人血小板体外抗菌活性研究

目的探讨洗涤人血小板在体外对革兰阳性球菌代表菌(金黄色葡萄球菌)及革兰阴性杆菌代表菌(大肠埃希菌)活性的影响。方法分离和洗涤人血小板,加入20倍的细菌共孵育3h。将共孵育后的反应液梯度稀释后涂平板,计数细菌菌落形成单位(CFU)来判断活菌数量;将共孵育后反应液离心,检测上清液中Th1/Th2/Th17细胞因子,分析与细菌作用后的血小板细胞因子释放情况。结果金黄色葡萄球菌和大肠埃希菌与血小板共孵育后,细菌CFU数目均明显下降。血小板对金黄色葡萄球菌和大肠埃希菌的杀菌率分别为96.8%和40.5%。金黄色葡萄球菌可以促进血小板产生γ-干扰素和白细胞介素-17a,而大肠埃希菌没有显示出明显作用。结论洗涤血小板能够明显抑制金黄色葡萄球菌和大肠埃希菌的活性,并释放出较高浓度的炎性细胞因子。     

TaqMan探针法荧光定量PCR检测脑脊液细菌方法的建立及应用

目的 建立TaqMan探针法荧光定量PCR检测方法,用于脑脊液标本细菌的快速检测.方法 针对细菌的16S rRNA基因,设计合成细菌的通用引物和革兰阳性菌、革兰阴性菌分型探针,通过构建质粒和制作脑脊液模拟标本来研究引物和探针的检测特异度和敏感度.结果 两种革兰分型探针只对相应的细菌可以检测到荧光信号,乙肝病毒DNA、白色念珠菌及人基因组DNA检测结果均为阴性,此方法最低可以检测到10个拷贝的质粒DNA以及102CFU/ml的金黄色葡萄球菌和大肠埃希菌.结论 TaqMan探针法荧光定量PCR检测细菌的特异度和敏感度高,检测快速,对脑脊液细菌的早期诊断有重要意义.     

非特异磁性分离法在血小板制品污染菌富集中的应用研究

目的建立非特异磁性分离法富集血小板制品中的大肠杆菌和金黄色葡萄球菌,以提高应用核酸扩增方法检测血小板制品污染菌的效率。方法应用6种磁珠(80 nm羧基、200 nm羧基、2.8μm氨基、2.8μm羧基、2.8μm环氧基、2.8μm甲苯磺酰基磁珠),分别对高浓度(102—103CFUs.ml-1)、低浓度(<102CFUs.ml-1)血小板制品中的大肠杆菌进行非特异性富集,选取富集效率最高的磁珠,进一步对不同稀释浓度的大肠杆菌、金黄色葡萄球菌(10-1、10-2、10-3、10-4、10-5、10-6)进行富集,与直接离心法进行对比,比较不同细菌富集方法对后续聚合酶链式反应(polymerase chain reaction,PCR)检测灵敏度的影响。结果 6种磁珠对高、低浓度的血小板制品中大肠杆菌均有富集作用,其中80 nm羧基磁珠的富集效率最高(P<0.05);应用直接离心法和80 nm羧基磁珠富集样品后,PCR检测大肠杆菌的灵敏度分别为588 CFUs.ml-1、1 CFUs.ml-1,检测金黄色葡萄球菌的灵敏度分别为1 130 CFUs.ml-1、40 CFUs.ml-1。结论应用磁性分离法富集血小板制品中污染菌,其操作简单,所获细菌纯度高,为后续PCR检测方法在血小板制品污染菌检测中的临床应用提供帮助。     

PGD血小板细菌污染检测系统效果的评价

目的评价PGD检测系统用于混合血小板制品细菌污染检测的效果。方法分别将配制后经浓缩血小板稀释为101、103和105CFU/ml的大肠埃希菌和表皮葡萄球菌菌悬液进行PGD检测限的评估;将上述菌株分别接种于浓缩血小板中制成菌液浓度为101、103CFU/ml的模拟细菌污染浓缩血小板,经22℃振荡保存24、72、120 h后分别用PGD法和Bact/ALERT法进行细菌检测,比较2种方法的细菌污染检测限和阳性反应时间。结果 PGD法对大肠埃希菌的检测限为105CFU/ml,表皮葡萄球菌为103CFU/ml;将103CFU/ml的大肠埃希菌和表皮葡萄球菌分别接种到浓缩血小板,24 h后PGD检测均为阴性,72 h后均为阳性,而Bact/ALERT法检测在10 h均呈阳性反应;将105CFU/ml的大肠埃希菌和表皮葡萄球菌接种浓缩血小板4 h后PGD检测均为阳性,Bact/ALERT法在3—6 h内均呈阳性反应。结论 PGD检测系统对血小板细菌污染的检测限为(103—105)CFU/ml,适用于医院输血部门在血小板输血前的快速细菌检测。     

志贺菌TaqMan-MGB探针实时荧光聚合酶链反应快速检测方法的应用研究

目的建立志贺菌TaqMan-MGB探针实时荧光PCR快速检测技术,为从患者、食品和环境中快速分离和鉴定志贺菌提供技术支撑。方法根据志贺菌ipaH基因序列设计一对特异性引物和TaqMan-MGB探针;通过对PCR扩增体系和反应条件的优化,建立志贺菌TaqMan-MGB探针实时荧光PCR快速检测方法;用添加已知志贺菌浓度的样本验证方法敏感性;用志贺菌标准菌株、分离株以及大肠埃希菌、沙门菌、副溶血性弧菌、金黄色葡萄球菌等致病菌验证方法特异性。结果用本研究建立的志贺菌TaqMan-MGB探针实时荧光PCR检测方法检测志贺菌,其Ct值与模板浓度的对数值具有很好的对应关系（Y=-3.93×log（X）＋37.34,R=0.999）,最低检测浓度为30cfu/ml,3株志贺菌标准株,30株志贺菌分离株检测结果均为阳性;而沙门菌、副溶血性弧菌、大肠埃希菌、金黄色葡萄球菌等91株其他细菌的Ct值均〉35或扩增曲线成一平滑直线。与常规分离鉴定方法比较差异无统计学意义（P〉0.05,χ^2=0.27）。对于纯菌和食品样品整个检测过程仅需2h和10h。结论志贺菌TaqMan-MGB探针实时荧光PCR检测技术具有特异性强,敏感性高,易操作等优点,有很好的应用前景和研究价值。     

微孔板法评价碘伏的消毒效果研究

摘要 目的 利用微孔板建立微量试验法检测碘伏的抑菌和杀菌效果。方法用微孔板法鉴定中和剂对碘伏的中和效果;用微孔板悬液定量杀菌试验检测碘伏对金黄色葡萄球菌和大肠埃希菌的杀灭效果;用微量肉汤法检测碘伏对金黄色葡萄球菌和大肠埃希菌的最小抑菌浓度,平板划线后计算最小杀菌浓度。结果微孔板悬液定量杀灭实验显示,310～2 500 mg/L的碘伏随作用时间（30 s～5 min）延长,对金黄色葡萄球菌和大肠埃希菌的杀灭作用逐渐增强。碘伏对两种细菌的最小抑菌浓度和最小杀菌浓度均为625 mg/L。结论微孔板法可用于检测碘伏对常见细菌的抑菌和杀菌效果。     

血小板制品细菌污染的核酸检测方法评价

目的探讨实时荧光定量PCR法检测血小板制品中细菌污染的C t值用于血液细菌污染筛查的可行性。方法用实时荧光定量PCR技术检测单采血小板样本740份,包括420份阳性样本及320份阴性样本。通过受试者工作特征曲线(ROC)分析检测结果,确定最佳工作点的特异性和灵敏度。结果通过对检测结果C t值分析,确定最佳工作点即C t值在31.97时灵敏度和特异性分别为99.4%和99.3%。通过对不同诊断点的结果分析初步确定C t值<31.97时发阳性报告,>33.00时发阴性报告,之间则为疑似样本。结论通过ROC曲线分析证实血小板制品细菌污染的实时荧光定量PCR检测法是一种高效、可靠、便于临床应用的检测手段。     

16S rRNA基因荧光定量PCR与芯片杂交法快速诊断儿童化脓性脑膜炎

目的探索快速诊断儿童化脓性脑膜炎的新技术。方法通过设计细菌16SrRNA基因高度保守区的引物和探针,对疑似化脑的21份脑脊液(CSF)进行荧光定量PCR及基因芯片杂交检测,同时与脑脊液细菌培养结果比较。结果(1)荧光定量PCR发现21份脑脊液标本有8份阳性,阳性率为38.095%(8/21),明显高于脑脊液培养的阳性率19.047%(4/21),差异具有显著意义(P<0.01)。(2)基因芯片杂交结果8份阳性,其中大肠埃希菌探针阳性5份,肠道杆菌科探针阳性2份,金黄色葡萄球菌探针阳性1份;脑脊液培养4份阳性,分别为肺炎克雷伯菌培养阳性1份,阴沟肠杆菌阳性1份,大肠埃希菌阳性2份;基因芯片杂交阳性率为38.095%(8/21),芯片杂交阳性结果与荧光定量PCR大体一致。结论荧光定量PCR结合基因芯片杂交技术检测儿童化脑具有快速、敏感、特异的特点,对儿童化脓性脑膜炎的早期诊断有一定的应用价值。     

16S rDNA实时荧光定量PCR法与全自动培养法在血小板细菌污染检测中的应用比较

目的比较16S rDNA实时荧光定量PCR法与全自动培养法在血小板制品细菌污染检测中的灵敏度和特异性,评价2种方法的应用前景。方法将血小板制品污染中常见的6种细菌用浓缩血小板悬液进行稀释,并选取浓度为102、101、100的菌悬液,分别用实时荧光定量PCR法和全自动培养法进行检测。结果用16S rDNA实时荧光定量PCR法对6株细菌检测,其灵敏度和特异性均为100%,Κ=1.000。用全自动培养仪对6株细菌检测,其特异性均为100%;灵敏度分别为:金黄色葡萄球菌需氧瓶95.5%,Κ=0.886,厌氧瓶90.9%,Κ=0.787;表皮葡萄球菌及大肠杆菌2种培养瓶均为83.3%,Κ=0.667;蜡样芽孢杆菌2种培养瓶均为86.7%,Κ=0.684;铜绿假单胞杆菌需氧瓶度为100%,Κ=1.000,厌氧瓶为44.4%,Κ=0.286;痤疮丙酸杆菌需氧瓶为16.7%,Κ=0.105,厌氧瓶为91.7%,Κ=0.886。结论实时荧光定量PCR法检测血小板制品中的细菌污染,灵敏度高,特异性好,且省时、经济,能应用于临床上血液样本的大规模筛查。     

2010年某院临床常见细菌分布及耐药性分析

目的 了解2010年临床分离菌株对抗菌药物的耐药情况.方法 细菌鉴定采用手工法,药敏试验采用纸片扩散法.结果 2010年各临床科室送检标本中,真菌、克雷伯菌、大肠埃希菌、假单胞菌、肠球菌、凝固酶阴性葡萄球菌、金黄色葡萄球菌为主要病原菌.革兰阴性菌中以假单胞菌耐药性最高,其次为大肠埃希菌、克雷伯菌.革兰阳性菌中金黄色葡萄球菌感染呈下降趋势,而凝固酶阴性葡萄球菌感染呈上升趋势,耐药率也随之增长.结论 临床科室应注意微生物实验室所提供的细菌耐药情况,合理使用抗生素.     

Evaluation of two detection methods of microorganisms in platelet concentrates

Background: The performance of a bacterial 16S ribosomal DNA real‐time polymerase chain reaction (PCR) assay was evaluated and validated with an automated culture system to determine its use for screening of platelet concentrates (PCs). Study Design and Methods: PCs were spiked with suspensions of Escherichia coli, Serratia marcescens, Staphylococcus epidermidis and St. aureus at 1, 10, and 100 colony‐forming units (CFUs) mL and stored for 5 days. DNA amplification was performed using real‐time PCR. The BacT/ALERT was used as a reference method and samples were inoculated into an aerobic culture bottle; for the PCR assay, aliquots were drawn from all (spiked) PCs on days 0 to 5 of storage. Results: Real‐time PCR detected only the gram‐positive bacteria in PCs spiked with low bacterial titres (1 CFU mL) after 48 h; however, it was able to detect all positive samples in PCs spiked with 10 CFU mL of either gram‐positive or gram‐negative bacteria after 48 h. In addition, real‐time PCR detected all positive samples in PCs spiked with high gram‐positive bacterial titres (100 CFU mL) after 24 h. On the other hand, the BacT/ALERT system showed positive results in all samples within 24 h. Conclusion: The BacT/ALERT method is more sensitive and should continue to be the gold standard for identifying bacterial contaminations in blood samples. The real‐time PCR approach can be used for the screening of PCs for microbial detection before they are released from blood centres or shortly before they are used in blood transfusion, and thus allow an extended shelf life of the platelets.     

Real-time polymerase chain reaction in transfusion medicine: applications for detection of bacterial contamination in blood products

Bacterial contamination of blood components, particularly of platelet concentrates (PCs), represents the greatest infectious risk in blood transfusion. Although the incidence of platelet bacterial contamination is approximately 1 per 2,000 U, the urgent need for a method for the routine screening of PCs to improve safety for patients had not been considered for a long time. Besides the culturing systems, which will remain the criterion standard, rapid methods for sterility screening will play a more important role in transfusion medicine in the future. In particular, nucleic acid amplification techniques (NATs) are powerful potential tools for bacterial screening assays. The combination of excellent sensitivity and specificity, reduced contamination risk, ease of performance, and speed has made real-time polymerase chain reaction (PCR) technology an appealing alternative to conventional culture-based testing methods. When using real-time PCR for the detection of bacterial contamination, several points have to be considered. The main focus is the choice of the target gene; the assay format; the nucleic acid extraction method, depending on the sample type; and the evaluation of an ideal sampling strategy. However, several factors such as the availability of bacterial-derived nucleic acid amplification reagents, the impracticability, and the cost have limited the use of NATs until now. Attempts to reduce the presence of contaminating nucleic acids from reagents in real-time PCR have been described, but none of these approaches have proven to be very effective or to lower the sensitivity of the assay. Recently, a number of broad-range NAT assays targeting the 16S ribosomal DNA or 23S ribosomal RNA for the detection of bacteria based on real-time technology have been reported. This review will give a short survey of current approaches to and the limitations of the application of real-time PCR for bacterial detection in blood components, with emphasis on the bacterial contamination of PCs.     

A comparison of three rapid bacterial detection methods under simulated real-life conditions

BACKGROUND: Bacterial screening of all produced platelet concentrates (PCs) is implemented in many countries to reduce the risk of transfusion-transmitted sepsis. This study compares three rapid bacterial detection methods by imitating real-life conditions. STUDY DESIGN AND METHODS: The sensitivity of a solid-phase scanning cytometer (optimized Scansystem, Hemosystem), fluorescence-activated cell sorting (FACS) analysis, and 16S RNA in-house nucleic acid testing (NAT) was evaluated by spiking PCs with four transfusion relevant bacteria (Staphylococcus aureus, Bacillus cereus, Klebsiella pneumoniae, and Escherichia coli ). Two different inocula (10 colony-forming units [CFUs]/mL and 10 CFUs/bag) were used to simulate real-life conditions. Samples were taken at 12, 16, 20, and 24 hours after spiking. RESULTS: With the high inoculum, NAT had a 100 percent rate of positive testing for all four types of bacteria (10/10 replicates) at each time point. With the exception of E. coli, the sensitivity of FACS and optimized Scansystem was comparable for the high inoculum. With the low inoculum, 60 percent of E. coli, 80 percent of B. cereus, 90 percent of K. pneumoniae, and 100 percent of S. aureus were NAT-positive 12 hours after spiking. In contrast, only 20 percent of E. coli, 10 percent of B. cereus, and 70 percent of K. pneumoniae were FACS-positive with the low inoculum 12 hours after spiking. CONCLUSIONS: In summary, the preliminary data revealed a higher sensitivity for NAT in comparison to FACS and optimized Scansystem under the defined study conditions. To imitate real-life conditions, further spiking studies with a low inoculum (10 CFUs/bag) and slower growing organisms should be conducted to examine the sensitivity of available detection systems.     

Evaluation of an automated microbiologic blood culture device for detection of bacteria in platelet components

BACKGROUND: Automated culture methods have been used by several investigators to detect bacterial contamination of cellular blood components. We investigated several factors affecting detection by automated culture of bacteria in platelet concentrates (PCs).These factors included the initial contamination level in PCs, the PC sample volume, the PC sample time, and the white cell level in relation to bacteria levels in the PCs. STUDY DESIGN AND METHODS: Staphylococcus epidermidis or Escherichia coli was inoculated into freshly prepared PCs or white cell-reduced PCs to yield colony-forming unit (CFU) levels of 10, 1, or 0.1 per mL. At the time of inoculation (t=0) and at t=6, t=24, and t=48 hours, 0.5, 1.0, and 2.0 mL samples of the contaminated PCs were transferred into culture bottles. The presence of bacteria in the culture bottles was subsequently monitored by an automated blood culturing instrument. Bacteria levels in the PC at the time of first automated culture detection were determined by quantitative plating. RESULTS: E. coli was detected in 92 percent of experiments when 1.0- or 2.0-mL samples were taken at t=6 hours. At t=24 hours, 100-percent detection was observed with all tested inoculation volumes; however, by that time,>10(7) CFU per mL of bacteria were present in every PC. For S. epidermidis, 89 percent and 83 percent of contaminated PCs were detected with a t=24 hour sampling time and 2.0- or 1.0-mL sampling volume. Seven of 36 PCs with a 2.0-mL sampling volume and 10 of 36 PCs with a 1.0-mL sampling volume contained>10(6) CFU per mL of S. epidermidis at the time of first detection. CONCLUSION: Data from this preliminary evaluation suggest that sampling times of 24 hours or more would be necessary to provide confidence in detection of E. coli or S. epidermidis in PCs using this culture method.     

多种一步法快速提取DNA对肺炎支原体PCR检测的影响

目的 对比不同一步法提取DNA在肺炎支原体PCR检测中的效果及应用范围,为临床标本中肺炎支原体的检测提供依据。方法 以两种经典的DNA提取一步法（ROSE和Chelex-100法）及其优化方案和商品化DNA提取试剂盒,提取肺炎支原体纯培养菌株及30份肺炎支原体阳性临床咽拭子标本DNA,分别进行普通PCR和荧光PCR检测,对比不同方法提取的DNA对PCR检测结果的影响。结果 ROSE法和Chelex-100一步法中的SDS严重影响Taq酶活性,提取物稀释100倍方可进行荧光PCR扩增,稀释10倍可用于普通PCR扩增。改良的Chelex-100一步法提取肺炎支原体纯菌DNA产量最高,且PCR扩增效果最好。对于30份临床标本来说,试剂盒法、改良Chelex-100法和水煮法的阳性率分别为100.00%（30/30）、80.00%（24/30）和43.33%（13/30）,且对相同肺炎支原体阳性标本提取的DNA,试剂盒法检测的循环阈值（Ct值）比上述两种方法分别小1.95和2.38。结论 经典一步法提取的DNA必须经稀释后方可作为扩增模板。改良的Chelex-100法操作简便,可用于纯菌培养的DNA提取。临床标本中肺炎支原体DNA提取以试剂盒法最优,改良的Chelex-100法提取的DNA也可用于临床标本中肺炎支原体的检测,但不可用于定量分析。     

Detection of bacteria in platelet concentrates: comparison of broad-range real-time 16S rDNA polymerase chain reaction and automated culturing

BACKGROUND: Based on real-time polymerase chain reaction (PCR) technology, a broad-range 16S rDNA assay was validated and its performance was compared to that of an automated culture system to determine its usefulness for rapid routine screening of platelet concentrates (PCs). STUDY DESIGN AND METHODS: The presence of bacteria in pooled PCs was routinely assessed in an automated culturing system (BacT/ALERT, bioMerieux). The PCR assay was performed with DNA extracted from the same samples as used for culturing. DNA extraction was performed with a automated extraction system (MagNA Pure, Roche Diagnostics). PCR amplification was performed with a set of universal primers and probe targeting eubacterial 16S rDNA. RESULTS: A total of 2146 PCs were tested. Eighteen (0.83%) samples were found to be contaminated. These samples were positive for the presence of bacteria by both methods. All contaminants were identified as bacteria belonging to the common human skin flora. These included Propionibacterium spp. (n = 7), Staphylococcus spp. (n = 6), Bacillus spp. (n = 2), Micrococcus spp. (n = 2), and Peptostreptococcus spp. (n = 1). Estimation of the bacterial load in PCs by real-time PCR showed that the initial levels of contamination varied between 13.6 and 9 x 10(4) colony-forming unit equivalents per PCR procedure. CONCLUSIONS: Compared to culture in the BacT/ALERT system, the PCR assay had a sensitivity of 100 percent and a specificity of 100 percent. This real-time PCR assay has a much shorter turnaround time of 4 hours, which offers the possibility to test and obtain results on PCs before release or the day they are transfused. This would permit the withdrawal of contaminated PCs before transfusion.     

Application of real-time PCR and RT-PCR assays for the detection and quantitation of VT 1 and VT 2 toxin genes in E. coli O157:H7

Real-time PCR assays, based on hybridisation probes and LightCycler technology, were developed for VT 1 and VT 2 genes and applied to the detection and quantitation of DNA and mRNA of Escherichia coli O157:H7. The qualitative consensus PCR assay for the detection of VT 1 and/or VT 2 genes had a detection limit of 100 fg of E. coli O157:H7 genomic DNA and did not detect DNA from 13 non-VTEC isolates. When E. coli O157:H7 was inoculated into minced beef, enriched and recovered by immunomagnetic separation, the real-time consensus PCR assay had a detection limit of log(10)3.5 ml(-1) E. coli O157:H7 cells. Nineteen E. coli O157:H7 isolates, derived from food, bovine samples and human faeces, were analysed and compared for mRNA expression of three genes, VT 1, VT 2 and gapA (housekeeping gene), using quantitative real-time PCR assays. While there was no statistically significant difference for the expression of the VT 1 (p=0.134) or VT 2 (p=0.52) mRNA in the E. coli O157:H7 isolates from food, bovine and human sources, three clinical isolates did show lower expression of VT 2 compared to other isolates in the study. The study indicates that the consensus qualitative real-time PCR assay for VT 1 and VT 2 is rapid and sensitive and that the quantitative assays reported here have the potential to be used as an alternative method to more conventional methods for studying VT 1 and VT 2 virulence gene expression in E. coli O157:H7 with potential application in other pathogenic E. coli species.     

Effect of platelet additive solution on bacterial dynamics and their influence on platelet quality in stored platelet concentrates

BACKGROUND: Platelet additive solutions (PASs) are an alternative to plasma for the storage of platelet concentrates (PCs). However, little is known about the effect of PAS on the growth dynamics of contaminant bacteria. Conversely, there have been no studies on the influence of bacteria on platelet (PLT) quality indicators when suspended in PAS. STUDY DESIGN AND METHODS: Eight buffy coats were pooled, split, and processed into PCs suspended in either plasma or PAS (SSP+, MacoPharma). PCs were inoculated with 10 and 100 colony‐forming units (CFUs)/bag of either Serratia liquefaciens or Staphylococcus epidermidis. Bacterial growth was measured over 5 days by colony counts and bacterial biofilm formation was assayed by scanning electron microscopy and crystal violet staining. Concurrently, PLT markers were measured by an assay panel and flow cytometry. RESULTS: S. liquefaciens exhibited an apparent slower doubling time in plasma‐suspended PCs (plasma‐PCs). Biofilm formation by S. liquefaciens and S. epidermidis was significantly greater in PCs stored in plasma than in PAS. Although S. liquefaciens altered several PLT quality markers by Days 3 to 4 postinoculation in both PAS‐ and plasma‐PCs, S. epidermidis contamination did not produce measurable PLT changes. CONCLUSIONS: S. liquefaciens can be detected more quickly in PAS‐suspended PCs (PAS‐PCs) than in plasma‐PCs by colony counting. Furthermore, reduced biofilm formation by S. liquefaciens and S. epidermidis during storage in PAS‐PCs increases bacteria availability for sampling detection. Culture‐based detection remains the earliest indicator of bacterial presence in PAS‐PCs, while changes of PLT quality can herald S. liquefaciens contamination when in excess of 10⁸ CFUs/mL.