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.     

儿童肺炎的肺炎支原体病因学分析

目的:明确肺炎支原体在儿童肺炎致病微生物中的地位,探讨聚合酶链反应(PCR)技术在儿童肺炎病原检测中的使用价值。方法:用PCR扩增技术,对278例肺炎患儿进行MPDNA检测。结果:MPDNA检测阳性患儿68例,阳性率为24.5%,其中<6岁儿童占64.7%。结论:MP是儿童肺炎的主要致病微生物之一,且发病年龄明显前移。PCR用于检测儿童肺炎病原体,灵敏、特异、迅速。     

寡核苷酸阵列芯片快速鉴定大肠杆菌O157:H7初步研究

目的设计、制作一种微型化寡核苷酸阵列芯片，评价快速鉴定肠出血性大肠杆菌O157：H7的效果。方法多重PCR扩增大肠杆菌O157：H7七个特异性基因位点（rfbE、flicH7、intimin、Shiga-like toxins Ⅰ and Ⅱ、hemolysin A和uidA）．通过PCR反应掺入SpectrumOrang^TM-dUTP获取荧光标记的靶序列，与制备的芯片寡核苷酸探针杂交。结果寡核苷酸阵列芯片检测结果与试验预期相符，获取的杂交图分辨效果明显优于多重PCR琼脂糖凝胶电泳。结论基于玻片的寡核苷酸阵列芯片制作简便，鉴定病原菌检测细菌毒力因子快速、灵敏、特异，有良好的应用前景。     

Progress in diagnosing herpesvirus infections

As molecular biology has developed, several new diagnostic techniques have found application in the clinical setting. The use of the polymerase chain reaction (PCR) assay to study the molecular biology of microbial organisms has led to unparalleled advances, largely due to the rapidity with which results can be obtained. The sensitivity and specificity of PCR detection of viral DNA for diagnostic purposes are remarkable. With such excellent sensitivity, PCR is destined to become a useful diagnostic tool in herpesvirus infections. However, it is well known that herpesviruses establish latency after primary infections and that they can often be reactivated under various conditions. Because of the high sensitivity of PCR, detection of virus sequences by this method does not necessarily imply a disease state. We must be careful not to overdiagnose conditions in a clinical setting based on a PCR assay.     

Utility of multiplex PCR in detecting the causative pathogens for pediatric febrile neutropenia

Febrile neutropenia (FN) is a life-threatening complication, and the primary cause of FN is considered to be microbial infection. Therefore, prompt and appropriate antimicrobial therapy is crucial. Clinicians usually prescribe antimicrobial therapy on the basis of presumptive and empirical data. This is because the causative pathogen for FN in blood culture (BC) analysis is detected several days after sampling. Polymerase chain reaction (PCR) analysis has been used for detecting the causative bacteria of infections. Here, we examined whether multiplex PCR is useful for detecting the causative pathogens for FN patients. We extracted DNA from the patients' whole blood and performed multiplex PCR. In total, 128 samples of 40 patients clinically diagnosed with FN were used in this study. Multiplex PCR analysis revealed the causative pathogen in 3 patients with FN; the DNA fragments amplified were those of Pseudomonas aeruginosa in 2 cases and Psedomonas putida in 1 case. These patients could be started on appropriate antimicrobial therapy a few hours after sampling. However, the DNA fragment of the causative pathogen could not be amplified by PCR in 2 patients, although BC analysis did detect the causative bacteria. Thus, we conclude that multiplex PCR is serviceable in case of FN because of its rapidness. However, BC is also indispensable to treating FN owing to its high sensitivity.     

Detection of Bacterial Pathogens in Cerebrospinal Fluid using Restriction Fragment Length Polymorphism

Background

Polymerase chain reaction (PCR) is useful for rapid microbial detection in body fluids with low microbial load. It is easier to use universal or broad range primers for the amplification of conserved stretches of DNA common to all bacteria like 16S rRNA gene, followed by restriction fragment length polymorphism (RFLP) of PCR products.

Methods

Forty samples of cerebrospinal fluid were collected. After DNA extraction, universal or broad range PCR was performed using two universal primers U1-5''-CCAGCAGCCGCGGTAATACG-3'', corresponding to nucleotides 518 to 537 of the Escherichia coli 16S rRNA gene, and U2 − 5''-ATCGG(C/T)TACCTTGTTACGACTTC-3'', corresponding to nucleotides 1513 to 1491 of the same gene. The PCR product was subjected to digestion by endonucleases- HaeIII, Mn11, BstB1 and Alu1. Restriction pattern obtained was compared with that of standard organisms to identify the pathogen. The results were compared with conventional methods.

Result

Universal PCR could detect pathogens in 20% samples within 13-18 hours as compared to 16% by conventional methods. The analytical sensitivity was 10 Gram negative and 250 Gram positive organisms per 200 μl sample. Overall sensitivity was 83.3% and specificity was 91.2%.

Conclusion

Universal PCR followed by RFLP of PCR product is a good alternative to conventional diagnosis of bacterial pathogens.Key Words: Body fluids, Polymerase chain reaction, Restriction fragment length polymorphism     

阴道分泌物念珠菌感染检测方法对比分析

目的 分析常规镜检、荧光PCR核酸扩增法和真菌显色培养法在阴道分泌物真菌检测结果的相关性.方法 选取2014-2016年于该院就诊的疑似阴道炎患者,收集镜检阴道分泌物真菌阳性和阴性标本各500例,用荧光PCR核酸扩增法鉴定念珠菌类型,并将100份荧光PCR核酸扩增法检测结果阳性标本进行真菌微生物培养,验证分型结果的正确率.结果 荧光PCR核酸扩增法和常规镜检一致性检验Kappa值为0.632,二者一致性差.500例患者阴道分泌物常规镜检阳性标本,荧光PCR核酸扩增法测得白色念珠菌感染382例(76.4％),光滑假丝酵母菌感染73例(14.6％),热带假丝酵母菌感染10例(2.0％),白色念珠菌合并光滑假丝酵母菌感染3例(0.6％),其他真菌感染32例(6.4％).500例患者阴道分泌物常规镜检阴性标本,荧光PCR核酸扩增法鉴定阳性共152例,其中白色念珠菌130例,光滑假丝酵母菌16例,热带假丝酵母菌6例.荧光PCR核酸扩增法与CHROMAgar快速显色法检测结果比较,差异无统计学意义(x2 =0.131,P=0.936).结论 对于有临床表现而镜检阴性的患者,建议行荧光PCR核酸扩增快速分型鉴定或真菌培养鉴定.     

菌液SYBR实时荧光PCR快速检测金黄色葡萄球菌

目的建立菌液SYBR荧光PCR法快速检测金黄色葡萄球菌的方法。方法根据金黄色葡萄球菌的特异基因耐热核酸酶nuc设计引物,PCR方法和SYBR荧光PCR溶解曲线法验证引物的特异性;建立菌液SYBR荧光PCR快速检测方法,奶粉加标实验和菌株特异性实验评价方法的特异性,制备基因组浓度梯度和菌落梯度检验方法的灵敏性。结果利用引物对实验室的3个标准菌株进行PCR扩增,出现的条带均为单一条带,且与目的片段长度一致。16株金黄色葡萄球菌菌株的熔解曲线均呈单峰,特异性良好,无非特异性扩增。建立的菌液SYBR荧光PCR方法对金黄色葡萄球菌有很好的特异性和灵敏性,灵敏度达1pg／山DNA浓度,并可检测1 CFU／ml的复杂样品。结论本研究建立的菌液SYBR荧光PCR方法快速、特异性好、灵敏性高,对快速检测样品中金黄色葡萄球菌有重要意义。     

Culture shock. Molecular methods for diagnosis of infectious diseases

Nucleic acid detection methods, such as polymerase chain reaction (PCR), can often detect specific microbial pathogens, virulence markers and antimicrobial resistance genes more rapidly and with greater sensitivity and specificity than culture and conventional identification and susceptibility testing. Multiplex PCR can detect multiple genes in a single assay; this capability will be greatly extended by new techniques such as the DNA chip. However, limitations and pitfalls of nucleic detection methods remain.     

泉州市6例布鲁菌病的微生物生化鉴定及分子生物学确认

目的 验证布鲁菌的微生物鉴定和PCR检测结果的一致性. 方法 利用全自动细菌鉴定仪对血（骨髓）培养阳性的标本进行微生物鉴定,提取细菌DNA,应用布鲁菌通用引物BCSP31进行PCR扩增,产物进行测序. 结果 6例培养阳性的病原菌经鉴定为布鲁菌.经荧光PCR检测、测序结果在线blast分析确认为布鲁菌. 结论 细菌的分离培养结合PCR方法可以提高布鲁菌的检测.     

头孢噻肟处理的城市河流沉积物微生物群落结构及blaCTX-M多样性的研究

目的研究抗生素(头孢噻肟)作用下河流沉积物微生物群落结构的变化及耐药基因blaCTX-M多样性。方法应用基于16SrRNA的末端限制性酶切片段长度多态性(T-RFLP)技术,分析分别在0、6.4、64和320mg的头孢噻肟作用下,城市河流底泥细菌群落结构变化;并通过巢式PCR,分析其耐药基因blaCTX-M的多样性变化。结果T-RF特征峰统计分析表明:不同抗生素浓度处理,与底泥细菌群落结构多样性变化之间无显著相关性;但实验室处理时间会导致群落结构产生显著变化。PCR结果表明:随实验室处理时间的延长,耐药基因blaCTX-M多样性下降。结论对已承受排放废水污染的河流沉积物,由于其中富含大量的耐药菌群,其总体菌群结构对抗生素的作用不敏感。同时,对非原位开展的菌群结构研究,实验室处理时间是重要的影响因素。     

Rapid detection of coliforms in drinking water of Arak city using multiplex PCR method in comparison with the standard method of culture (Most Probably Number)

ObjectiveTo analyse molecular detection of coliforms and shorten the time of PCR.MethodsRapid detection of coliforms by amplification of lacZ and uidA genes in a multiplex PCR reaction was designed and performed in comparison with most probably number (MPN) method for 16 artificial and 101 field samples. The molecular method was also conducted on isolated coliforms from positive MPN samples; standard sample for verification of microbial method certificated reference material; isolated strains from certificated reference material and standard bacteria. The PCR and electrophoresis parameters were changed for reducing the operation time.ResultsResults of PCR for lacZ and uidA genes were similar in all of standard, operational and artificial samples and showed the 876 bp and 147 bp bands of lacZ and uidA genes by multiplex PCR. PCR results were confirmed by MPN culture method by sensitivity 86% (95% CI: 0.71-0.93). Also the total execution time, with a successful change of factors, was reduced to less than two and a half hour.ConclusionsMultiplex PCR method with shortened operation time was used for the simultaneous detection of total coliforms and Escherichia coli in distribution system of Arak city. It's recommended to be used at least as an initial screening test, and then the positive samples could be randomly tested by MPN.     

耐亚胺培南鲍曼不动杆菌碳青霉烯酶检测及同源性分析

目的 了解临床分离亚胺培南耐药鲍曼不动杆菌(IRAB)碳青霉烯酶的基因型及同源性.方法 采用MicroScan WalkAway-40微生物分析仪进行菌株鉴定和药敏试验,聚合酶链反应(PCR)检测OXA-23、OXA-24、OXA-51、OXA-58组碳青霉烯酶基因型,肠杆菌科重复序列PCR(ERIC-PCR)检测耐药基因阳性菌株的同源性.结果 64株IRAB均为多重耐药菌株,其中10株(15.6%)为泛耐药菌株,51株(79.7%)blaOXA-23阳性,4株(6.3%)blaOXA-58阳性,57株(89.1%)blaOXA-66/blaOXA-51组阳性.ERIC-PCR结果 显示blaOXA-23阳性菌株中47株为同一基因谱.结论 产OXA-23型碳青霉烯酶是我院鲍曼不动杆菌对亚胺培南耐药的重要机制,菌株间可能存在克隆传播.     

Identification of a New Peptide Deformylase Gene From Enterococcus faecium and Establishment of a New Screening Model Targeted on PDF for Novel Antibiotics

INTRODUCTION Vancomycin is the drug of the last resort for treating resistant Gram-positive bacterial infections, and the emergence of vancomycin-resistance Enterococci (VRE) presents a serious threat to public health[1]. Till now there has been no effect…     

耐亚胺培南铜绿假单胞菌CARB-3基因的发现

目的分析我院2005年1~12月临床样本中分离的34株耐亚胺培南铜绿假单胞菌CARB基因携带情况,并对CARB基因分型。方法应用BD phoenix 100全自动微生物分析仪鉴定临床分离的铜绿假单胞菌并筛选耐亚胺培南的铜绿假单胞菌。采用PCR方法筛查耐亚胺培南铜绿假单胞菌的CARB基因,用DNA测序法对阳性扩增产物进行分型。结果34株耐亚胺培南的铜绿假单胞菌中,有32.4%携带CARB基因,测得DNA序列进行BLASTn比对,基因型为CARB-3型。结论我院临床样本中分离的耐亚胺培南的铜绿假单胞菌携带CARB-3型基因,携带率较高,医务工作者应重视和加强耐亚胺培南的铜绿假单胞菌的防治和耐药监测。     

Identification of a new peptide deformylase gene from enterococcus faecium and establishment of a new screening model targeted on PDF for novel antibiotics

Objective To identify a new peptide deformylase (PDF) gene (Genebank Accession AY238515) from Enterococcus faecium and to establish a new screening model targeted on PDE Methods A new PDF gene was identified by BLAST analysis and PCR and was subsequently over-expressed in the prokaryotic expression host E.coli B121(DE3). Over-expressed protein was purified for enzymatic assay by metal affinity chromatography and a new screening model was established for novel antibiotics. Result A new PDF gene of Enterococcus faecium was identified successfully. Ten positive samples were picked up from 8000 compound library and the microbial fermentation broth samples. Conclusion A new PDF of gene Enterococcusfaecium was first identified and the model had a high efficacy. Positive samples screened may be antibacterial agents of broad spectrum.     

The role of DNA amplification technology in the diagnosis of infectious diseases

Nucleic acid amplification and detection methods developed in the past decade are useful for the diagnosis and management of a variety of infectious diseases. The most widely used of these methods is the polymerase chain reaction (PCR). PCR assays can detect rapidly and accurately the presence of fastidious and slow-growing microorganisms, such as Chlamydia, mycoplasmas, mycobacteria, herpesviruses and enteroviruses, directly from clinical specimens. Commercial PCR assays for the diagnosis of tuberculosis and genital C. trachomatis infection are now routinely used in many diagnostic laboratories. Assays have also been developed that can detect antimicrobial resistance and are used to identify the cause of infection by organisms that cannot be cultivated. The value of viral load measurement by nucleic acid amplification in the management of patients with HIV infection or hepatitis C has also been well established. However, evaluations of this technology for rapid microbial diagnosis have generally been limited by small samples, and the cost of these assays may be as high as Can$125 per test. As nucleic acid amplification methods continue to evolve, their role in the diagnosis and management of patients with infectious diseases and their impact on clinical outcomes will become better defined.     

Detection of Tumor Markers in Prostate Cancer and Comparison of Sensitivity between Real Time and Nested PCR

The objective of this study is to investigate and compare the sensitivity in conventional PCR, quantitative real time PCR, nested PCR and western blots for detection of prostate cancer tumor markers using prostate cancer (PCa) cells. We performed conventional PCR, quantitative real time PCR, nested PCR, and western blots using 5 kinds of PCa cells. Prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), and androgen receptor (AR) were compared for their detection sensitivity by real time PCR and nested PCR. In real time PCR, there was a significant correlation between cell number and the RNA concentration obtained (R2=0.9944) for PSA, PSMA, and AR. We found it possible to detect these markers from a single LNCaP cell in both real time and nested PCR. By comparison, nested PCR reached a linear curve in fewer PCR cycles than real time PCR, suggesting that nested PCR may offer PCR results more quickly than real time PCR. In conclusion, nested PCR may offer tumor maker detection in PCa cells more quickly (with fewer PCR cycles) with the same high sensitivity as real time PCR. Further study is necessary to establish and evaluate the best tool for PCa tumor marker detection.     

Endodontic bacteria from primary and persistent endodontic lesions in Chinese patients as identified by cloning and 16S ribosomal DNA gene sequencing

Background Few literatures pertain to the 16S ribosomal DNA (16S rDNA) analysis of bacteria contributing to primary and persistent endodontic lesions,with no information available for the Chinese popul...     

宏基因组测序在临床重症感染患者寻找病原体中的应用

  目的  应用宏基因组测序检测重症患者感染病原体,了解在重症患者中的应用对寻找感染病原体的帮助。  方法  对1例表现为腹痛、逐渐进展至意识障碍合并严重感染的急性重症胰腺炎患者,用传统方法检测病原体的同时应用宏基因组测序。传统方法包括:常规涂片、培养,VitekII Compact全自动微生物鉴定和药敏分析,实时荧光定量PCR检测病毒载量,以及免疫组织化学等病理检查。宏基因组测序则采用基于BGISEQ-100高通量测序平台的二代测序,剔除人类宿主序列后,将保留下来的序列与包含病毒、细菌、真菌和寄生虫基因组序列的微生物基因组数据库进行比对,确定标本中所有的病原体。  结果  传统检验发现该患者感染了耐甲氧西林的金黄色葡萄球菌、耐碳青霉烯的肺炎克雷伯菌和耐碳青霉烯的鲍曼不动杆菌。宏基因组测序结果不仅检出了前述病原体,还检查出了加重疾病恶化的巨细胞病毒（CMV）,耗时约5 d。实时荧光定量PCR补充病毒载量检测证实其CMV载量为189 000 copies/mL。  结论  在本例重症患者中,宏基因组测序一次性快速准确检出了3种病原菌和1种病毒,可辅助重症患者感染病的快速诊断。