筛选与确诊试验在检测香港海鸥菌中的建立与应用

目的建立简便、快速、准确、特异的香港海鸥菌实验室诊断技术。方法采集淡水鱼类、胃肠炎腹泻患者新鲜粪样,接种于改良头孢哌酮MacConkey（CMA）培养基,对典型菌落分别进行氧化酶、触酶、三糖铁（TSI）3项筛选试验和常规生化鉴定,凡氧化酶、触酶试验阳性以及三糖铁（TSI）试验阴性的菌株,采用特异性PCR检测确诊;通用引物扩增16SrRNA并与香港海鸥菌HKU1株进行同源性分析。结果在92份淡水鱼样品和211份胃肠炎腹泻患者粪样中,共检出香港海鸥菌11株和1株,阳性率分别为11.96%（11/92）和0.48%（1/211）。筛选与确诊试验和常规生化试验的检测结果一致,符合率为100%（12/12）。分离的香港海鸥菌16SrRNA序列与HKU1株同源性在99.5%～100%之间,仅差1～2个碱基。结论本文建立的筛选与确诊试验适用于香港海鸥菌的诊断。     

香港海鸥形菌DNA脉冲场凝胶电泳的技术条件

目的 探索香港海鸥形菌（Laribacter hongkongensis,LH）DNA脉冲场凝胶电泳技术的条件。方法 从2株LH参考株中提取DNA,经过限制性内切酶SpeI的切割,再用脉冲场凝胶电泳仪进行DNA分子分型,比较5种条件的分型效果。结果 在OD值为1.8、细胞裂解4 h、SpeI 20U酶切20 h、电泳脉冲时间为2.2～54.2 s、电泳25 h的条件下,LH基因组DNA片段得到良好分离。结论 上述条件产生的LH DNA指纹图谱图像清晰,分辨率高,在没有图像分析仪的情况下,肉眼即可进行判断。证实所建方法可行,条件适合。     

香港海鸥形菌对理化生因子抵抗力的实验研究

目的了解香港海鸥形菌（LH）对理化生因子的抵抗力。方法用紫外线、高温、干燥、pH、常用多种化学消毒剂及部分抗菌药物处理两株分离于病人的香港海鸥形菌,定量观察其生长情况。结果温度≥56℃即可灭活香港海鸥形菌;紫外线照射30s杀灭率〉90%,照射20min可完全灭菌;菌株在pH〉8.5或〈5.0条件下不能存活,最适生存pH范围为5.5～8.5;在3%以上盐浓度的肉汤中不生长;对化学消毒剂和干燥较敏感,除对2%碳酸氢钠、2%来苏儿、0.1%迭氮钠和70%乙醇有一定耐受力外,其余消毒剂均可在30s内杀灭该菌;香港海鸥形菌在自然干燥后15min即全部死亡;在18种常见抗菌药物中,仅对头孢噻吩、头孢哌酮、氨苄西林、头孢唑啉、头孢他啶、克林霉素耐药,其余均敏感。结论香港海鸥形菌对多数理化生因子抵抗力较弱,常用的消毒剂能够有效杀灭LH。     

深圳市酒店三文鱼和北极贝生食中致病菌污染状况调查

目的了解酒店、日韩料理餐厅冰鲜海产品（三文鱼、北极贝）的微生物污染状况,为预防病原微生物食物中毒提供科学依据。方法抽取酒店、日韩料理餐厅、超市商场等冰鲜海产品共168份．依照国家标准方法和荧光PCR方法对样品进行检测。结果168份样品中共检出不合格大肠菌群101份,阳性率为60．12％：菌落总数29份,阳性率为17．26％;致病菌：检出副溶血性弧菌5份,阳性率为2．98％;金黄色葡萄菌38份,阳性率为22．62％。沙门氏菌和志贺氏菌致病菌未检出。结论冰鲜海产品三文鱼和北极贝受到病原微生物的污染相当严重,各部门要加强监管监测,积极做好预防控制工作,减少食原性食物中毒发生的危险。     

深圳市肉类食品空肠弯曲菌的污染情况调查

目的了解我市常见肉类食品中空肠弯曲菌的污染状况,为控制和预防空肠弯曲菌食物中毒提供科学依据。方法在食品污监测点随机抽检肉类食品样品117份,用传统国标法和荧光PCR法进行检测。结果从117份肉类食品中检出4份空肠弯曲菌,阳性检出率为3.4%。结论深圳市肉类食品中存在空肠弯曲菌的污染,如果加工、销售不当,及监督不严,会对群众健康构成潜在威胁。     

实时荧光PCR同时检测丙型副伤寒沙门菌和猪霍乱沙门菌方法的建立和应用

目的建立丙型副伤寒沙门菌和猪霍乱沙门菌的实时荧光PCR快速检测方法,用于沙门菌属内的分型鉴定。方法根据GenBank公布的丙型副伤寒沙门菌和猪霍乱沙门菌的保守序列,设计引物和改良分子信标探针,建立实时PCR检测方法。结果检测体系灵敏度高,纯DNA和菌液的最低检出限分别可达10fg和20CFU/反应体系;特异性好,对71株细菌的检测符合率达100%。20株沙门菌采取盲号模拟血培养标本进行血培养检测及鉴定,检出5株丙型副伤寒沙门菌和4株猪霍乱沙门菌,与试验的菌株相符。70份食品中用实时荧光PCR同时检测丙型副伤寒沙门菌和猪霍乱沙门菌均为阴性,而用传统方法分离培养未检出。结论建立的实时PCR检测方法可以快速、特异、灵敏地检测出丙型副伤寒沙门菌和猪霍乱沙门菌。     

招远市口腔诊所污染状况调查

为了解我市口腔诊所消毒工作现状,规范口腔诊所消毒技术规范,预防医源性感染的发生,我们于2006年4月-5月对辖区内的32个口腔诊所进行了细菌污染状况的调查.现将结果报告如下:     

单核细胞增生李斯特菌与志贺菌双重实时PCR检测技术的建立

目的 建立一种快速、特异、灵敏、准确定量的单核细胞增生(单增)李斯特菌(Listeriamonocytogenes)与志贺菌(Shigella)同步检测方法.方法 分别根据单增李斯特菌溶血素O基因hly与志贺菌侵袭性质粒抗原H基因ipaH设计合成引物和探针.构建重组质粒pGEM-T-hly与pGEM-T-ipaH,并以EcoR I单酶切使环状重组质粒线性化作为标准品.优化反应体系,分析特异性.双重荧光定量PCR对人工污染的脱脂灭菌乳进行检测.结果 成功构建了重组质粒标准品,并运用5'、3'端分别标记FAM、TAMRA的hly基因探针和5'、3'端分别标记HEX、TAMRA的ipaH基因探针成功建立了单增李斯特菌与志贺菌同步荧光定量PCR检测方法.结论 建立的方法有较强的特异性,线性范围好(105～101copies/μl,R2≥0.998),灵敏度为10 copies/PCR,同步检测人工污染脱脂灭菌乳的灵敏度为102CFU/ml.     

实时荧光RT-PCR检测活性单核细胞增生李斯特菌方法的建立

目的 采用逆转录结合实时荧光定量PCR技术,建立一种快速、准确、特异甄别单核细胞增生李斯特菌(Listeria monocytogenes,简称单增李氏菌)死活状态的定量方法.方法 根据单增李氏菌hlyA基因序列设计引物和探针;对实时荧光PCR反应体系进行优化后,提取菌体mRNA,通过随机引物进行逆转录反应;产生的cDNA通过实时荧光定量PCR进行鉴定.进一步评价逆转录结合实时荧光定量PCR方法的特异性、灵敏度、重复性后,对20份模拟双盲样本进行检测.结果 本实验所建立的逆转录结合实时荧光定量PCR方法可准确、特异地检测单增李氏菌,其他菌株和失活的单增李氏菌均无阳性结果出现;该方法检测纯菌和模拟样本的灵敏度分别可达到10 CFU/ml和1000CFU/ml;定量检测的批间和批内的变异系数均小于5%;对20份模拟样本进行检测,其中10份含有活性单增李氏菌样本的检测结果均为阳性,其他含有失活单增李氏菌的5份样本和其他致病菌的5份样本检测结果为阴性.结论 本文建立的检测活性单增李氏菌实时荧光定量PCR方法快速、准确,结果可靠,实用性强,可进行定量分析,为食品安全监测和现场流行病学调查提供较好的分析手段和完整的数据.     

罗湖区常见可生食蔬果寄生虫污染状况调查

目的 了解罗湖区常见可生食蔬果和即食凉菜等食品的寄生虫污染情况,以评价直接生食此类食品的安全性,为采取有效措施防治寄生虫、保障食品安全提供科学依据.方法 按照监测工作要求,在罗湖区部分农贸市场、超市和餐厅,抽取常见可生食蔬果和即食凉菜等的成品或半成品,每份样品经去离子水清洗,洗液离心后比较不同种类蔬果中寄生虫和虫卵的阳性率差异.结果 检测350份可生食蔬果,检出寄生虫或寄生虫卵者共77份,阳性率22.00%,样品类别(χ2=173.34,P<0.01),蔬菜种类χ2=92.33,P<0.001),是否水洗(χ2=124.23,P<0.01)等因素和寄生虫检出情况可能有关.结论 罗湖区内市售与土壤直接接触的蔬菜寄生虫污染状况不容忽视,提示在食品卫生学上,生食该类蔬菜有一定危险性,需进一步重点调查该类食品,明确其危险性,及时采取有效措施.     

Bacteremia caused by Laribacter hongkongensis misidentified as Acinetobacter lwoffii: report of the first case in Korea

Laribacter hongkongensis is an emerging pathogen in patients with community-acquired gastroenteritis and traveler's diarrhea. We herein report a case of L. hongkongensis infection in a 24-yr-old male with liver cirrhosis complicated by Wilson's disease. He was admitted to a hospital with only abdominal distension. On day 6 following admission, he complained of abdominal pain and his body temperature reached 38.6℃. The results of peritoneal fluid evaluation revealed a leukocyte count of 1,180/μL (polymorphonuclear leukocyte 74%). Growth on blood culture was identified as a gram-negative bacillus. The isolate was initially identified as Acinetobacter lwoffii by conventional identification methods in the clinical microbiology laboratory, but was later identified as L. hongkongensis on the basis of molecular identification. The patient was successfully treated with cefotaxime. To the best of our knowledge, this case is the first report of hospital-acquired L. hongkongensis bacteremia with neutrophilic ascites.     

细胞培养中细菌类微生物污染的快速检测

目的 检测细胞培养中细菌类(包括支原体)微生物的污染情况。方法 用细胞培养中常见的培养物人为污染He-La细胞，设计细菌类微生物16S rRNA基因序列通用引物，PCR扩增16SrRNA基因序列片断，建立PCR检测培养细胞污染的方法，并用该方法检测本室保存的细胞株的污染情况。结果 人为污染绿脓杆菌、大肠杆菌、白色葡萄球菌和支原体M.fernentans的Hela细胞的培养上清中均扩增出大小的片段，与目的片段相符。本室所收藏的15个细胞株有3株的培养上清中扩增出大小的片段。结论 本实验建立了：16SrRNA基因序列通用引物PCR法，可用于快速检测培养细胞中细菌类微生物的污染。     

Use of a polymorphic dinucleotide repeat sequence to detect non-blastomeric contamination of the polymerase chain reaction in biopsy samples for preimplantation diagnosis

Using the polymerase chain reaction (PCR), amplification oftwo different target DNA sequences has been achieved with highfrequency using single human blastomeres as template for theduplex reaction. One sequence is located within the –globingene and contains the sickle cell locus, the other is a polymorphicdinucleotide repeat, which, as well as acting as a positivecontrol for amplification, was used to check the origin of theamplified DNA. A comparison of the sequences amplified fromthe blastomere with sequences amplified from parental samplesconfirmed that amplification of blastomeric sequences, but notextraneous contaminating DNA, had taken place in most cases.The efficacy of this system for detecting extraneous DNA waschecked by deliberately contaminating single blastomeres withforeign cells. The presence of contamination was detected bythe amplification of sequences not present in blastomeric DNAand which therefore must have been amplified from extraneouscontaminating DNA.     

广州市亚运接待宾馆酒店淋浴热水军团菌污染状况分析

目的对广州市亚运接待宾馆酒店淋浴热水的军团菌污染状况进行调查分析,提出相应的对策。方法采集广州市48间亚运接待宾馆酒店淋浴热水108份,按照卫生部《公共场所集中空调通风系统卫生规范》2006年附录A进行军团菌的检测。结果 108份淋浴热水中,10份淋浴热水共检出11株嗜肺军团菌,阳性率为9.3%;其中LP1、LP3、LP6血清型分别占45.5%、18.2%、36.4%;三星级、四星级和五星级宾馆酒店淋浴热水军团菌的阳性率分别为15.2%、11.1%和2.6%。结论广州市亚运接待宾馆酒店的淋浴热水存在嗜肺军团菌的污染,建议定期清洗消毒淋浴设施,并加强对淋浴热水中军团菌的监测,以防范军团菌病的发生。     

Dedicated decontamination: A necessity to prevent cross contamination in high throughput mycobacteriology laboratories

Unrecognized cross-contamination has been known to occur in laboratories frequently, especially with sensitive recovery system like BACTEC 460 TB. In March 2001, we investigated a pseudo-outbreak of Mycobacterium tuberculosis isolates in three smear negative clinical specimens and would like to present our experience in this communication. Methods: All suspected cases were confirmed by checking the drug susceptibility and DNA fingerprints using spoligotyping as well as restriction fragment length polymorphism. Results: On investigation, the most likely cause was found to be the use of common decontamination reagents and phosphate buffer. Conclusions: To avoid erroneous diagnosis, we have devised a dedicated decontamination procedure, which includes separate aliquoting of phosphate buffer and decontamination reagents per patient. Timely molecular analysis and appropriate changes to specimen processing have been identified as useful measures for limiting laboratory cross contamination.     

A simple and rapid approach to the problem of tissue contamination and patient identity in histopathologic specimens

Individual pathologists deal with thousands of diagnostic biopsy and excision specimens annually. At each stage, procedures are in place to limit the possibility of human error, which could result in specimen transposition or contamination. One specimen contaminating another is usually easily identified and rarely causes diagnostic difficulty; however, when it does, the consequences can be very serious. We discuss 5 cases in which concerns over specimen identity and tissue contamination arose and the methodology by which we resolved those concerns. Polymerase chain reaction analysis of each case was carried out using a panel of 12 polymorphic microsatellite markers, specific for chromosomes 13, 18, and 21. These markers are routinely used in the molecular genetics diagnostic laboratory for rapid trisomy screening. In each case, the question of error was satisfactorily resolved. Using this approach, we prevented the real possibility of patients undergoing second invasive procedures. We suggest that this or a similar methodology become a routine part of pathology practice.     

Comparison of the Cobas 4800 HPV Test and the Seeplex HPV4A ACE with the Hybrid Capture 2 Test

Background: It is well-known that persistent cervical infections with high-risk human papillomavirus (HPV) are related to the development of high-grade cervical intraepithelial neoplasia and invasive cervical cancer and that infection with HPV 16 and HPV 18 accounts for approximately 70% of all cases of invasive cervical cancer.Methods: We performed 3 HPV molecular tests―the Cobas 4800 HPV test, the Seeplex HPV4A ACE, and the hybrid capture 2 (HC2) test―in 146 cervical swab samples to compare between these three tests.Results: There was a concordance rate of 82.8% between the results of the Cobas 4800 HPV and the HC2 test and a concordance rate of 84.9% between the results of the Seeplex HPV4A ACE and the HC2 test. Between the Cobas 4800 HPV test and the Seeplex HPV4A ACE, there was a concordance rate of 89.6% in the detection of high-risk HPV between the results and a concordance rate of 98.7% in the detection of HPV 16 or 18. When an abnormal Pap test was defined as ≥low grade squamous intraepithelial lesion (LSIL), the sensitivity of the Cobas 4800 HPV test, the Seeplex HPV4A ACE and the HC2 test were 71.1%, 80.0%, and 88.9%, respectively, while their specificities were 76.4%, 74.5%, and 67.9%, respectively.Conclusions: The results of this study suggest that the Cobas 4800 HPV test and the Seeplex HPV4A ACE may be as effective as the HC2 test in detecting HR HPV and that the concordance between the results of the Cobas 4800 HPV test and the Seeplex HDV4A ACE may be higher in the detection of HPV 16 and HPV18 than concerning high-risk HPV.     

Comparison of real-time PCR and conventional hemi-nested PCR for the detection of Bordetella pertussis in nasopharyngeal samples

We directly compared a conventional hemi-nested PCR assay with a real-time (LightCycler) PCR assay for the detection of Bordetella pertussis in nasopharyngeal samples. Of the 152 samples tested, 49 (32%) were positive by first-round conventional PCR, 56 (37%) were positive by the hemi-nested PCR assay, and 39 (26%) were positive by the real-time assay. All samples testing positive with the real-time assay were also positive by the hemi-nested assay (both first- and second-round PCR), and all culture-positive samples tested positive by both PCR assays. These results suggest that the hemi-nested assay is more sensitive than the real-time assay for detecting B. pertussis .