端螺旋体病的流行病学调查研究

目的用Dotblotting杂交技术检测蛙类钩端螺旋体（钩体）,为动物钩体的流行病学监测和调查提供一种比较理想的方法。方法根据钩体赖株DNA合成1对flaB引物,用PCR技术对钩体菌株、疫区现场蛙肾材料等进行flaB基因扩增,用地高辛（DIG）标记flaB基因探针,用斑点杂交技术进行检测。结果结果表明用DIG标记的flaB探针可以检测到5fg及以下的DNA扩增产物。疫区70份蛙肾标本,斑点杂交检测阳性19份,阳性率为27．14％。而钩体细菌分离阳性者只有8份,阳性率11．43％,PCR扩增阳性者14份,阳性率20．00％。结论Dotblotting杂交是一种灵敏、特异、快速的钩体检测方法,可用于两栖类钩体的流行病学监测和调查。     

聚合酶链反应和地高辛标记的核酸杂交技术用于钩端螺旋体的检测

目的为钩端螺旋体快速诊断和流行病学调查建立一种比较理想的方法.方法根据钩端螺旋体赖株DNA合成一对flaB引物,用PCR技术对钩端螺旋体菌株、疫区现场动物标本等进行flaB基因扩增,用地高辛（DIG）标记flaB基因探针,用琼脂糖凝胶电泳和斑点杂交技术进行检测.结果纯化钩端螺旋体DNA 5pg经flaB-PCR扩增后,琼脂糖凝胶电泳可以目测.用DIG标记的flaB探针可以检测到5fg及以下的DNA扩增产物.疫区70份蛙肾标本,分离细菌8株,阳性率11.43%.flaB扩增阳性14份,阳性率20%;DIG标记探针斑点杂交检测,阳性19份,阳性率为27.14%.结论PCR-斑点杂交是一种灵敏、特异、快速的钩端螺旋体检测方法,既可用于快速检测和早期诊断,也可用于疫情监测和流行病学调查.     

应用PCR技术进行健康孕妇及新生儿中HHV-6感染状况的初步研究

目的：为了探索人类疱疹病毒－６型（ＨＨＶ－６）在健康孕妇及新生儿中的感染状况。方法：采用聚合酶链反应（ＰＣＲ）技术首次对济南地区３６例健康孕妇的外周血白细胞和４０例健康产妇顺产的新后儿脐带血白细胞进行了ＨＨＶ－６ＤＮＡ的检测。结果：前者有７例检出ＨＩＶ－６ＤＮＡ，其阳性率为１９．４％。而后者无一例检测到ＨＨＶ－６ＤＮＡ。结论：据此初步认为在健康孕妇中存在ＨＨＶ－６潜伏感染，而新生儿没有发现ＨＨＶ－     

Comparison of HHV-6 antibody titers in West Africa and the Caribbean

Human herpesvirus-6 (HHV-6) infection seems to be ubiquitous early in life, but antibody responses vary by geographic area. We compared HHV-6 antibody titer in 123 West African and 122 Caribbean serum samples. A quantitative immunofluorescence assay (IFA) using antigens derived from an HSB-2 cell line was used to test for IgG HHV-6 (GS strain) antibodies. The prevalence of HHV-6 antibodies was high (98%) in both sites. African samples had a significantly higher geometric mean titer (GMT: 697) than did Caribbean samples (GMT: 99). There was no difference between males (GMT: 260) and females (GMT: 270) overall. Children up to and including 9 years old had significantly higher titers (GMT: 483) than did all others (GMT: 237), and female children tended to have higher titers than did male children. In both areas there was a trend towards highest titer at younger age, followed by a decrease in titer during adulthood and middle age, and a secondary rise in titer in the oldest age group. Environmental and host factors may explain these geographic differences in antibody responses between two groups of African origin.     

人类疱疹病毒7型在血液肿瘤患者中感染情况

人类疱疹病毒7型(Human Herpsevirus 7,HHV—7)是90年代新发现的主要感染人体淋巴细胞的一类DNA病毒。为深入探讨HHV—7在不同人群中的感染机制,山东大学等单位在济南地区采用PCR技术检测了健康人群与血液肿瘤患者中HHV—7的感染状况。结果表明,50例健康成人和73例血液肿瘤患者外周血淋巴细胞中HHV—7DNA检出事为32％与42.5％,抽样血清中为0与6.7％,显示了白血病HHV—7感染率略高于健康人群,且可能在白血病急性期参与病理过程。     

HHV-6 in Djibouti--an epidemiological survey in young adults

Human herpesvirus type six (HHV-6), previously called human B-cell lymphotropic virus (HBLV), was first isolated in 1986 from patients with various lymphoproliferative disorders, some related to the acquired immunodeficiency syndrome. In order to investigate the epidemiology of HHV-6 in the Horn of Africa, we studied 281 young adults living in the city of Djibouti during June 1988. Of these, 181 belonged to various groups at risk for human immunodeficiency virus (HIV), while 100 represented the normal young adult population. Sera were screened and titrated for antibodies against HHV-6 by an indirect fluorescent antibody assay. The percentage seropositivity for HHV-6 was 71 in the normal population, 75 in the population at risk for HIV, and 93 in the population of subjects with a confirmed positive HIV Western blot. Mean titres of positive sera were similar in all population groups. No correlation existed between HHV-6 seropositivity and age, sex, tribe, habitat, and risk factors for HIV. A positive correlation was noted between HHV-6 and patients complaining of fatigue.     

Detection of bovine herpesvirus-1 infection in breeding bull semen by virus isolation and polymerase chain reaction

Serum samples from 51 apparently healthy breeding bulls were screened for bovine herpesvirus-1 (BHV-1) antibodies using an avidin-biotin enzyme-linked immunosorbent assay, revealing a sero-positive prevalence rate of 45.09%. Semen samples were then collected from 12 of the sero-positive and 12 of the sero-negative bulls and tested for BHV-1 antigen using both a virus isolation assay and a polymerase chain reaction (PCR) assay; PCR was applied to detect BHV-1 deoxyribonucleic acid by using primers selected from the relatively conserved sequence of the gl glycoprotein gene to amplify a 468 base pair fragment. The PCR-amplified products were confirmed as BHV-1 by restriction enzyme, Dde 1, which produced fragments of predictable sizes, namely 340 and 128 base pairs. Positive virus isolation test results, confirmed by virus neutralisation, found BHV-1 antigen in the semen of five sero-positive and six sero-negative bulls. In comparison, positive PCR results found BHV-1 genome in the semen of six sero-positive and eight sero-negative bulls. From the 24 semen samples tested, 14 were shown to be positive by PCR and 11 by virus isolation. The sensitivity and specificity of virus isolation were 57.14% and 70% respectively, and were significantly lower than PCR. In the semen samples taken from sero-negative bulls, BHV-1 was detected more often by PCR methods than by virus-isolation, suggesting that PCR is a more sensitive method for BHV-1 screening in bulls.     

Update on infections with human herpesviruses 6A, 6B,and 7

Human herpesviruses 6A, 6B, and 7 (HHV-6A, HHV-6B, HHV-7) are genetically related to cytomegalovirus. They belong to the Roseolovirus genus and to the Betaherpesvirinae subfamily. They infect T cells, monocytes-macrophages, epithelial cells, and central nervous system cells. These viruses are ubiquitous and are responsible for lifelong chronic infections, most often asymptomatic, in the vast majority of the general adult population. HHV-6B is responsible for exanthema subitum, which is a benign disease of infants. HHV-6A and HHV-6B also cause opportunistic infections in immunocompromised patients: encephalitis, hepatitis, bone marrow suppression, colitis, and pneumonitis. Their etiological role in chronic diseases such as multiple sclerosis, cardiomyopathy, and thyroiditis is still controversial. The pathogenicity of HHV-7 is less clear and seems to be much more restricted. Chromosomal integration of HHV-6A and HHV-6B is transmissible from parents to offspring and observed in about 1% of the general population. This integration raises the question of potential associated diseases and can be a confounding factor for the diagnosis of active infections by both viruses. The diagnosis of HHV-6A, HHV-6B, and HHV-7 infections is rather based on gene amplification (PCR), which allows for the detection and quantification of the viral genome, than on serology, which is mainly indicated in case of primary infection. Ganciclovir, foscarnet, and cidofovir inhibit the replication of HHV-6A, HHV-6B, and HHV-7. Severe infections may thus be treated but these therapeutic indications are still poorly defined.     

Serum IgG antibodies to human herpesvirus-6 (HHV-6) do not predict the progression of HIV disease to AIDS

Objectives: To evaluate if different levels of human herpesvirus 6 (HHV-6) antibodies can predict HIV disease progression. Design: Longitudinal study of individuals with a documented date of HIV seroconversion. Setting: Clinical centers located throughout Italy. Patients: Individuals who serconverted for HIV between 1983 and 1995 in Italy. Methods: Sera were tested for IgG antibodies to HHV-6 using a commercial enzyme immunoassay. A serum sample with an optical density (OD) 242 (i.e. the mean value of 10 negative controls+ 4×standard deviation) was considered as HHV-6 positive; the progression of HIV disease was evaluated estimating the relative hazards (RH) of AIDS (by Cox models) for individuals with higher levels vs. lower levels of HHV-6 antibodies or considering levels of antibodies based on 10% increase of the distribution (deciles). Rates of CD4 decline fitting linear regression were also estimated. Results: A total of 381 persons were followed for a median time of 4 years (range: 0.15–9 years) following the date of collection of the serum sample. The median OD value of HHV-6 antibodies was 306, with an interquartile range of 241–440 and a range of 48–2330. A slight inverse correlation was found between HHV-6 antibody levels and age of the individual at the time of serum collection (Spearman rank correlation coefficient, –0.16; p = 0.0013). No association was found between HHV-6 and CD4 level or between HHV-6 and CD8 level at the date of serum collection. The unadjusted RH of progression to AIDS was 0.63 (95% CI: 0.42–0.96) for HHV-6 positive individuals vs. HHV-6 negative; when adjusting for possible confounders (CD4, age, pre-AIDS HIV-related pathologies at the date of sera collection, and previous anti-herpes treatment), the RH of AIDS increased to 0.80 (95% CI: 0.51–1.23). No particular association with HIV disease progression was found when using the deciles of the distribution of HHV-6 antibodies. The median CD4 cell loss was 5.0 × 106 cells/l per month among HHV-6 positive individuals and 5.7 × 106 cells/l per month among the others. Conclusions: The presence of high levels of HHV-6 antibodies does not seem to predict the clinical or immunologic progression of HIV disease.     

Fatal Infection of a Pet Monkey with Human herpesvirus 1

Concerns have been raised about pet monkeys as a potential threat to humans. We report the opposite situation, a danger to pets that arises from humans. Similar to herpesvirus B (Cercopithecine herpesvirus 1), which endangers humans but not its host species, Human herpesvirus 1 can act as a “killer virus” when crossing the species barrier to New World monkeys.Key words: primate, marmoset, Callithrix, Human herpesvirus, HHV-1, zoonosisA man was bitten by a marmoset (genus Callithrix) that had stomatitis. For exclusion of possible zoonotic pathogens, virus culture was performed on a specimen obtained from the marmoset’s oral mucosa. Virus isolation and typing with antibodies revealed Human herpesvirus 1 (HHV-1) infection, confirmed by type-specific polymerase chain reaction (PCR). Despite treatment, the monkey died 2 days after the sample was drawn. Standard veterinary practice is to consider whether diseases of primates that have been in close contact with humans might have been caused by human viruses. Acute stomatitis in pet monkeys can suggest HHV-1 infection, among other diseases, and systemic treatment with acyclovir may be appropriate.