Development of a membrane-array method for the detection of human intestinal bacteria in fecal samples

A membrane-array method was developed for the detection of human intestinal bacteria in fecal samples without using the expensive microarray-arrayer and laser-scanner. The 16S rDNA sequences of 20 predominant human intestinal bacterial species were used to design oligonucleotide probes. Three 40-mer oligonucleotides specific for each bacterial species (total 60 probes) were synthesized and applied to nitrocellulose membranes. Digoxigenin (DIG)-labeled 16S rDNAs were amplified by polymerase chain reaction (PCR) from human fecal samples or pure cultured bacteria using two universal primers, and were hybridized to the membrane-array. Hybridization signals were read by NBT/BCIP color development. The 20 intestinal bacterial species tested were Bacteroides thetaiotaomicron, B. vulgatus, B. fragilis, B. distasonis, Clostridium clostridiiforme, C. leptum, Fusobacterium prausnitzii, Peptostreptococcus productus, Ruminococcus obeum, R. bromii, R. callidus, R. albus, Bifidobacterium longum, B. adolescentis, B. infantis, Eubacterium biforme, E. aerofaciens, Lactobacillus acidophilus,Escherichia coli, and Enterococcus faecium. The two universal primers were able to amplify full size 16S rDNA from all of the 20 bacterial species tested. The hybridization results indicated that the membrane-array method is a reliable technique for the detection of predominant human intestinal bacteria in the fecal samples. The result was also confirmed by using specific PCR methods for these bacteria.     

In vitro alterations of intestinal bacterial microbiota in fecal samples during storage

The human gastrointestinal tract harbors an extremely diverse and complex microbial ecosystem. Most of the existent data about the enteric microflora have been generated using stool samples, but the collection and storage of fecal samples are often problematic. The influence of the storage of stool samples on the bacterial diversity and the degradation of bacterial DNA was analysed in this study. Stool samples from 5 healthy volunteers were exposed to different storage temperatures and durations. The bacterial diversity and the amount of intact bacterial DNA were analysed by single-stranded conformation polymorphism analysis (SSCP) and real-time polymerase chain reaction (PCR), both using a 16S rDNA approach. Additionally, biopsy specimens were taken from 3 of the 5 individuals to compare fecal and mucosal flora. The bacterial diversity of the fecal flora and the total number of bacteria were significantly reduced after 8 and 24 hours at both room temperature and 4°C. The mucosa-associated bacterial microflora showed substantial differences compared with the fecal flora. The observed alterations of fecal flora during storage point to the difficulty of the molecular analysis of the bacterial diversity and the enumeration of bacterial cells in fecal samples.     

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...     

Enhanced retrieval of DNA from human fecal samples results in improved performance of colorectal cancer screening test

Colorectal cancer accounts for more than 10% of all cancer deaths but is curable, if detected early. We reported previously on a stool-based screening test in which DNA from stool samples is subjected to genome analysis; sensitivity of the test has been limited in part by inefficiency of retrieving DNA from stool. Our aim was to test the impact of a new purification method that would increase the yield of human DNA from stool. DNA from 86 cancer and 100 non-cancer subjects (diagnosed by colonoscopy) were purified from stool with a new method for DNA recovery based on sequence-specific capture with acrylamide gel immobilized capture probes as well as with a previously developed magnetic bead-capture procedure. The new purification method gives an average 5.4-fold increase in the quantity of human DNA that can routinely be retrieved from fecal samples. The increased recovery of DNA corresponds with an increase in assay sensitivity from 53% (CI: 42 to 64%) to 70% (CI: 59 to 79%); P = 0.0005 (by McNemar's test), with no change in specificity. The newly developed sample preparation method mitigates a major problem in detecting rare cancer-associated genetic changes in heterogeneous clinical samples such as stool.     

Effect of oral ofloxacin on fecal bacteria in human volunteers.

Intestinal members of the family Enterobacteriaceae were eliminated in five human volunteers treated with oral ofloxacin for 5 days. No emergence of resistant Enterobacteriaceae was observed. Counts of group D streptococci were significantly reduced. Colonization by Candida sp. was observed in all five volunteers during ofloxacin treatment. The anaerobic flora was fairly stable from one sample to another before treatment and was not substantially modified by ofloxacin.     

Mosaic tetracycline resistance genes are widespread in human and animal fecal samples

Mosaic tetracycline resistance genes comprising tet(O), tet(W), and tet(32) sequences were abundant in DNA extracted from pig and human fecal samples, accounting for 78% (50/64) and 46% (37/80) of genes amplified with a tet(O) primer set, respectively, in two samples. The nonmosaic tet(32) gene was isolated from a human saliva bacterium.     

2451份不同类型标本中人巨细胞病毒DNA的检出情况分析

目的 对孕妇和婴儿不同类型标本中人巨细胞病毒(HCMV)DNA的检出情况进行回顾性分析,为尽早有效地检出HCMV DNA提供参考依据.方法 收集2017年5月至2020年7月在该院检验科采用荧光定量PCR法进行HCMV DNA检测的临床标本共2451份,对不同标本来源人群、不同类型标本的HCMV DNA检出率进行回顾性分析.结果 孕妇的HCMV DNA的总体检出率为3.80％,<35岁和≥35岁两个年龄段孕妇的检出率比较,差异无统计学意义(P>0.05).0～6个月婴儿的总体检出率为9.63％;不同性别婴儿的检出率比较差异无统计学意义(P>0.05);除母亲乳汁外,全血、血清、尿液标本的检出率在新生儿(<28 d)和28 d至6个月婴儿间差异均有统计学意义(P<0.05).不同类型的标本中,乳汁的检出率最高(78.49％),其次为脐血(20.00％)和尿液(6.82％),而羊水检出率最低(1.17％),脑脊液标本仅有1份且未检出.若同一婴儿采集多种类型的标本,母亲乳汁标本的检出率明显高于尿液和血清,差异有统计学意义(P<0.05).若同一孕妇中采集多种类型的标本,乳汁标本的检出率也高于其他类型标本(P<0.05).结论 孕妇和婴儿各年龄组的各类标本中,母亲乳汁标本的HCMV DNA检出率均明显高于其他类型的标本.因此对于血、尿HCMV DNA阴性而又高度疑似HCMV感染的孕妇或婴儿,建议采集乳汁标本进行检测,这有助于临床早期对HC-MV DNA的检出.     

Frequent isolation of extended-spectrum beta-lactamase-producing bacteria from fecal samples of individuals with severe motor and intellectual disabilities

Extended-spectrum beta-lactamase (ESBL) producing bacteria spread worldwide and became major concern for antibiotic treatment. Although surveillance reports in general hospitals and long-term care facilities are increasing, their frequencies in individuals with severe motor and intellectual disabilities (SMID) are so far unknown. In this study, we examined the frequency of ESBL in stool samples collected from 146 asymptomatic SMID subjects hospitalized in a single institution. With their clinical information, we evaluated possible risk factors for ESBL colonization. From 146 fecal samples, ESBL-producing bacteria were isolated in 45 cases (31%). Drug sensitivity testing showed that 82% of the isolates were resistant to levofloxacin but were sensitive to tazobactam/piperacillin and cefmetazole. The most frequent genotype was CTX-M-9 detected in 36/45 (80%). A high degree of disability, antibiotic use within three months before sampling and post-tracheostomy were statistically significant risk factors. Tube feeding was also strongly correlated with ESBL colonization (p < 0.001) and associated with lower micro-organismic diversities. Our findings are the first to reveal a high prevalence of ESBL in the fecal samples of SMID individuals and suggest possible relationships between high degree disability, tube feeding and latest histories of antibiotic use.     

DNA microarray analysis for the detection of mutations in hemophilia A

BACKGROUND: Congenital deficiency of factor (F) VIII results in the inherited X-linked bleeding disorder hemophilia A. More than 900 different mutations are reported in the hemophilia A mutation database with the largest number of mutations being single nucleotide substitutions distributed throughout the gene. Complicating the molecular characterization of this disease is the complexity of the F8 gene, the mutational heterogeneity, and technical limitations of the current mutation detection techniques. OBJECTIVE: Development of a DNA oligonucleotide microarray-based technique for F8 gene analysis to detect hemophilia A mutations. METHODS: To construct the oligonucleotide DNA microarray system: a total of 720, one base pair overlapping, 25-mer perfect match probes were designed from six exons of the F8 gene. Twenty-two different F8 gene mutations previously identified by CSGE and DNA sequence analysis were tested by using a loss-of-signal analysis approach. Differentially labeled wild type and hemophilic samples were co-hybridized to the array. Sequence alterations were detected by quantifying relative losses of test sample hybridization signals to the perfectly matched probes. RESULTS: A total of 22 different F8 mutations were tested. To test the sensitivity of the system, a blinded study was performed on 16 of the samples. F8 gene mutations can be detected with 96% efficiency with this microarray system. CONCLUSION: This proof-of-principle study has demonstrated that a F8 DNA microarray platform is an alternative gene mutation analysis approach that has a high sensitivity, and reproducibility. The methodology is, however, expensive and time consuming, and with the reduction in sequencing costs, direct sequencing is now the most cost and time efficient strategy for hemophilia A mutation analysis.     

Ecological studies of intestinal bacteria. Relation between the specificity of fecal ABO blood group antigen-degrading enzymes from enteric bacteria and the ABO blood group of the human host

This report presents evidence for enteric bacterial adaptation to genetically controlled environmental factors in the individual human host. Human feces contains bacterial enzymes that degrade water-soluble A, B, and H antigens, and both the presence and the specificity of ABH blood group antigens in human gut mucous secretions are genetically determined for each individual. In this study, partially purified fecal blood group antigen-degrading enzymes from 31 subjects of known blood group and secretor status were obtained and their relative specificity for A, B, and H antigen was measured.     

革兰氏阴性菌耐药基因检测芯片的研制及应用

目的探讨应用基因芯片对革兰氏阴性菌耐药基因进行快速检测的方法,并进行鉴定。方法根据GenBank上已发表的革兰氏阴性菌耐药基因盒不同亚型的基因序列,参考各亚型cDNAs区域的保守序列设计了针对16种耐药基因的特异探针,制备了革兰氏阴性菌耐药基因分型鉴定基因芯片。从16株临床分离的菌株中提取细菌基因组DNA,随机引物标记后与芯片进行杂交,同时与DNA测序法比较。结果用于评价革兰氏阴性菌耐药基因芯片的阳性菌株均出现良好的特异性杂交信号。结论该基因芯片可实现对革兰氏阴性菌耐药基因的快速检测。     

The sensitivity of PCR detection of Cryptosporidium oocysts in fecal samples using two DNA extraction methods.

BACKGROUND: The implementation of cost-effective intervention strategies for zoonotic protozoa relies on the development of sensitive and accurate diagnostic methods. We carried out a study to evaluate the accuracy of a PCR method for the detection of Cryptosporidium spp. oocysts in fecal samples from cattle. METHODS: Fecal samples were spiked with different numbers of oocysts and the limit of detection of the method was determined. Two methods of DNA extraction were assessed: glass beads and freeze-thawing using liquid nitrogen. A nested PCR approach was developed targeting the Cryptosporidium SSU rRNA and TRAP-C2 genes. Agreement between the diagnosis of Cryptosporidium spp. at the SSU rRNA and TRAP-C2 loci was quantified using the kappa-coefficient. RESULTS: Compared with the freeze-thawing method, the glass beads method was found to be a better way of extracting DNA from Cryptosporidium oocysts (sensitivities were 83 and 100%, respectively). The limits of detection for glass beads and freeze-thaw were low, 1 and 10 oocyst/g fecal samples, respectively. Forty-six percent of the field samples previously classified as negative for Cryptosporidium parvum by the flotation-concentration and enzyme-linked immunosorbent assay methods showed DNA with the PCR protocol. CONCLUSION: Primers for SSU rRNA are more successful in producing an amplification than primers for the TRAP-C2 gene which makes the former PCR protocol the approach of choice for detecting Cryptosporidium parvum oocysts in field samples.     

利用基因芯片技术分析空肠弯曲菌的遗传特征

目的 为分析我国弯曲菌遗传特征,本研究根据已发表多株弯曲菌的全基因组测序特征及比对结果自行设计基因芯片,利用芯片对我国不同宿主来源菌株进行遗传特异性分析。方法 根据前期基因组水平比对分析的结果,利用Combimatrix tilingCustomArrayTM 90K芯片,设计DNA芯片。芯片包含已测序菌株 ICDCCJ07001、269.97、NCTC11168、81-176、81-116和RM1221共3384个CDS的探针序列,以及空肠弯曲菌耐药及致病性相关2个质粒共80个CDS的探针序列,与脂寡糖的合成相关基因簇16种共219个CDS的探针序列、荚膜多糖合成相关基因簇7种共160个CDS的所有序列。对我国不同宿主来源27株分离菌株提取DNA,利用芯片进行杂交,获得杂交信息并分析不同菌株CDS分布特征分析及聚类特点。结果 中国菌株的主要变异区域主要存在于与脂寡糖、荚膜多糖合成相关的基因簇、鞭毛修饰相关的基因簇、DNA限制/修饰相关的基因簇以及空肠弯曲菌Mu样噬菌体基因簇。基因组水平不同来源菌株CDS分布的聚类结果没有发现显著的宿主归因特点,但GBS相关菌株脂寡糖合成相关基因组成具有共性特征。结论 通过验证以及与过去研究的比较,本次研究中的基因芯片技术结果准确可信,本研究所用基因芯片在分析空肠弯曲菌基因多态性方面具有很好的优势,可用于弯曲菌遗传特征和重要毒力因子的分析和检测。     

Detection of multiple human herpes viruses by DNA microarray technology.

BACKGROUND: The detailed characterization of virus DNA is a challenge, and the genotyping that has been achieved to date has only been possible because researchers have sent a great deal of time and effort to do so. Instead of the simultaneous detection of hundreds of viruses on a single high-density DNA-chip at very high costs per chip, we present here an alternative approach using a well-designed and tailored microarray which can establish whether or not a handful of viral genes are present in a clinical sample. METHODS: In this study we applied a new concept of microarray-based, optimized and robust biochemistry for molecular diagnostics of the herpesviruses. For comparison, all samples were genotyped using standard procedures. RESULTS: The biochemical procedure of a knowledge-based, low-density microarray was established based on the molecular diagnostics of human herpes viruses: herpes simplex virus (HSV) HSV-1, HSV-2, varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and HHV-6. The study attempted to optimize parameters of microarray design, surface chemistry, oligonucleotide probe spotting, sample labeling and DNA hybridization to the developed DNA microarray. The results of 12 900 hybridization reactions on about 150 configured herpes virus microarrays showed that the established microarray-based typing procedure was reproducible, virus-specific and sufficiently sensitive with a lower limit of 100 viral copies per mL sample. CONCLUSIONS: The developed method utilizes low-fluorescence background coverslips, epoxy surface chemistry, standardized oligonucleotide probe spotting, PCR-labeling with Cy3 of isolated DNA, array hybridization, and detecting of specific spot fluorescence by an automatic microarray reader. We expect the configured microarray approach to be the method for high-throughput associated studies on human herpes viruses.     

Direct identification of chlamydiae from clinical samples using a DNA microarray assay: a validation study

While DNA microarrays have become a widely accepted tool for mRNA expression monitoring, their use in rapid diagnosis of bacterial and viral pathogens is only emerging. So far, insufficient sensitivity and high costs have been the major limiting factors preventing more widespread use of microarray platforms in direct testing of clinical samples. In the present study, a total of 339 samples, among them 293 clinical specimens from animals and humans, were examined by the ArrayTube (AT) DNA microarray assay to detect chlamydial DNA and identify the species of Chlamydia and Chlamydophila involved. Samples included nasal and conjunctival swabs, formalin-fixed, paraffin-embedded and fresh organ tissue, milk, feces and cell culture. Notably, the AT test was shown to detect mixed infections in clinical samples. The calculated median sensitivity of 0.81 over the entire panel of clinical samples was comparable to conventional 16S PCR, but slightly lower than real-time PCR and other PCR assays. However, when a panel of long-time stored swab samples was excluded from the calculation, the sensitivity was clearly higher (0.87) and equivalent to that of real-time PCR. Altogether, the data demonstrate the suitability of this DNA microarray assay for routine diagnosis.     

Genotyping of Chlamydia trachomatis strains from culture and clinical samples using an ompA-based DNA microarray assay

Current typing methods of Chlamydia (C.) trachomatis are mainly based on the diversity of the ompA gene, which is coding for the major outer membrane protein A. The present study aimed at facilitating genotyping of strains of this obligate intracellular human pathogen by developing a DNA microarray assay using the ArrayTube? format for individual samples and the ArrayStrip? format for higher throughput.The new test is exploiting multiple discriminatory sites by involving a total of 61 oligonucleotide probes representing genotype-specific polymorphisms in variable domains 1, 2 and 4 of the ompA gene. After multiplex amplification of these domains using biotinylated primers, the sample is hybridized in the microarray vessel under highly stringent conditions. The resulting binding pattern is genotype specific, thus allowing direct identification. We were able to show that DNA from each of the currently accepted genotypes (serovars) yielded a unique, theoretically expected and distinct hybridization pattern. The assay was also shown to be highly sensitive as a dilution containing the equivalent of 1 inclusion-forming unit was still correctly genotyped. In addition, when 62 clinical samples were examined and compared to PCR-RFLP typing results, the genotype was correctly identified by the DNA microarray in all cases.The present test is easy to handle and economically affordable, and it allows genotyping of C. trachomatis to be accomplished within a working day, thus lending itself for epidemiological studies and routine diagnosis.     

导流杂交基因芯片检测人乳头状瘤病毒21种亚型的应用评价

目的了解基因芯片检测人乳头状瘤病毒(HPV)21种亚型感染的临床应用价值。评价导流杂交基因芯片技术(凯普导流杂交)HPV DNA检测法(HybriMax)在女性生殖道HPV感染检测中的临床效用,初步探讨最常见的HPV基因型。方法利用标记有生物素的HPV通用引物进行PCR扩增,扩增产物变性后与固定在尼龙膜上21种HPV特异性分型探针进行反向斑点杂交检测HPV亚型。结果 591例患者标本中,基因芯片阳性率69.2%,其中单一亚型感染266例,重叠感染143例。共检出亚型20种。结论基因芯片一次试验可联合检测多种HPV亚型感染并分型,可研究HPV感染型别的分布,提高由HPV引起的肿瘤防治水平。     

Effects of roxithromycin on fecal bacteria in human volunteers and resistance to colonization in gnotobiotic mice.

The ecological impact of roxithromycin given orally at 300 mg/day on the intestinal floras in six human volunteers was studied. The resulting fecal concentrations of active roxithromycin were in the range of 100 to 200 micrograms/g of feces. Consecutive modifications in the composition of the fecal floras were limited to a decrease in counts of total members of the family Enterobacteriaceae. The rest of the intestinal floras, including the predominant anaerobic floras, changed little. No overgrowth of Pseudomonas aeruginosa, staphylococci, fungi, or highly erythromycin-resistant strains of the family Enterobacteriaceae was observed. The strains of Enterobacteriaceae and of anaerobes isolated during treatment were not markedly more resistant to roxithromycin than those isolated before treatment started. Changes in intestinal resistance to colonization by exogenous microorganisms in gnotobiotic mice inoculated with human fecal flora were studied and were also found to be minimal. The impact of oral roxithromycin on the intestinal microbiota appears to be weaker than that previously observed with oral erythromycin, perhaps because the concentrations of roxithromycin in the feces were lower than those previously found for erythromycin.