实时荧光PCR在呼吸道感染耐甲氧西林金黄色葡萄球菌检测中的临床应用

目的评价实时荧光PCR(RT-PCR)用于检测呼吸道感染耐甲氧西林金黄色葡萄球菌(MRSA)中的方法学特性,为临床快速诊断呼吸道MRSA定植或感染提供可靠循证依据。方法通过RT-PCR技术对痰液标本中金黄色葡萄球菌(SA)特异性核酸酶编码基因nuc和MRSA的特异性耐药基因mec A进行定量检测,并同时以细菌培养、鉴定和MRSA耐药表型确诊实验作为参考方法,评价其灵敏度、特异度、阴阳性预测能力;用10倍梯度稀释法配制已知标准菌株菌悬液评估RT-PCR的最低检出限。结果 RT-PCR法较细菌培养法对MRSA检测的总符合率为97.5%,检测SA的敏感度为97.2%、特异度为98.2%、阳性预测值为92.1%、阴性预测值为99.4%,检测甲氧西林耐药性的敏感度为100%、特异度为97.7%、阳性预测值为85.2%、阴性预测值为100%;RT-PCR对nuc基因和mec A基因的最低检出限均为103/m L。结论RT-PCR法与细菌培养法对于检测MRSA差异无统计学意义,且灵敏度优于后者,是用于临床快速排除性筛查呼吸道感染MRSA的较佳检测方法。     

四种方法检测乙型肝炎病毒基因型的比较

目的比较四种方法检测乙型肝炎病毒基因型的差异性。方法对36例乙型肝炎患者的血清分别采用测序技术,荧光定量PCR技术,恒温扩增技术,基因芯片技术进行乙肝病毒基因型的检测。结果36份血清以测序技术为金标,观测荧光定量PCR技术特异性100%,敏感性83%,恒温扩增技术特异性100%,敏感性89%,基因芯片技术特异性100%,敏感度92%。结论目前临床采用的四种方法检测乙型肝炎病毒基因型的特异性相同,敏感度以测序为金标准,基因芯片技术最灵敏,其次为恒温扩增技术,最后为荧光定量PCR技术。     

基于LAMP技术建立解脲支原体基因试纸条检测方法

目的建立一种灵敏度高、特异性强、检测速度快的方法检测解脲支原体。方法基于环介导恒温扩增技术（LAMP）,根据解脲支原体序列特征设计3对引物进行解脲支原体DNA切口酶核酸恒温扩增,扩增过程在一对引物中标记生物素,随着扩增的进行生物素直接引入扩增片段中,扩增结束后产物在密闭装置中进行免疫试纸条显色反应,根据显色卡的颜色判定结果的阴阳性。结果该技术检测解脲支原体较实时荧光PCR技术灵敏度要高10倍以上,其它病原体检测均阴性该方法特异性与实时荧光PCR技术相当。结论恒温扩增联合试纸条技术检测解脲支原体具有较高的敏感性和特异性,检测速度快,适合各医院开展。     

基于锁核酸及等位位点特异性扩增技术的KRAS基因突变检测荧光定量PCR方法研究

基于等位位点特异性扩增的原理,设计锁核酸修饰KRAS基因突变特异性扩增引物,结合封阻探针技术,建立检测KRAS基因突变的荧光定量PCR方法。结果发现,锁核酸修饰的引物及探针可显著提高等位位点特异性扩增技术用于复杂样本中的微量基因突变检测的敏感度,该技术检测KRAS基因突变的敏感性可达0.01%~0.1%。进一步用建立的荧光定量PCR方法检测52例结直肠癌患者血浆标本,并用DNA测序法作为对照,同时用健康人血浆标本建立阴性检测结果判读标准,以初步评价该方法应用于循环DNA中KRAS基因突变检测的可行性。结果发现结直肠癌患者KRAS基因突变主要是G12C、G12A和G12R,而且q PCR法的阳性检出率为46.15%,高于DNA测序法(13.46%),阴性结果与DNA测序法的符合率为100%。此外,结直肠癌患者外周血KRAS基因的突变检出率与文献报道组织标本中的突变检出率及常见突变类型基本相符。上述结果说明该方法检测循环肿瘤DNA(circulating tumor DNA,ct DNA)具有较高的可靠性,可以用于肿瘤患者循环血液中KRAS基因突变的检测。     

PCR/16sRNA联合核苷酸测序法检测化脓性脑膜炎病原菌的临床价值

目的:探讨PCR/16sRNA联合核苷酸测序法在化脓性脑膜炎病原菌检测中的临床诊断价值。方法:选择2016年4月至2017年2月上海儿童医学中心临床考虑中枢感染的43例化脓性脑膜炎患儿的脑脊液标本,所有患儿标本同时进行培养,并行PCR/16sRNA联合核苷酸测序法检测,记录检测结果,并统计检测方法的灵敏度和特异度,以脑脊液培养检测结果为金标准,对比脑脊液培养和PCR/16sRNA联合核苷酸测序法的灵敏度和特异度。结果:脑脊液培养的灵敏度为21.7%,特异度为100.0%;PCR/16sRNA联合核苷酸测序的灵敏度为69.6%,特异度为95.0%;两者的灵敏度比较差异具统计学意义(P0.05),而两者特异性比较差异无统计学意义(P0.05)。PCR/16sRNA联合核苷酸测序可检出脑脊液培养阴性的病原体。结论:PCR/16sRNA联合核苷酸测序具有较高的灵敏度,可检出脑脊液培养阴性的病原体,且受抗菌药物影响小,可为临床早期提供化脓性脑膜炎的病原学依据,降低致死率及致残率。     

大鼠冠状病毒和仙台病毒的双重PCR检测方法的建立与应用

目的建立快速检测实验大鼠冠状病毒和仙台病毒的双重PCR方法。方法根据大鼠冠状病毒N基因、仙台病毒L基因设计特异性引物;经过双重PCR优化,特异性和敏感性的检测,建立双重PCR体系。应用该PCR体系检测人工感染仙台病毒组织DNA样本和实验动物组织样本,并与ELISA方法比对。结果双重PCR扩增出大鼠冠状病毒(168 bp)和仙台病毒(262 bp)目的条带,PCR扩增产物测序结果利用核酸BLAST功能进行同源序列对比,仙台病毒和大鼠冠状病毒同源性分别为100%和99%。仙台病毒和大鼠冠状病毒的检测下限为1.56×10~2 copies/μL。特异性检测对小鼠肝炎病毒扩增,产生片段大小近似大鼠冠状病毒产物。应用建立的双重PCR体系检测人工感染仙台病毒组织DNA样本,30份DNA标本均被检出;检测94份实验动物肺组织样本,结果均阴性。结论建立的双重PCR方法操作简单、快速、特异性强、灵敏度高,能够实现对实验动物仙台病毒和大鼠冠状病毒病原体的快速检测。     

RNA实时荧光核酸恒温扩增检测技术 检测解脲脲原体的Meta分析

目的评估RNA实时荧光核酸恒温扩增(simultaneous amplification and testing,SAT)检测技术检测泌尿生殖道拭子及尿液样本中解脲脲原体(Ureaplasma urealyticum,UU)阳性率。方法检索中国学术期刊全文数据库(CNKI)、万方数据库、维普数据库、PubMed、ScienceDirect和Cochrane图书馆等,收集有关探讨实时荧光核酸恒温扩增检测UU阳性率的所有文献,检索起止时间为1990年1月至2018年8月。拟定入选标准和排除标准,2名研究者分别独立进行文献资料提取及文献质量评估,采用Stata 13.0软件进行Meta分析。结果共检索出57篇文献,依据入选标准和排除标准,纳入了21篇文献进行分析,包含了33 176例尿液及拭子样本量,所有标本均运用实时荧光核酸恒温扩增对检测样本进行UU检测。Meta分析结果显示,实时荧光核酸恒温扩增检测UU阳性率的汇总检出率(95%CI)为0.44 (0.39-0.49)。结论实时荧光核酸恒温扩增检测UU具有快速、灵敏的特点,很好的判断预后效果,适合临床实验室泌尿生殖道患者UU检测,值得临床进一步推广应用。     

ChromID ESBL选择性显色平板对产ESBLs肠杆菌科细菌筛选效果的评价

目的评价ChromID ESBL选择性显色平板对产超广谱β-内酰胺酶(ESBLs)肠杆菌科细菌的筛选效果。方法选取临床分离的371株肠杆菌科细菌进行ESBLs的检测,用ChromID ESBL选择性显色平板做筛选试验,同时用纸片确证法做确证试验,采用Kappa检验对两者结果进行一致性分析。结果两种方法对371株肠杆菌科细菌ESBLs检测的总Kappa值为0.761。ChromID ESBL选择性显色平板法的总灵敏度为95.2%,总特异度为83.5%。对大肠埃希菌、肺炎克雷伯菌、奇异变形杆菌分别统计,其Kappa值分别为0.832、0.514、0.778;ChromID ESBL选择性显色平板法对其检测灵敏度分别为95.7%、91.3%、100.0%,特异度分别87.6%、72.9%、90.9%。另外有6株大肠埃希菌在该显色平板上显非特征色。结论 ChromID ESBL选择性显色平板对产ESBLs肠杆菌科细菌的筛选效果与纸片确证法总体的一致性较好,其灵敏度和特异度较高,可应用于临床。但该显色平板对产ESBLs肺炎克雷伯菌的筛选效果与纸片确证法一致性一般,其特异度也较低;对大肠埃希菌存在一定的假阴性,对于该显色平板上生长的绿色和白色菌落需要作进一步鉴定确认。     

高灵敏可视化核酸试纸条法快速检测HBV DNA

目的：本研究旨在建立一种基于试纸条的快速、灵敏及可视化检测乙型肝炎病毒核酸的方法。方法：利用聚合酶链反应扩增乙肝病毒的保守区,其中上、下游引物的5＇端分别修饰异硫氰酸荧光素和生物素。核酸试纸条上的胶体金以及检测线处分别标记有链霉亲和素以及抗荧光素抗体。将扩增产物与展开液混合后点样,10 min后即可用肉眼判读结果。在优化了展开液成分、上样体积以及上样浓度之后,对该方法的灵敏度进行了评价。最后收集15例阴性样本及33例HBsAg阳性样本,按血清标志物结果进行分类后使用核酸试纸条进行检测,并与实时荧光PCR的结果进行了比较。结果：试纸条检测乙肝病毒核酸的灵敏度为250copies/mL。在临床样本的测定中,该方法与实时荧光定量PCR的特异性均为100%。且两种方法检测不同血清标志物类型的阳性检出率无差异。结论：核酸试纸条技术能够用于乙肝病毒核酸的可视化检测,与实时荧光PCR相比检测速度快,具有较好的灵敏度和特异性,适合流行病学调查以及在基层医院体检使用。     

多酶恒温扩增核酸试纸条法快速检测肺炎支原体方法的建立及应用

【背景】肺炎支原体是导致儿童和青少年呼吸道感染的重要病原体,长期以来由于其临床表现不特异而容易错过最佳治疗时期。【目的】结合多酶恒温扩增(multienzyme isothermal rapid amplification,MIRA)技术和核酸试纸条建立一种快速检测肺炎支原体的方法。【方法】以肺炎支原体社区获得性肺炎呼吸窘迫综合征(community acquired respiratory distress syndrome, CARDS)毒素编码基因为靶基因设计引物和探针,对反应体系的温度、时间等进行优化,评估其敏感性,通过检测肺炎支原体和其余7种病原体分析其特异性,并对35份临床样本进行验证。【结果】MIRA核酸试纸条法在37℃条件下,15 min内便可完成对肺炎支原体的检测,最低检出限为10 copies/μL;除肺炎支原体外,其余7种病原体均不能扩增,特异性较好。以实时荧光PCR检测为标准,MIRA核酸试纸条法对35份临床样本检测后的诊断特异度为100.00%、灵敏度为96.15%、阴性预测值为90.00%、阳性预测值为100.00%。【结论】本研究建立了MIRA核酸试纸条法...     

Detection of methicillin-resistant <Emphasis Type="BoldItalic">Staphylococcus aureus</Emphasis> (MRSA) from nasal samples by multiplex real-time PCR based on dual priming AT-rich primers

In this study, we reported on the design of a multiplex real-time PCR assay based on SYBR Green I, incorporating dual priming adenine-thymine (AT)-rich primers for direct detection of MRSA from nasal samples. The multiplex real-time polymerase chain reaction (RT-PCR) assay reported in this study is based on SYBR Green I with incorporation of six dual priming AT-rich primers designed from the SCCmec/orf junction. A string (4–6 bp) of low-melting bases, such as adenine and thymine, was incorporated into the primers, which virtually divided a single primer in two functional regions, thus decreasing non-specific PCR products. The analytical sensitivity and specificity of the RT-PCR assay was determined with genomic DNA of reference strains (MRSA, MSSA, and MRCoNS). RT-PCR assay was performed for analysis of 72 nasal swab specimens, and the results were confirmed by use of a culture method. Furthermore, the results of RT-PCR were compared with LightCycler MRSA advance test. The multiplex RT-PCR assay reproducibly detected a minimum of 1 pg genomic DNA (31.5 copy of genome) of MRSA reference strains and clinical isolates, with a specific melting peak at 83.5 ± 1.5°C, and neither fluorescence nor a melting peak was detected in non-target isolates. The concordance rate between RT-PCR assay and culture method was 87.5% with Cohen's kappa value (κ) 0.75, which showed good agreement between the two assays. The sensitivity, specificity, positive predictive value, and negative predictive value of the assay were 93.5%, 82.9%, 80.5%, and 94.4%, respectively. In a comparative study for the detection of 72 nasal samples, the sensitivity, specificity, positive predictive value, and negative predictive value of the multiplex RT-PCR assay with respect to LightCycler MRSA advance test was 84.2%, 88.2%, 89%, and, 83.3%, respectively. The results of RT-PCR assay demonstrated high specificity (88.2%) and positive predictive value (89%) for the direct detection of MRSA from nasal samples.     

Evaluation of a rapid culture-based screening test for detection of methicillin resistant Staphylococcus aureus

The performance of a culture based assay, BacLite Rapid MRSA for the rapid detection (5 hours) of methicillin resistant Staphylococcus aureus (MRSA) from specimens (n = 377) obtained from nares, throat, wounds and perineum was investigated. Compared to culture based reference methods (chromogenic MRSA ID (bioMerieux)), selective enrichment broth, PBP2' latex agglutination (Oxoid) and VITEK 2 identification (bioMerieux), an overall sensitivity of 71% with a 82% specificity and a negative predictive value (NPV) of 95% was provided. The Baclite test is rapid and easy to use and has the advantage of a culture-based detection method for MRSA.     

实时荧光核酸恒温检测技术（SAT）在甲型流感病毒动物模型检测中的应用

评价实时荧光核酸恒温检测技术（SAT）对H1N1和H3N2两种甲型流感病毒小鼠模型肺组织匀浆液的检测效能。小鼠滴鼻感染不同浓度H1N1、H3N2病毒液30μL,感染96h后取肺组织匀浆液分别进行SAT检测及抗原血凝效价测定。结果表明,SAT检测的病毒拷贝数基本与抗原血凝效价测定结果一致,但灵敏度提高10^4倍。与血凝实验比较,SAT检测具有灵敏度高、特异性强、结果准确、高通量自动化的优势,有利于甲型流感动物模型的建立,为实验室筛选抗甲型流感病毒药物奠定基础。     

Characterization of the substitution pattern of cellulose derivatives using carbohydrate-binding modules

Background

Methicillin-resistant Staphylococcus aureus (MRSA) has become one of the most prevalent pathogens responsible for nosocomial infections throughout the world. As clinical MRSA diagnosis is concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for MRSA detection. This study aimed at developing a simple loop-mediated isothermal amplification (LAMP) assay targeting on orfX for the rapid detection of methicillin-resistance Staphylococcus aureus (MRSA).

Results

The protocol was designed by targeting orfX, a highly conserved open reading frame in S. aureus. One hundred and sixteen reference strains, including 52 Gram-positive and 64 Gram-negative isolates, were included for evaluation and optimization of the orfX-LAMP assay. This assay had been further performed on 667 Staphylococcus (566 MRSA, 25 MSSA, 53 MRCNS and 23 MSCNS) strains and were comparatively validated by PCR assay using primers F3 and B3, with rapid template DNA processing, simple equipments (water bath) and direct result determination (both naked eye and under UV light) applied. The indispensability of each primer had been confirmed, and the optimal amplification was obtained under 65°C for 45 min. The 25 μl reactant was found to be the most cost-efficient volume, and the detection limit was determined to be 10 DNA copies and 10 CFU/reaction. High specificity was observed when orfX-LAMP assay was subjected to 116 reference strains. For application, 557 (98.4%, 557/566) and 519 (91.7%, 519/566) tested strains had been detected positive by LAMP and PCR assays. The detection rate, positive predictive value (PPV) and negative predictive value (NPV) of orfX-LAMP were 98.4%, 100% and 92.7% respectively.

Conclusions

The established orfX-LAMP assay had been demonstrated to be a valid and rapid detection method on MRSA.     

Simultaneous detection of six human diarrheal pathogens by using DNA microarray combined with tyramide signal amplification

Multiplex PCR and DNA microarray were combined with tyramide signal amplification (TSA) to develop a reliable method suitable for simultaneous detection of six species of human diarrheal pathogens (Yersinia enterocolitica, Shigella spp, Salmonella typhi, Brucella spp, Vibrio cholera and Escherichia coli O157:H7). Meanwhile, our method could distinguish V. cholera serotype O1 from O139, and O157:H7 from O157: non-H7. This assay conferred a specificity of 100% for target pathogens. The limit of detection was 103 degrees CFU/mL approximately. The results of 98.6% (357/362) clinical specimens and 100% (5/5) mocked double-blind samples were the same to that from conventional assay. Consequently this assay is sensitive and a specific tool suitable for diagnostic detection and surveillance of multiple human pathogens.     

炭凝集试验快速检测呼吸道合胞病毒的研究

用炭凝集试验（ＣＡＴ）检测呼吸道合胞病毒（ＲＳＶ）,结果表明该法是一种简便、快速、特异的诊断方法。用ＣＡＴ对１６株ＲＳＶ和８株其它病毒做试验,结果仅ＲＳＶ凝集,而其它病毒均阴性。用该法与细胞培养法检测８３份临床呼吸道感染幼儿鼻咽吸出物,结果ＣＡＴ法阳性率为６９．８８％（５３／８３）,细胞培养法为３９．７５％（３３／８３）,两者阳性检出率相差极显著。阻断试验证明ＣＡＴ是高度特异的。结果证明ＣＡＴ具有较高的敏感性与特异性,可用于临床ＲＳＶ标本的快速检测。     

A rapid immunochromatographic assay for the detection of Mycobacterium tuberculosis antigens in pulmonary samples from HIV seropositive patients and its comparison with conventional methods

As tuberculosis generates a highly heterogeneous antibody repertoire, its diagnosis requires tests based on cocktails of antigens. We describe a new, rapid method called rapid immunochromatographic assay (RICA) for cocktail-based diagnosis, which can detect Mycobacterial antigens in sputum specimens. Six antigenic fractions of pathogenic Mycobacterium tuberculosis were used in combination as the capture antigens in the control line of the flow-through assay. Antigen detection of 200 sputum samples from HIV seropositive patients by RICA assay gave a sensitivity of 97.9%, specificity of 99.0%, positive predictive value of 98.9%, negative predictive value of 98.0%, false positive rate of 0.9%, false negative rate of 2.0%, prevalence rate of 49%, likelihood ratio for positive results 97 and likelihood ratio for negative results 0.02. The combination of RICA and AFB staining gave a sensitivity of 100%, specificity of 100%, positive predictive value of 100%, negative predictive value of 100%, false positive rate of 0%, false negative rate of 0%, likelihood ratio for negative results 0. The assay was simple, rapid and economical for the detection of M. tuberculosis infection and suitable for large scale screening of samples in endemic areas without any sophisticated equipment. The results of the assay proved to be superior to conventional methods and combined with clinical data, could form the basis for starting an earlier course of treatment.     

Detection of methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> using double duplex real-time PCR and dye Syto 9

A screening method for methicillin-resistant Staphylococcus aureus (MRSA) using real-time polymerase chain reaction (PCR) and dye Syto 9 was developed and evaluated. The assay was based on the two duplex reactions run simultaneously. The detection reaction amplified staphylococcal cassette chromosome mec (SCCmec) right extremity sequences and S. aureus-specific 442-bp DNA (Sa442). The control reaction amplified S. aureus-specific nuclease gene nuc and a marker of methicillin resistance, mecA. The method was evaluated by analyzing 214 clinical S. aureus isolates yielding 98.7 % sensitivity, 100 % specificity, 100 % positive predictive value and 96.6 % negative predictive value for detection of MRSA. The detection limit was determined to be 15–80 genome copies per real-time PCR. It was able to discriminate between MRSA, methicillin resistant coagulase negative staphylococci and methicillin susceptible S. aureus (MSSA) isolates containing only small fragments of the right extremity of the SCCmec (MSSA revertants).