建立检测血液中EC、KP、SA和MRSA的多重PCR方法

目的建立快速检测血液中常见致病菌包括大肠埃希菌(EC)、肺炎克雷伯菌(KP)、金黄色葡萄球菌(SA)及耐甲氧西林SA(MRSA)的多重聚合酶链反应(PCR)方法,有利于败血症的及时治疗。方法建立多重PCR方法对EC phoA基因、KP mdh基因、SA femA基因和MRSA mecA基因进行检测,并将16SrDNA作为致病菌感染对照。结果采用建立的多重PCR方法进行检测的特异性为100%,EC检测限为2.75×102CFU/mL,KP为2.43×103 CFU/mL,SA为3.13×102 CFU/mL,MRSA为3.03×102 CFU/mL;用多重PCR方法和传统血培养方法对300个血标本进行检测,传统血培养方法检测出187个血标本内阳性,多重PCR方法有4个血标本为假阴性,未能被检出。结论建立的多重PCR方法可简便、及时地检测血流感染中的EC、KP、SA和MRSA。     

三种食源性致病菌多重PCR检测方法的建立及初步应用

目的建立快速检测沙门菌、变形杆菌和金黄色葡萄球菌的多重聚合酶链反应(PCR)方法。方法本研究依据沙门菌侵袭基因正调节蛋白(hilA)基因、变形杆菌溶血素(hpmA)基因和金黄色葡萄球菌特异性pSa-442序列,运用Primer Premier 5.0分别设计3对特异性引物,预计PCR扩增的目的基因片段分别为为580bp、401 bp、256 bp。通过对单管多重PCR扩增的特异性、敏感性分析以及建立L16(43)正交试验对单管多重PCR扩增条件如引物浓度、dNTP浓度和Tm值等的优化,建立了快速同时检测3种食源性致病菌的单管多重PCR方法。结果该方法检测的灵敏度分别为:94.07 pg沙门菌DNA,140.85 ng变形杆菌DNA,1.41 ng金黄色葡萄球菌DNA。模拟检测食品中的混合3种菌,4 h培养后样品的最低检测限度分别为:沙门菌100菌落形成单位(CFU)/mL、变形杆菌101CFU/mL、金黄色葡萄球菌100CFU/mL。结论该方法特异性和灵敏度高,检验周期短,可用于对食品中多种致病菌的快速诊检和监控。     

三种食源性致病菌多重PCR检测方法的建立及初步应用

目的建立快速检测沙门菌、变形杆菌和金黄色葡萄球菌的多重聚合酶链反应（PCR）方法。方法本研究依据沙门菌侵袭基因正调节蛋白（hilA）基因、变形杆菌溶血素（hpmA）基因和金黄色葡萄球菌特异性pSa-442序列,运用PrimerPremier5．0分别设计3对特异性引物,预计PCR扩增的目的基因片段分别为为580bp、401bp、256bp。通过对单管多重PCR扩增的特异性、敏感性分析以及建立L16（4^3）正交试验对单管多重PCR扩增条件如引物浓度、dNTP浓度和Tm值等的优化,建立了快速同时检测3种食源性致病菌的单管多重PCR方法。结果该方法检测的灵敏度分别为：94．07pg沙门菌DNA,140．85ng变形杆菌DNA,1．41ng金黄色葡萄球菌DNA。模拟检测食品中的混合3种菌,4h培养后样品的最低检测限度分别为：沙门菌10^0菌落形成单位（CFU）／mL、变形杆菌10^1CFU／mL、金黄色葡萄球菌10^0CFU／mL。结论该方法特异性和灵敏度高,检验周期短,可用于对食品中多种致病菌的快速诊检和监控。     

实时定量PCR在血流感染病原体快速检测中的应用

目的探讨实时定量PCR在血流感染病原体检测中的临床应用价值。方法选取收治的80例患者共92份血液标本进行实时定量PCR检测,同时进行血液培养,比较两种方法的特异度和敏感度。结果在92份标本当中,两种方法共同阴性标本66份(71.7%),两种方法共检测出病原体10种。实时定量PCR和血培养共同检出阳性标本7例,两种方法的一致性为79.3%。实时定量PCR的阴性预测值是0.94,敏感度是0.64,特异度是0.82。其中15份标本实时定量PCR阳性而血培养阴性,4份标本血培养阳性而实时定量PCR阴性。其中2份标本所培养出的病原体不在实时定量PCR的检测范围内,且实时定量PCR也不能检测光滑念珠菌。结论实时定量PCR是快速检测血液感染标本的有价值方法,但不能完全替代血培养。     

利用实时荧光定量-聚合酶链反应方法检测粪便标本中的伤寒沙门菌

目的 建立针对粪便标本中伤寒沙门菌的检测方法,评价该方法的特异性、敏感性及检测下限,以提高感染人群粪便标本中伤寒沙门菌的检出率。 方法 根据伤寒沙门菌特异基因STY1633设计特异性引物,优化反应条件,建立实时荧光定量-聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction,qPCR)反应体系。利用92株常见的非伤寒病原菌及44株伤寒沙门菌的染色体DNA评价该方法的特异性和灵敏性,并对伤寒沙门菌的粪便模拟标本进行检测下限评价,同时利用10份伤寒患者粪便标本及48份其他病原所致发热和/或伴腹泻的患者粪便标本进行特异性、敏感性验证。 结果 利用本方法检测的伤寒沙门菌纯菌及伤寒患者标本均扩增阳性,其余的非伤寒沙门菌、致腹泻的其他5种肠道致病菌及引起发热症状的8种常见非肠道病原菌纯菌及相应患者标本均扩增阴性。在对纯伤寒沙门菌DNA检测中,qPCR法的最低检测限为500 fg/反应,相当于97个拷贝/反应。以粪便模拟样品提取DNA为模板的检测中,增菌前检测下限达104 cfu/g,增菌后可达50 cfu/g。 结论 基于STY1633基因的实时荧光定量PCR方法在检测粪便中的伤寒沙门菌中具有很好的特异性、灵敏度,为伤寒沙门菌的快速诊断及某些不明原因发热及腹泻症状的病原初步鉴定提供了新的简便型手段,对于伤寒的早期诊断及预防控制提供了技术支持。     

实时荧光定量PCR检测金黄色葡萄球菌方法的试验研究

目的建立金黄色葡萄球菌实时荧光定量PCR的快速检测方法,探讨该方法的可行性和应用价值。方法根据金黄色葡萄球菌femB基因序列设计引物和探针,采用基因重组技术构建用于金黄色葡萄球菌检测的定量标准品,建立实时荧光定量PCR检测金黄色葡萄球菌的方法。结果成功构建了金黄色葡萄球菌重组质粒标准品和金黄色葡萄球菌实时荧光定量PCR方法;通过特异性、敏感性、稳定性和重复性试验,结果表明具有较好的特异性、敏感性、稳定性和重复性;将模拟标本与分离培养对比,两者符合率为100%。结论金黄色葡萄球菌实时荧光定量PCR检测方法的建立,为金黄色葡萄球菌感染诊断及食源性金黄色葡萄球菌污染的快速检测提供依据,可用于临床感染诊断及食品卫生监管、商品检验检疫等。     

血培养标本阳性报警时间分析

目的分析本院检验科血培养仪检测阳性标本报警时间,寻找缩短血培养阴性报告时间的方法。方法收集长治市人民医院2018年6月—2019年1月住院时间为8个月患者的4 318份血液培养标本,采用法国梅里埃Bact/Alert 3D 60全自动血培养仪培养,统计阳性标本,记录所有阳性标本的阳性报警时间(TTP)并计算中位值,统计病原菌的种类及构成比。结果血培养阳性标本共475份,阳性率为11.0%;其中革兰阳性(G+)菌112份(占23.6%),革兰阴性(G-)菌361份(占76.0%),真菌2份(占0.4%)。G+菌中,葡萄球菌89份(18.7%)〔包括凝固酶阳性葡萄球菌51份(10.7%)、凝固酶阴性葡萄球菌38份(8.0%)〕,中位时间36.00 h;肠球菌17份(3.6%),中位时间14.40 h;链球菌6份(1.3%),中位时间36.48 h。G-菌中,大肠杆菌163份(34.3%),中位时间11.04 h;肺炎克雷伯菌91份(19.2%),中位时间11.43 h;非发酵菌99份(20.8%),包括铜绿假单胞菌67份(14.1%)、鲍曼不动杆菌32份(6.7%),中位时间17.04 h;其他G-菌8份(1.7%),中位时间26.32 h。真菌2份(0.4%),中位时间55.20 h。TTP最早为0.4 h,最迟为86.4 h,97.68%病原菌在72 h内阳性报警,100%病原菌在96 h内阳性报警。结论血培养阳性病原菌TTP由快到慢依次为大肠杆菌、肺炎克雷伯菌、肠球菌、非发酵菌、葡萄球菌、链球菌、真菌。所有病原菌均在4 d内报阳,因此考虑可将血培养阴性报告时间缩短为5 d,以节约医疗成本。     

血培养污染菌鉴别分析与对策

目的：回顾分析血培养病原菌分布构成,探讨污染菌的判定与对策,通过双侧采血及联合C-反应蛋白(CRP)、降钙素原(PCT)、内毒素等辨别污染菌,评价该方法对血培养污染菌判定的价值。方法回顾性统计该院2013年8月至2014年8月的血培养病原菌分布,同时检测双侧培养与单侧培养生长凝固酶阴性葡萄球菌(CNS)患者血标本中PCT、WBC、PALB、CRP以及内毒素水平。结果血培养总体阳性检出率为8.7％,主要为凝固酶阴性葡萄球菌;双侧血培养阳性检出率达3.6％,其中一侧生长的占10.2％;检测结果发现单侧培养生长CNS群体血液患者的PCT、白细胞(WBC)、前清蛋白(PALB)、CRP以及内毒素水平与双侧分离到同一病原菌群体比较有明显差异。结论血培养污染菌的辨别需要通过双侧采血送检及联合CRP、PCT、内毒素等检测,加强临床沟通,结合患者具体情况综合判断。     

荧光原位杂交法快速鉴定金黄色葡萄球菌和凝固酶阴性葡萄球菌

目的 建立一种新的荧光原位杂交法,快速鉴定血培养中的金黄色葡萄球菌(金葡菌)和凝固酶阴性葡萄球菌。方法 将荧光同位素标记的针对金葡菌和凝固酶阴性葡萄球菌的核糖体16SrRNA序列的互补的DNA寡核苷酸探针,在同一张玻片上50℃杂交1 h,用杂交缓冲液50℃洗涤10min,干燥后置荧光显微镜下观察,全过程大约2 h。结果 探针的特异性经标准菌株和临床分离菌株证实。将259份临床标本测试与培养法比较,金葡菌的特异性和敏感性均为100％,CoNs的特异性和敏感性分别为100％和95.5％,荧光原位杂交法的最低检出限为103CFU/ml。结论 本方法适用于血培养中的金葡菌和凝固酶阴性葡萄球菌的快速鉴定。     

实时荧光PCR快速检测耐甲氧西林金黄色葡萄球菌的临床评估

目的:采用实时荧光PCR检测耐甲氧西林金黄色葡萄球菌(MRSA),并评价该方法在临床上的应用价值。方法:将临床分离的62株金黄色葡萄球菌(金葡菌)和35株非金黄色葡萄球菌同时采用分离培养及药物敏感试验(药敏试验)和实时荧光PCR法进行检测并比较,对2种方法检测结果不符的菌株辅以测序法进行最后鉴定,并确定实时荧光PCR法的检测灵敏度。结果:分离培养法与实时荧光PCR法对金葡菌检测的一致率为100%,都检测出了62株金葡菌阳性株。实时荧光PCR法与分离培养药敏试验法MRSA检测的符合率为96.77%,而分离培养药敏试验法检测为阴性而实时荧光PCR法检测为阳性的2株待检菌的测序结果显示为MRSA阳性株。最后确定实时荧光PCR法检测MRSA的临床灵敏度为1×103 cfu/mL。结论:实时荧光PCR法可用于临床对MRSA的快速检测,具有较高的临床价值。     

DNA microarray-based identification of bacterial and fungal pathogens in bloodstream infections

The accurate and rapid identification of pathogens in blood is a major challenge in clinical pathogen diagnostics because of the high mortality of sepsis. Here we report the development of DNA microarray for the identification of pathogens causing bloodstream infections. Species-specific and bacteria- and fungi-broad-ranged probes were designed to identify 50 bacteria and 7 fungi. The specificities and sensitivities of the selected probes were successfully validated by applying reference strains. To assess the performance of the DNA microarray in a clinical setting, blind tests were performed using 112 blood culture specimens that showed preliminary presence of pathogenic microorganisms by culture-based method, resulting in the correct identification of pathogens in 104 samples showing the sensitivity of 93%. In addition, closely-related species could be discriminated by the distinct hybridization patterns. This DNA microarray-based pathogen diagnosis takes approximately 10 h starting from a positive blood culture, considerably reducing time required to sufficiently identify pathogens by subsequent agar-culture and biochemical tests which requires altogether at least 1–3 days. Also, the amount of sample required for the identification of pathogens is much less than that required for biochemical assays. Thus, the DNA microarray reported here should be useful for the effective identification of microbial pathogens in blood cultures from septicemic patients.     

The usefulness of multiplex PCR for the identification of bacteria in joint infection

Background: The diagnosis of septic arthritis (SA) relies on synovial analysis and conventional culture. But, these methods lack of sensitivity and culture is time consuming to establish a definite diagnosis. This study evaluated a new multiplex PCR assay which entailed screening PCR for Gram typing and identification PCR for species identification using two primer mixes. Methods: A total of 80 synovial fluid samples from patients with suspected SA were collected. Culture, multiplex PCR, and 16S rRNA gene PCR were performed. Results: The analytical sensitivity of multiplex PCR assay was 10¹ CFU/ml for each type of bacteria. There was no cross‐reactivity with common bacterial pathogens. Bacteria were detected in 20, 25, and 26 of 80 samples for culture, multiplex PCR, and 16S rRNA gene PCR, respectively. Nineteen (95%) of 20 culture‐positivesamples and 6 (10%) of 60 culture‐negative samples were positive for the multiplex PCR. Five of six samples which were positive only from multiplex PCR were also positive in 16S rRNA gene PCR. The multiplex PCR showed 2 false‐negative in 27 true‐positive samples but no false‐positive. The sensitivity and specificity of the multiplex PCR were 92.6 and 100%, and the agreement with culture and 16S rRNA gene PCR were 91.3 and 96.3%, respectively. The time to detection for multiplex PCR was a maximum of 6 hr. Conclusion: This multiplex PCR assay offers high sensitivity and improved detection speed relative to culture. The appropriate combination of this new multiplex PCR assay with culture may contribute to the accurate and rapid diagnosis of SA. J. Clin. Lab. Anal. 24:175–181, 2010. © 2010 Wiley‐Liss, Inc.     

The role of a real-time PCR technology for rapid detection and identification of bacterial and fungal pathogens in whole-blood samples

The rapid diagnosis of pathogens and prompt initiation of appropriate antibiotic therapy are critical factors to reduce the morbidity and mortality associated with sepsis. In this study, we evaluated a multiplex polymerase chain reaction (PCR-M) test that detects bacteria and fungi in whole-blood specimens, comparing its features to those of a blood culture (BC). Following evaluation of the performance for sensitivity and specificity of PCR-M, 78 blood samples from 54 patients with suspected bacterial infections were evaluated. Whole-blood samples for PCR-M were collected at the same time as BC, and PCR-M results were compared with BC results. As a result, minimum sensitivity of the kit was 1–100 cfu/ml. The PCR-M test correctly identified specificity for 13 out of 14 strains blinded to the assay analyst. Of 78 blood samples examined, 56 (72%) were negative by both methods, and 22 (28%) were positive by at least one of the two methods. PCR-M detected organisms in 21 cases (27%) compared with 12 cases (15%) in BC. The correlation of positives between PCR-M and BC was 92% (11/12), and both methods identified the same organisms in these 11 cases. With higher positive rate compared with BC, PCR-M could detect and identify potentially significant microorganisms within a few hours by using a small volume of a single whole-blood sample. Early detection of microorganisms has the potential to facilitate early determination of appropriate treatment and antimicrobial selection.     

广东台山HIV携带者 艾滋病患者血培养阳性病原菌种类及药敏分析

目的了解2009年1月至2011年10月广东台山人类免疫缺陷病毒（HIV）携带者和艾滋病（AIDS）患者血培养阳性病原菌种类及耐药情况,认真执行血培养的标准化操作。方法血培养用美国BD9050全自动血培养仪、法国生物梅里埃公司血培养双相瓶,鉴定、药敏用法国生物梅里埃公司ATB Expression自动细菌鉴定仪。结果 2009年1月至2011年10月46例血培养阳性的HIV携带者和AIDS患者病原菌种类包括22株革兰阳性球菌、10株革兰阴性杆菌、14株真菌,分别占47.8%、21.7%、30.5%;革兰阳性球菌、革兰阴性杆菌、真菌对抗菌药物基本敏感。结论 HIV携带者血培养阳性病原菌主要是金黄色葡萄球菌,AIDS患者血培养阳性病原菌主要是马尓尼菲青霉菌。     

宏基因组学第二代测序技术对比传统实验室微生物培养在脓毒症病原学诊断中的优势

目的通过对宏基因组学第二代测序技术(mNGS)获得的病原体与实验室培养结果进行对比,了解mNGS在脓毒症病原学诊断中的优势及其临床指导意义。方法将入选的脓毒症患者的标本(肺泡灌洗液、痰液、血液、脑脊液、胸水、腹水、分泌物等)同时送检mNGS和实验室细菌培养,对结果进行对比分析,评价mNGS在脓毒症病原学诊断方面的临床价值。结果mNGS的阳性率为78.9%;细菌培养的阳性率为40.4%(P<0.05)。通过mNGS共检出致病病原体57种,其中细菌31种,真菌16种,病毒7种,非典型病原体3种;细菌培养共检出病原体24种,其中细菌18种,真菌6种。以培养结果为金标准,mNGS的敏感度为76.2%,特异度为29.8%,阳性预测值为42.3%,阴性预测值为64.8%。根据病原学结果的抗生素调整将患者分为三组:按mNGS调整为mNGS组、经验性调整为经验组、按培养结果调整为传统培养组。mNGS组ICU住院时间更短(P<0.05),培养组降钙素原下降更明显(P<0.05)。结论mNGS在感染性疾病病原体的诊断方面较传统微生物培养时间更短,阳性率更高,在少见病原体、罕见病原体诊断方面有显著优势,可缩短患者ICU住院时间。     

厦门地区血培养病原菌分布及耐药性分析

目的了解厦门地区血培养检出的病原菌分布及其对抗菌药物的耐药情况，为临床合理选择抗菌药物提供依据。方法2107例血标本经BacT／ALERT 120全自动血培养仪培养检测，检出的病原菌采用VITEK 2 compact全自动微生物鉴定药敏仪进行细菌鉴定及药敏分析。结果共检出230株病原菌，阳性率为10．9％，其中革兰阳性球菌占38．7％，革兰阴性杆菌占51．7％，真菌占9．6％，葡萄球菌属和肠杆菌科细菌是厦门地区菌血症或败血症的主要病原菌。有43、6％的大肠埃希菌和26．7％的肺炎克雷伯菌产超广谱β-内酰胺酶。结论血培养病原菌种类复杂，耐药率较高，应重视血培养及对血培养病原菌耐药性的监测，合理使用抗菌药物。     

弥散黏附性大肠埃希菌多重PCR检测方法的建立及其在感染性腹泻患者中的流行情况

目的 建立一种快速检测弥散黏附性大肠埃希菌（DAEC）的普通多重PCR方法,了解DAEC在腹泻患者中的流行情况。方法 根据DAEC黏附基因afaB、afaC、afaD、daaE及16S rRNA基因rrs,建立4重PCR反应体系,对PCR引物、反应体系和反应条件进行优化,评价敏感性和特异性,并应用于389份腹泻患者粪便标本的筛查。结果 4重PCR反应可以特异性扩增DAEC菌株afaB/C、afaD、daaE基因片段,除2株肠集聚性大肠埃希菌（EAEC）外,检测引物对其他致泻性大肠埃希菌及常见肠道病原菌均无特异性扩增,此多重PCR方法的检测下限可达3.20 101 CFU/反应（8.01 103 CFU/ml）。利用本研究建立的多重PCR方法对腹泻患者粪便标本进行检测,发现DAEC菌株分离率为6.2%（24/389）。结论 本研究建立的多重PCR方法可用于DAEC菌株的快速鉴别,也可用于人粪便标本DAEC的初步筛查。     

High-throughput identification of clinically important bacterial pathogens using DNA microarray

Rapid and accurate detection of pathogenic bacteria is important for the treatment of patients with suitable antibiotics. Here we report the development of a diagnostic DNA microarray for the high-throughput identification of 39 pathogenic bacteria selected based on their high prevalence rate and/or difficulty of cultivation. The 23S ribosomal DNA and 16S–23S rDNA intergenic spacer region were used as target DNAs for pathogen detection. Universal- and species-specific probes were designed based on the unique and common sites within the target DNA sequences. New target DNA sequences were determined for the detection of 19 bacterial pathogens. The usefulness of the designed probes was validated using 39 reference bacteria and also with 515 clinical isolates from various clinical samples including blood, stool, pus, sputum, urine and cerebrospinal fluid. The DNA microarray developed in this study allowed efficient detection of bacterial pathogens with the specificities of 100%. The sensitivities were 100% as well except for the two pathogens, Enterobacter cloacae (75%) and Enterococcus faecium (85%). These results suggest that the DNA microarray-based assay developed in this study outperforms current diagnostic systems with respect to sensitivity, specificity, and high-throughput detection, and thus should be useful in pathogen diagnosis in the clinical setting.     

6种食品致病菌的多重PCR检测

目的寻求食物中毒诊断及食品检测的有效方法。方法建立多重PCR体系一次检测多种食品致病菌方法。结果可以一次检测出肠毒性大肠埃希菌(E.coli-ETEC),蜡样芽胞杆菌(Bacilluscereus),沙门菌属(Salmonellaspp),大肠埃希菌O157:H7(E.coli-O157:H7),志贺菌属(Shigellaspp),霍乱弧菌(Vibriocholerae)等菌属或菌。结论多重PCR体系检测6种菌DNA的灵敏度最低为10.30fg,与单独的PCR检测的灵敏度相同。将它做成体外基因诊断试剂盒,将成为细菌性食物中毒诊断及食品检测的有效工具。