DNA microarray-based detection of nosocomial pathogenic Pseudomonas aeruginosa and Acinetobacter baumannii

Infection by nosocomial pathogenic bacteria is increasingly becoming a major threat to the patients in the hospital. We have developed a diagnostic DNA microarray for the detection of two important nosocomial pathogens, Pseudomonas aeruginosa and Acinetobacter baumannii. The diagnostic DNA microarray contains the species-specific probes of 15mer oligonucleotides designed based on the sequences of 23S ribosomal DNA. The performance of DNA microarray in diagnosing P. aeruginosa and A. baumannii was evaluated using reference bacteria as well as clinical specimens such as blood, stool, pus, sputum, urine and cerebrospinal fluid. Using this DNA microarray, A. baumannii could be successfully detected in 11 out of 13 clinical specimens, thus giving the sensitivity of 84.6% with the specificity of 100% and the positive predictive value of 100%. P. aeruginosa could also be detected in 25 out of 26 clinical specimens, showing the sensitivity of 96.2%, the specificity of 100%, and the positive predictive value of 100%. These results suggest that two nosocomial pathogens, P. aeruginosa and A. baumannii, can be efficiently diagnosed by using the DNA microarray developed in this study.     

Sequence-specific identification of 18 pathogenic microorganisms using microarray technology

We have developed a Multi-Pathogen Identification (MPID) microarray for high confidence identification of eighteen pathogenic prokaryotes, eukaryotes and viruses. Analysis of amplified products from pathogen genomic DNA using microarray hybridization allows for highly specific and sensitive detection, and allows the discrimination between true amplification products and false positive amplification products that might be derived from primers annealing to non-target sequences. Species-specific primer sets were used to amplify multiple diagnostic regions unique to each individual pathogen. Amplified products were washed over the surface of the microarray, and labelled with phycoerythrin-streptavidin for fluorescence detection. A series of overlapping 20-mer oligonucleotide probes hybridize to the entire diagnostic region, while parallel hybridizations on the same surface allow simultaneous screening for all organisms. Comparison to probes that differ by a single mismatch at the central position reduced the contribution of non-specific hybridization. Samples containing individual pathogens were analyzed in separate experiments and the corresponding species-specific diagnostic regions were identified by fluorescence among their highly redundant probe sets. On average, 91% of the 53 660 pathogen probes on the MPID microarray performed as predicted. The limit of detection was found to be as little as 10 fg of B. anthracis DNA in samples that were amplified with six diagnostic primer-pairs. In contrast, PCR products were not observed at this concentration when identical samples were prepared and visualized by agarose gel electrophoresis.     

Multiplex PCR amplification and immobilized capture probes for detection of bacterial pathogens and indicators in water

Detection of pathogens (Legionella species) and indicator bacteria (coliform bacteria) was achieved by multiplex (simultaneous) PCR amplification of diagnostic gene sequences and by hybridization to immobilized poly-dT-tailed capture probes using a dot- or slot-blot approach. Complex manipulations of primer concentrations and staggered additions of primers were required in order to achieve equal amplification of multiple genes. Multiplex PCR amplification of two different Legionella genes, one specific for L. pneumophila (mip) and the other for the genus Legionella (5S rRNA), was achieved by staggered amplification. Multiplex PCR amplification using differing amounts of primers specific for lacZ and lamB genes permitted the detection of coliform bacteria and those associated with human faecal contamination, including the indicator bacterial species E. coli and enteric pathogens Salmonella and Shigella. Hybridization of biotin-labelled amplified DNA, in which the biotin was incorporated during PCR amplification from biotinylated-dUTP, to immobilized 400-dT-tailed capture probes permitted specific and sensitive detection of target gene sequences. The sensitivity of colorimetric detection achieved by PCR amplification of target DNA was at a level equivalent to 1-2 bacterial cells, which is the same level of sensitivity obtained with radioactive detection. The simultaneous amplification of several genes and hybridization to immobilized capture probes with colorimetric detection is an effective, efficient and rapid detection method for various human bacterial pathogens.     

Detection and identification of intestinal pathogens in clinical specimens using DNA microarrays

The detection and identification of intestinal pathogens is critical for clinical patient diagnosis and antimicrobial therapy. No currently available assays with DNA microarrays can simultaneously detect and identify multiple intestinal pathogens, because there is no appropriate method for choosing target probes. To solve the problem we have experimented for facilitating screening of specific probes and developed a rapid (<3h) and reliable assay for simultaneous detection of intestinal pathogens using two universal PCR primers to amplify two variable regions of bacterial 16S and 23S ribosomal DNA (rDNA) genes, and then applied to DNA microarrays, hybridization between probes and amplicons occurred. Through this idea for screening of probes the assay was successful in discriminating 15 genera or species of intestinal pathogens. The limit of detection was approximately 10(3)CFU/mL for one species of pathogen and 10(5)CFU/mL for six species pathogens existing simultaneously in stool. When this assay was applied directly to identify 99 clinical specimens, 80(80.8%) were correctly analyzed, including four with mixed pathogens; 8(8.08%) received negative results due to no corresponding probes in this array and 11(11.11%) belonging to our targets were misidentified due to low-level pathogens and other factors. This approach is also convenient to obtain specific and proper probes while establishing assays for the applications in other aspects using DNA microarrays. In addition, the more species may be added to this system easily and endlessly by screening of candidate target probes in order to increase the power of simultaneous detection.     

多重PCR/反向线点杂交（RLB）检测细菌性脑膜炎病原体的研究

目的建立快速、高通量的多重PCR/反向线点杂交（RLB）的方法检测3种细菌性脑膜炎病原体--肺炎链球菌、流感嗜血杆菌和脑膜炎奈瑟菌。方法针对肺炎链球菌、流感嗜血杆菌和脑膜炎奈瑟菌的特异性基因序列设计引物和相应的特异性探针,优化多重PCR/RLB检测中的引物浓度、dNTP浓度、探针的浓度等的条件。结果多重PCR/RLB检测对肺炎链球菌DNA最低检测极限可达6.2×10-1ng/μl,对流感嗜血杆菌DNA最低检测极限可达8.5×10-8ng/μl,对脑膜炎奈瑟菌DNA最低检测极限可达8.3×10-8ng/μl。结论多重PCR/RLB检测体系能够正确的鉴定3种细菌性脑膜炎病原体,用于临床样本的高通量筛查细菌性脑膜炎病原体,特异性好,灵敏度高。     

细菌性肺炎快速诊断基因芯片的初步构建

目的：根据细菌16 S rRNA基因特点设计常见病原菌的特异性探针,采用酶显色技术构建基因芯片,探讨其临床应用的可能性。方法选取肺炎链球菌、流感嗜血杆菌及铜绿假单胞菌等8种细菌性肺炎常见的病原菌的标准菌株作为研究对象,并选择12份患者的痰液标本进行检测。在16 S rRNA基因保守区设计 PCR反应的通用引物及革兰阳性菌、革兰阴性菌的通用探针,利用可变区的差异设计合成特异性探针,构建基因芯片。利用细菌16S rRNA基因设计的PCR通用引物进行扩增,所有8种细菌均获得350 bp的扩增产物。以地高辛标记特异性探针,构建完成可用于8种常见病原菌检测的基因芯片,结果8种标准菌株基因芯片检测均取得了预期效果,对12份痰标本中常规培养阳性7份,其对应芯片检测结果均成阳性,5份常规培养阴性的标本中,芯片结果提示阳性的有3份,其中1份为嗜肺军团菌,2份为使用抗生素后的患者标本。结论设计合成的 PCR通用引物对扩增细菌的16 S rRNA基因具有较高的特异性及灵敏度。构建的基因芯片可用于常见细菌性肺炎病原菌的检测鉴定,且对抗生素使用后的临床标本及苛养菌有一定的诊断价值。本研究所获得基因芯片对于细菌性肺炎的检测具有简单、快速、特异性及敏感性高的特点。     

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.     

High-throughput screening of bacterial pathogens in clinical specimens using 16S rDNA qPCR and fragment analysis

Molecular-based detection of bacterial pathogens directly from clinical specimens permits rapid initiation of effective antimicrobial treatment and adequate patient management. Broad-range polymerase chain reaction (PCR) amplification of the 16S rRNA gene (16S rDNA qPCR) is used in many diagnostic laboratories as a complement to cultural identification of bacterial pathogens. However, efforts for automation of 16S rDNA PCR workflows are needed in order to reduce turnaround times and to enhance reproducibility and standardization of the technique. In this retrospective method evaluation study, clinical specimens (N?=?499) from patients with suspected bacterial infections were used to evaluate 2 diagnostic semiautomated workflows for rapid bacterial pathogen detection. The workflows included automated DNA extraction (QIASymphony), 16S rDNA qPCR, fragment or melting curve analysis, and amplicon sequencing. Our results support the use of the 16S rDNA qPCR and fragment analysis workflow as it enabled rapid and accurate identification of bacterial pathogens in clinical specimens.     

Development of a microarray for identification of pathogenic Clostridium spp.

In recent years, Clostridium spp. have rapidly reemerged as human and animal pathogens. The detection and identification of pathogenic Clostridium spp. is therefore critical for clinical diagnosis and antimicrobial therapy. Traditional diagnostic techniques for clostridia are laborious, are time consuming, and may adversely affect the therapeutic outcome. In this study, we developed an oligonucleotide diagnostic microarray for pathogenic Clostridium spp. The microarray specificity was tested against 65 Clostridium isolates. The applicability of this microarray in a clinical setting was assessed with the use of mock stool samples. The microarray was successful in discriminating at least 4 species with the limit of detection as low as 10⁴ CFU/mL. In addition, the pattern of virulence and antibiotic resistance genes of tested strains were determined through the microarrays. This approach demonstrates the high-throughput detection and identification of Clostridium spp. and provides advantages over traditional methods. Microarray-based techniques are promising applications for clinical diagnosis and epidemiologic investigations.     

Rapid Polymerase Chain Reaction-based Screening Assay for Bacterial Biothreat Agents

Objectives:  To design and evaluate a rapid polymerase chain reaction (PCR)-based assay for detecting Eubacteria and performing early screening for selected Class A biothreat bacterial pathogens.
Methods:  The authors designed a two-step PCR-based algorithm consisting of an initial broad-based universal detection step, followed by specific pathogen identification targeted for identification of the Class A bacterial biothreat agents. A region in the bacterial 16S rRNA gene containing a highly variable sequence flanked by clusters of conserved sequences was chosen as the target for the PCR assay design. A previously described highly conserved region located within the 16S rRNA amplicon was selected as the universal probe (UniProbe, Integrated DNA Technology, Coralville, IA). Pathogen-specific TaqMan probes were designed for Bacillus anthracis, Yersinia pestis, and Francisella tularensis. Performance of the assay was assessed using genomic DNA extracted from the aforementioned biothreat-related organisms (inactivated or surrogate) and other common bacteria.
Results:  The UniProbe detected the presence of all tested Eubacteria (31/31) with high analytical sensitivity. The biothreat-specific probes accurately identified organisms down to the closely related species and genus level, but were unable to discriminate between very close surrogates, such as Yersinia philomiragia and Bacillus cereus .
Conclusions:  A simple, two-step PCR-based assay proved capable of both universal bacterial detection and identification of select Class A bacterial biothreat and biothreat-related pathogens. Although this assay requires confirmatory testing for definitive species identification, the method has great potential for use in ED-based settings for rapid diagnosis in cases of suspected Category A bacterial biothreat agents.     

Development of an oligonucleotide-based microarray to detect multiple foodborne pathogens

Escherichia coli O157:H7, Salmonella enterica, Listeria monocytogenes and Campylobacter jejuni are considered important pathogens causing the most food-related human illnesses worldwide. Current methods for pathogen detection have limitations in the effectiveness of identifying multiple foodborne pathogens. In this study, a pathogen detection microarray was developed using various 70-mer oligonucleotides specifically targeting the above pathogens. To reduce the cost of detection, each microarray chip was designed and fabricated to accommodate 12 identical arrays which could be used for screening up to 12 different samples. To achieve high detection sensitivity and specificity, target-specific DNA amplification instead of whole genome random amplification was used prior to microarray analysis. Combined with 14-plex PCR amplification of target sequences, the microarray unambiguously distinguished all 4 pathogens with a detection sensitivity of 1 × 10^?4 ng (approximately 20 copies) of each genomic DNA. Applied the assay to 39 fresh meat samples, 16 samples were found to be contaminated by either 1 or 2 of these pathogens. The co-occurrences of Salmonella and E. coli O157:H7, Salmonella and L. monocytogenes in the same meat samples were also observed. Overall, the microarray combined with multiplex PCR method was able to effectively screen single or multiple pathogens in food samples and to provide important genotypic information related to pathogen virulence.     

应用基因芯片快速检测临床常见细菌

目的 应用基因芯片对临床常见的8种致病细菌进行检测.方法 选取8种临床常见的致病细菌,包括金黄色葡萄球菌、铜绿假单胞菌、肺炎克雷伯菌、大肠杆菌、奇异变形杆菌、产气肠杆菌、荧光假单胞菌、宋内志贺菌.以16S rRNA基因为目的基因,白行设计通用引物系列扩增目的片段,针对高变区域设计探针,建立基因芯片检测体系,并对所选细菌...     

Multiplex detection of CpG methylation using microarray combining with target-selection-padlock probe

BackgroundMicroarray technology combining with bisulfite-PCR offers a high-throughput approach for detection of DNA methylation. However, the use of microarray-based DNA methylation analysis has been limited by the low throughput of sample preparation due to the difficulty in simultaneous amplification of multiple targets.MethodsA set of target-selection-padlock probes was designed to capture the target sequences containing the queried CpG sites from bisulfite-treated genomic DNA. Then all targets were simultaneously amplified by a pair of common primers. The methylation status of multiple targets was detected by single base extension (SBE) on oligonucleotide microarray based on polyacrylic acid-covered surface.ResultsThis assay has been successfully applied to analyze promoter methylation of 8 tumor suppressor genes in 12 colorectal cancer samples and 2 normal control samples. The target-selection-padlock probe exhibited both high specificity and high efficiency for the parallel amplification of multiple genes. The accurate and high-throughput detection for DNA methylation was achieved by a combination of target-selection-padlock probes and microarray.ConclusionsThe present study provides a robust and accurate assay for DNA methylation status of multiple genes. This method may be useful for a large-scale screen of DNA methylation in cancer cell lines and clinical samples.     

Pyrosequencing-based validation of a simple cell-suspension polymerase chain reaction assay for Campylobacter with application of high-processivity polymerase and novel internal amplification controls for rapid and specific detection

Although Campylobacter is an important food-borne human pathogen, there remains a lack of molecular diagnostic assays that are simple to use, cost-effective, and provide rapid results in research, clinical, or regulatory laboratories. Of the numerous Campylobacter assays that do exist, to our knowledge none has been empirically tested for specificity using high-throughput sequencing. Here we demonstrate the power of next-generation sequencing to determine the specificity of a widely cited Campylobacter-specific polymerase chain reaction (PCR) assay and describe a rapid method for direct cell suspension PCR to quickly and easily screen samples for Campylobacter. We present a specific protocol which eliminates the need for time-consuming and expensive genomic DNA extractions and, using a high-processivity polymerase, demonstrate conclusive screening of samples in <1 h. Pyrosequencing results show the assay to be extremely (>99%) sensitive, and spike-back experiments demonstrated a detection threshold of <10² CFU mL⁻¹. Additionally, we present 2 newly designed broad-range bacterial primer sets targeting the 23S rRNA gene that have wide applicability as internal amplification controls. Empirical testing of putative taxon-specific assays using high-throughput sequencing is an important validation step that is now financially feasible for research, regulatory, or clinical applications.     

Distribution and drug resistance of pathogens in burn patients in China from 2006 to 2019

BACKGROUNDIn this study, recent trends in the distribution and drug resistance of pathogenic bacteria isolated from patients treated at a burn ward between 2006 and 2019 were investigated.AIMTo develop more effective clinical strategies and techniques for the prevention and treatment of bacterial infections in burn patients.METHODSClinical samples with positive bacteria were collected from patients at the burn ward in Beijing Jishuitan Hospital in China between January 2006 and December 2019. The samples were retrospectively analyzed, the distribution of pathogenic bacteria was determined, and the trends and changes in bacterial drug resistance during different period were assessed. Drug resistance in several main pathogenic bacteria from 2006 to 2011 and from 2012 to 2019 was comparatively summarized and analyzed.RESULTSSamples from 17119 patients were collected and analyzed from 2006 to 2019. Surprisingly, a total of 7960 strains of different pathogenic bacteria were isolated at this hospital. Among these bacteria, 87.98% (7003/7960) of the strains were isolated from burn wounds, and only 1.34% (107/7960) were isolated from the blood of patients. In addition, 49.70% (3956/7960) were identified as Gram-positive bacteria, 48.13% (3831/7960) were Gram-negative bacteria, and the remaining 2.17% (173/7960) were classified as fungi or other pathogens. Importantly, Staphylococcus aureus (21.68%), Pseudomonas aeruginosa (14.23%), and Staphylococcus epidermidis (9.61%) were the top three pathogens most frequently isolated from patients.CONCLUSIONIn patients treated at the burn ward in this hospital from 2006 to 2019, Staphylococcus aureus and Pseudomonas aeruginosa were the predominant clinical pathogens responsible for bacterial infections. The circumstantial detection and detailed monitoring of the intensity and growth of different pathogenic bacteria in clinical patients as well as tests of drug sensitivity during burn recovery are particularly important to provide guidelines for the application of antibiotics and other related drugs. Careful collection and correct, standard culture of bacterial specimens are also crucial to improve the efficiency of bacterial infection detection. Effective monitoring and timely clinical treatment in patients may help reduce the possibility and rate of infection as well as alleviate the effects of drug resistance among patients in burn centers.     

基因芯片用于病原细菌通用检测的研究进展

传统的检测技术一般是通过一次实验，检测一种或少数几种病原体。为了提高病原细菌检测的速度和准确性，建立一次实验能检测出一个样品中多个目标的通用检测技术，是临床微生物检测中重要的研究课题。理想的通用检测技术应具有较好的种属鉴别能力和检测灵敏度，并且能够平行、大量、快速、稳定和特异地实现多种细菌的检测。最近发展起来的基因芯片技术具有微型化、高通量、并行处理的特点，能够实现通用检测的目的，具有良好的应用前景。     

2016－2018年北京市通州区感染性腹泻细菌病原谱监测分析

目的了解北京市通州区感染性腹泻细菌多病原监测中致病菌的病原谱,为细菌性感染性腹泻的防控提供依据。方法收集2016 — 2018年北京市通州区哨点医院肠道门诊腹泻患者监测病例粪便标本,采用血清分型、毒力基因等方法对沙门菌、副溶血弧菌、致泻性大肠埃希菌进行分子生物学鉴定,并分析其流行特征。结果收集粪便标本1 269例,分离致病菌298株,检出率为23.48%,不同年龄组病例致病菌检出率差异有统计学意义（χ2＝33.584,P＜0.001）。 阳性菌株中致泻性大肠埃希菌检出最多（46.98%）,以肠道集聚性大肠埃希菌和产肠毒素大肠埃希菌为主;其次为副溶血弧菌（24.50%）和沙门菌（19.46%）。 细菌流行的季节性比较明显,以夏秋季为主。 沙门菌血清型以肠炎沙门菌和鼠伤寒沙门菌为主,副溶血弧菌血清型以O3∶K6为主。结论2016 — 2018年北京市通州区感染性腹泻致病菌以致泻性大肠埃希菌为主,副溶血弧菌和沙门菌感染也是腹泻常见病原菌。 应加强感染性腹泻致病菌常规监测,在流行季节做好重点人群健康宣教工作,同时进一步加强危险因素调查及其防控。     

2019-2021年广西壮族自治区桂林市腹泻病原谱分析

目的 了解广西壮族自治区桂林市腹泻病例的病原谱，为腹泻病防控提供依据。方法 对2019年1月至2021年6月桂林市腹泻病监测定点医院的腹泻病例进行监测，并采集病例的粪便标本进行5种致泻病毒、17种致病菌的病原学检测，分析腹泻病例的病原谱特征。结果 385份监测样本中，病原的总阳性检出率为37.40%，其中，细菌检出率为12.99%，病毒检出率为27.27%，混合感染检出率为9.61%，以病毒混合感染为主。各年龄组样本的阳性检出率差异无统计学意义（P=0.510）。检出的病原体9种，不同季节的病原体检出率的差异有统计学意义（P<0.05）。不同年龄组的样本病原阳性检出率以0～2岁组最高（43.48%），其次为15～35岁组（41.46%）和36～60组（38.78%）。主要以轮状病毒、诺如病毒和单胞菌感染为主。结论 桂林市区腹泻病原谱较为广泛，具有细菌性腹泻夏季高峰和病毒性腹泻春夏季高峰的特点，应在不同季节针对流行病原体采取防控措施。应考虑在临床诊疗时开展腹泻病原学检测，以便采取针对性措施防控腹泻传播与流行。     

高通量测序技术在尿路感染及腹膜透析相关腹膜炎病原学诊断中的价值

目的比较尿路感染、腹膜透析相关腹膜炎患者采用高通量测序技术、常规培养法对病原体的检出情况,探讨高通量测序技术在尿路感染、腹膜透析相关腹膜炎病原学诊断中的应用价值。方法收集77例尿路感染患者中段尿标本,36例腹膜透析相关腹膜炎患者透析流出液标本,分别应用高通量测序技术、常规培养法进行检测,记录病原体检出率及病原体分布情况,以培养法为金标准,计算高通量测序法对病原体检测的灵敏度。结果77例尿路感染患者中,高通量测序法检出病原体70例(90.9%),共分离出病原体169株;培养法检出病原体27例(35.1%),共分离出病原体27株;以培养法为金标准,高通量测序法对尿路感染患者病原体检出的灵敏度为96.3%。36例腹膜透析相关腹膜炎患者中,高通量测序法检出病原体28例(77.8%),共分离出病原体39株,培养法检出病原体11例(30.6%),共分离出病原体11株;以培养法为金标准,高通量测序法对腹膜透析相关腹膜炎患者病原体检出的灵敏度为72.7%。结论对尿路感染、腹膜透析相关腹膜炎患者,采用高通量测序技术检出的病原体株数多于培养法,可作为培养法的有效补充。     

16S rRNA 测序鉴定1株条件致病性人苍白杆菌

目的：探索一种基于16S rRNA 基因的细菌快速鉴定方法,为临床未知病原菌的诊断及治疗提供科学依据。方法对临床患者的痰标本分离培养纯菌落,直接以菌液为模板,以通用引物 PCR 扩增未知菌的16S rRNA 基因片段,产物直接测序。将测序结果进行 BLAST 比对,根据序列同源性鉴定病原细菌。结果未知病原菌经本实验鉴定为人苍白杆菌,经 ABI 细菌快速鉴定板条检测,确认结果一致。结论该研究简化了临床标本未知病原菌分离培养鉴定的步骤,建立了一种利用16S rRNA 基因扩增快速鉴定病原菌的简便方法。