可疑霍乱弧菌的实验鉴定

目的:对4株可疑霍乱弧菌进行种属鉴定。方法:对可疑菌落在传统血清学和系统生化鉴定的基础上进行细菌的分子鉴定:应用16S rDNA的序列分析进行细菌种属鉴定和实时荧光PCR检测O1、O139群霍乱弧菌。结果:16 s rDNA基因序列分析显示041和067为麦氏弧菌,059和074为霍乱弧菌;荧光定量PCR检测结果说明4株菌都不是O1和O139霍乱弧菌。结论:结合血清凝集和系统生化实验,可以确定041和067为麦氏弧菌,059和074为非O1/非O139霍乱弧菌。对于可疑和难鉴别的霍乱弧菌,不能单纯依靠血清学和表型鉴定方法,需要分子诊断方法,尤其是16SrDNA的序列分析,可以从分子水平为弧菌科细菌的种属鉴定提供更为直接的证据。     

Rapid identification of Vibrio cholerae by darkfield microscopy

This article describes a rapid, simple and reproducible method for detecting Vibrio cholerae in diarrhoeal patients. The method involves darkfield examination of a liquid stool specimen or a rectal swab immersed in broth and immobilization of V. cholerae by the addition of specific vibrio antisera. The authors state that in 80% of cases a definitive diagnosis is available within five minutes. There is no need for elaborate equipment or long training of technicians and the method is easily performed by one person in the field.     

霍乱弧菌多重PCR-DHPLC分型方法建立

目的 建立霍乱弧菌多重PCR-变性高效液相色谱(DHPLC)快速分型方法。方法 分别合成扩增霍乱弧菌胶原酶基因(vcc基因)、O1群和O139群的毒力基因(ctxA基因和tcpA基因)以及O139群毒力基因(LPSgt基因),并对4对引物的PCR退火温度进行优化,建立多重PCR-DHPLC分型方法。结果 4种基因可同时被扩增,应用多重PCR-DHPLC方法对霍乱弧菌进行分型的结果与预期的一致。结论 多重PCR-DHPLC分型方法可用于快速、准确地鉴定细菌纯培养物是否为霍乱弧菌以及具体的菌群。     

A simple and rapid molecular method for Leptospira species identification

Serological and DNA-based classification systems only have little correlation. Currently serological and molecular methods for characterizing Leptospira are complex and costly restricting their world-wide distribution and use. Ligation mediated amplification combined with microarray analysis avoids many of these drawbacks. We demonstrated that this approach used in the Check-Points (CP) assay can successfully applied for the generic detection of Leptospira and can discriminate between saprophytic, intermediate and pathogenic species. In addition, the CP assay could unambiguously detect strains of seven pathogenic species and revealed discrepancies in previous speciation and culture collections. The method provides a valuable tool adding to the molecular study of leptospires and their local and global distribution.     

TaqMan荧光定量PCR技术快速检测霍乱弧菌方法的建立

目的：建立特异、灵敏、快速的TaqMan荧光定量PCR方法用于快速检测霍乱弧菌。方法：根据GenBank上的霍乱毒素（cholera toxin，CT）基因序列，在其保守区域设计特异性引物与TaqMan探针，并对荧光定量PCR体系与反应条件进行优化，同时验证方法的特异性、敏感性和重复性。结果：本方法对霍乱弧菌的检测具有高度特异性，与副溶血性弧菌、沙门菌、志贺菌、变形杆菌、大肠埃希菌、蜡样芽孢杆菌、金黄色葡萄球菌等均无交叉反应；检测灵敏度达10cfu／ml；反应体系具有很高的稳定性；整个操作过程仅需2h。结论：本研究建立的TaqMan荧光定量PCR方法特异、灵敏、快速．适用于霍乱弧菌的日常监测和爆发疫情的应急诊断。     

霍乱弧菌NhaA基因的扩增、克隆和初步鉴定

目的 体外扩增、克隆霍乱弧菌O1群和O139群1149bp的NhaA基因片段。方法 采用PCR方法扩增NhaA基因,经限制性内切酶BamHI和EcoRI酶切的用低熔点琼脂糖法回收纯化,插入pcDNA3载体的多克隆位点,构建重组体,转化大肠杆菌,并对克隆基因进行酶切鉴定。结果 分别从霍乱弧菌O1群和O139群扩增出1149bp的NhaA基因片段,所构建的重组体经酶切分析和预期结果一致。结论 成功扩增并克隆我国散发霍乱O1群和O139群NhaA基因,为研究霍乱流行规律和基因疫苗提供了重要的实验依据。     

4重荧光PCR技术在霍乱监测与菌株鉴定的应用研究

目的 应用4重荧光PCR技术检测霍乱弧菌并对菌株进行鉴定.方法 用PCR法对2008-2010年监测的水样、水产品标本及食物中毒疑似菌株以进行检测.同时用细菌学方法分离菌株.再进行PCR检测.结果 1606份标本中.PCR法检出O1群霍乱弧菌阳性标本188份.从中分离到菌株150份(79.8％).O139群霍乱弧菌的P...     

Enzyme markers for Vibrio cholerae: identification of classical, El Tor and environmental strains

Enzyme electrophoretic variants were studied in 49 strains of Vibrio cholerae using zymovar analysis. The following seven enzymes were selected for use: alanine dehydrogenase (ADH), isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH), phosphoglucomutase (PGM), glucosephosphate isomerase (GPI), 6-phosphogluconate dehydrogenase (6PGDH) and glucose-6-phosphate dehydrogenase (G6PDH). The results indicated the presence of three main groups defined chiefly by their GPI and 6PGDH variants. The first group, defined by possessing the variants GPI-2 and 6PGDH-3, contained all the 01 serovar and E1T or biovar isolates from cholera cases. The second group, defined by possessing the variants GPI-3 and 6PGDH-2, contained all the 01 serovar and classical biovar isolates; the third group was heterogeneous and included the 01 serovar isolates from environmental sources as well as isolates of other serovars (the so called NAGs, non-agglutinable with 01 antisera or NCVs). It is thus now possible to separate the epidemic strains of 01 serovar from other members of this serovar isolated from the environment. Zymovar analysis deals with differences which are a direct expression of the genome and seems to be unaffected by gross phenotypic changes such as smooth-rough variation and phage resistance. It is a promising tool for investigating bacteriological and epidemiological questions, in particular the significance of an environmental reservoir of cholera.     

1株不典型非O1群霍乱弧菌的发现及鉴定

目的 对1株从肝硬化患者血液中分离的病原菌进行鉴定.方法 按霍乱弧菌的鉴定标准和方法进行细菌学表型特征(包括形态学、培养特性、生化特征)、血清学鉴定及16S rDNA序列分析.结果 从患者血液中分离获得1株病原菌HN 9837,该菌株的形态学、培养特性、生化特征、血清学诊断和分子生物学诊断结果基本符合非O1群霍乱弧菌定义;但与普通霍乱弧菌在甘露醇发酵及赖氨酸利用、柠檬酸盐利用试验结果存在差异.结论 海南省首次在肝硬化患者血液中分离出非O1群霍乱弧菌,该菌为1株不发酵甘露醇、赖氨酸且柠檬酸盐利用试验阴性的不典型霍乱弧菌.     

A simple and reliable method for estimating haemoglobin.

A new colour scale has been advised for estimating haemoglobin levels by matching the blood samples with ten levels of haemoglobin (3, 4, 5, 6, 7, 8, 9, 10, 12, and 14 g/dl) on the scale. Preliminary results show good correlations with spectrophotometric readings. The new device is being field tested and if the initial promise is confirmed, will provide a simple and reliable method for estimating haemoglobin where laboratory facilities are not available.     

A simple laboratory method for the diagnosis of V. cholerae.

In many developing countries where cholera occurs, bacteriological facilities at the local level are lacking and either cholera may be unreported or other diarrhoeal diseases may be mistakenly reported as cholera. A simple and practical method, suitable for use in small hospitals and health centres and by field teams of health workers is described. Swab samples are streaked on to various media (TTGA and TCBS were found to be the most satisfactory), incubated overnight at 35 degrees C and read. The characteristics which distinguish Vibrio cholerae colonies from others developing are described. In tests on 309 cholera patients, culture on TTGA gave 100% and on TCBS 96.7% positives.     

Vibrio cholerae O139 Bengal: a descriptive study

A prospective study was conducted to determine the clinical and laboratory characteristics and the clinical course of cholera due to Vibrio cholerae O139 Bengal. The study subjects included 22 adult males with stool culture-proven V. cholerae O139. On enrollment, mean +/- SD concentrations (mmol/L) of serum sodium, potassium, chloride, and bicarbonate were 134 +/- 3, 4 +/- 1, 102 +/- 4, and 13 +/- 4 respectively, and stool sodium, potassium, chloride, and bicarbonate concentrations were 120 +/- 24, 18 +/- 6, 93 +/- 16, and 37 +/- 9 respectively. Seventeen patients (7.8%) had faecal leukocytes ranging from 11 to 50 per high-power field. All V. cholerae O139 isolates (100%) were susceptible to tetracycline, erythromycin, and ciprofloxacin, 92% to furazolidine, and only 5% to trimethoprim-sulphamethaxazole. The median (interquartile) volume of liquid stool during the first 24 hours was 9 (5-12) litre. The median (interquartile) volume of liquid stool and the amounts of intravenous and oral rehydration fluids required during the entire study period were 16 (9-24) litre, 9 (6-18) litre, and 14 (9-20) litre respectively. The median (interquartile) duration of diarrhoea was 80 (48-104) hours. The median (interquartile) duration of excretion of V. cholerae O139 in stool was 5 (3-6) days. Clinical and laboratory features, and case management of cholera due to V. cholerae O139 are very similar to conventional cholera due to V. cholerae O1.     

Evaluating the indoor and outdoor environment: using a simple method

Housing is the conjunction of the dwelling, the home, the immediate environment and the community. Between 1960 and 1980, the urban population in developing countries more than doubled and is expected to reach 56% of the total population by the year 2025. In many cities, the development of squatter settlements and shanty towns had grown rapidly causing the destruction of green areas. The number of people living in urban slums and shanty towns is an indicator of conditions in the cities and the United Nations had estimated that about one-third of urban dwellers in developing countries live in such settlements. Poverty is highly prevalent among the residents of these areas. Outdoor environmental degradation, together with the social degradation affects the health of the urban population especially the poor causing a burden of ill-health, disability, poor indoor housing, and high maternal and infant mortality. The aim of the present study is to evaluate the indoor and outdoor environment using a simple method and to assess its reliability and validity. The scoring system, which was developed for the assessment of the indoor and outdoor environmental levels included 36 items (18 for the indoor and 18 for the outdoor) using a questionnaire. Results revealed that the proposed scoring system was able to reveal significance difference between served (water, electricity, and sewerage system were available) and unserved areas when using t-test, z-test, and chi-square testing. The proposed scoring system was reliable and valid especially in indoor assessment. Outdoor scores might need more modifications to improve its reliability.     

珠江河口水体霍乱弧菌污染状况调查研究

目的:了解珠江河口水体01/0139群霍乱弧菌的污染情况,提出改善水体霍乱弧菌监测的建议,为采取针对性措施预防控制霍乱的发生和流行提供依据.方法:根据历年霍乱监测资料和疫点分布状况,在珠江水系广州段选择设立24个采样点,每月采集水体标本进行01/0139群霍乱弧菌分离培养,对分离得到的阳性菌株进行血清分型、噬菌体一生物分型、PCR检测毒力基因、K-B法药敏试验.结果:2006年3月～2007年2月共采取862份水体样本,其中有67份检出01或0139群霍乱弧菌,检出率为7.77%,血清型以01群为主,尤以稻叶型占优;01群菌株均为非流行株,2株0139群菌株检出ctx毒力基因;菌株耐药分析发现不同血清型菌株耐药谱有所差异.结论:01/0139群霍乱弧菌在珠江河口水体环境中广泛存在,常年均可从水体中分离获得.必须加强环境水体及海水产品霍乱弧菌监测,及时了解其污染状况,以便采取针对性措施预防控制霍乱的发生和流行.     

Surveillance methodology for Vibrio cholerae in environmental samples

The purpose of this study was to examine the prevalence of Vibrio cholerae in environmental water samples by using a series of biochemical tests. A total of 223 V. cholerae-like bacteria were isolated from TCBS agar after spreading the alkaline peptone water enriched sewer (n = 21) and water (n = 16) samples. All oxidase positive isolates were subjected to confirmation for V. cholerae by seven other biochemical tests and polymerase chain reaction. Only 74.2% isolates were found to be V. cholerae by PCR using primers against an outer membrane protein (ompW) gene, out of which only 2 isolates were positive for cholera toxin (ctxAB) gene. Among the various biochemical tests studied, arginine hydrolysis, arabinose fermentation and string test showed 92 - 100% sensitivity and 42 - 67% specificity. Eight isolates including the toxigenic ones, showed agglutination with V. cholerae O1 antiserum. The present study showed that no biochemical test is 100% specific for V. cholerae. However, a few tests, if performed in a sequence after growing the alkaline peptone water enriched samples onto TCBS media can be used for screening of V. cholerae from the environmental samples. This study also showed that most of the environmental isolates are non-O1/non-O139 and the chances of presence of toxigenic V. cholerae are very rare in the environment.