A novel DNA microarray for rapid diagnosis of enteropathogenic bacteria in stool specimens of patients with diarrhea

A microarray technique for the detection and identification of enteropathogenic bacteria at the species and subspecies levels was developed in this study, and the target bacteria included pathogenic Escherichia coli, Vibrio cholerae, Vibrio parahaemolyticus, Salmonella enterica, Campylobacter jejuni, Shigellae, Yersinia enterocolitica, and Listeria monocytogenes. The virulence gene of each pathogen was chosen as the amplification target, labeled with a fluorescence dye by multiplex polymerase chain reaction (PCR), and hybridized to the specific virulence gene probes that had been immobilized on a microchip. Stool specimens from 34 patients with diarrhea were tested in this study. Five were positive for multiple genera. Nested PCRs and sequencing were used to amplify and identify the related genes, which were found to share 95.8% to 100% of the nucleotide identity with the corresponding regions in the Genbank database. Real-time PCR was used to determine the number of gene copies to determine the sensitivity of this technique, which was shown to be 58 copies/μl. The results indicated that the microarray technique which targets multiple virulence genes of enteropathogenic bacteria at the species and subspecies levels is an attractive diagnostic tool for rapidly and simultaneously identifying multiple enteropathogenic pathogens in clinical practice, especially in patients with infectious diarrhea.     

A novel method for rapid isolation of plasmid DNA

A new disposable chromatographic column, pZ523, has been developed for separating plasmid DNA from bacterial chromosomal DNA. Use of pZ523 spun columns eliminates the need for ethidium bromide-cesium chloride density gradients which require long centrifugation times. pZ523 purified plasmids have been shown to be of purity suitable for restriction analysis, ligation, transfection of mammalian cells and transformation of bacteria. Unlike the traditional ultracentrifugation method, pZ523 offers an extremely rapid alternative method for purifying large amounts of plasmid DNA (2.5 mg to 4.5 mg) from cleared bacterial lysates in only 25 minutes.     

A novel rapid DNA microarray assay enables identification of 37 Mycoplasma species and highlights multiple Mycoplasma infections

Mycoplasmas comprise a conglomerate of pathogens and commensals occurring in humans and animals. The genus Mycoplasma alone contains more than 120 species at present, and new members are continuously being discovered. Therefore, it seems promising to use a single highly parallel detection assay rather than develop separate tests for each individual species. In this study, we have designed a DNA microarray carrying 70 oligonucleotide probes derived from the 23S rRNA gene and 86 probes from the tuf gene target regions. Following a PCR amplification and biotinylation step, hybridization on the array was shown to specifically identify 31 Mycoplasma spp., as well as 3 Acholeplasma spp. and 3 Ureaplasma spp. Members of the Mycoplasma mycoides cluster can be recognized at subgroup level. This procedure enables parallel detection of Mollicutes spp. occurring in humans, animals or cell culture, from mono- and multiple infections, in a single run. The main advantages of the microarray assay include ease of operation, rapidity, high information content, and affordability. The new test's analytical sensitivity is equivalent to that of real-time PCR and allows examination of field samples without the need for culture. When 60 field samples from ruminants and birds previously analyzed by denaturing-gradient gel electrophoresis (DGGE) were tested by the microarray assay both tests identified the same agent in 98.3% of the cases. Notably, microarray testing revealed an unexpectedly high proportion (35%) of multiple mycoplasma infections, i.e., substantially more than DGGE (15%). Two of the samples were found to contain four different Mycoplasma spp. This phenomenon deserves more attention, particularly its implications for epidemiology and treatment.     

A rapid method for the isolation of plasmid DNA from bacteria

Summary A rapid method is described for the isolation of plasmid DNA from Escherichia coli and Pseudomonas putida. The effect of heating the cell preparation during plasmid extraction is discussed in relationship to the final plasmid yield.     

Prospects for using DNA probes for rapid diagnosis of antibiotic resistance in clinical strains of enteric bacteria

Aminoglucoside resistance patterns of clinical strains of enteric bacteria isolated from inpatients of Moscow clinics were determined. APH(3')-I and AAC(3)-II were shown to be the most frequent. The aphA1 and aacC2 genes encoding the enzymes were cloned from the R plasmid of the transconjugant of the E. coli clinical strains. DNA probes based on the determined nucleotide sequences of the cloned genes were constructed and used in DNA-DNA hybridization experiments. The results on the occurrence of APH(3')-I and AAC(3)-II in the strains tested were confirmed by the DNA-DNA hybridization. Prospects for developing a set of DNA probes for rapid diagnosis of antibiotic resistance are discussed.     

腹泻患儿粪便A群轮状病毒抗原检测的结果分析

目的:了解本地区腹泻婴幼儿的A群轮状病毒感染情况及其流行特点。方法:采用胶体金法对我院2009年10月-2010年9月2104例有腹泻和肠炎特征的婴幼儿粪便进行A群轮状病毒抗原检测。结果:在2104例受检者中,A群轮状病毒感染的总阳性率是24.71%,其中男性感染率24.17%,女性为25.40%。不同年龄组间以1-2岁婴幼儿感染率最高,为32.13%,0-1岁为20.72%,2-5岁为12.03%。感染的季节特征是秋末冬初季(10-12月)阳性率最高,为42.82%,春末夏初季(4-6月)最低,为8.81%。结论:由A群轮状病毒感染引发的急性腹泻主要发生在1-2岁的婴幼儿,各个季节均有发生,以秋末冬初季为高发。     

腹泻患儿粪便A群轮状病毒抗原检测的结果分析

目的：了解本地区腹泻婴幼儿的A群轮状病毒感染情况及其流行特点。方法：采用胶体金法对我院2009年10月-2010年9月2104例有腹泻和肠炎特征的婴幼儿粪便进行A群轮状病毒抗原检测。结果：在2104例受检者中,A群轮状病毒感染的总阳性率是24．71％,其中男性感染率24．17％,女性为25．40％。不同年龄组间以1—2岁婴幼儿感染率最高,为32．13％,0-1岁为20．72％,2-5岁为12．03％。感染的季节特征是秋末冬初季（10—12月）阳性率最高,为42．82％,春末夏初季（4．6月）最低,为8．81％。结论：由A群轮状病毒感染引发的急性腹泻主要发生在1-2岁的婴幼儿,各个季节均有发生,以秋末冬初季为高发。     

A method for assessment of Helicobacter pylori genotype using stool specimens

Helicobacter pylori infection has been regarded as a major factor associated with the development of gastric diseases. The characterization of infected H. pylori in asymptomatic individuals is important for the prediction of the onset of such diseases. However, because of the difficulty in obtaining gastric biopsy samples, H. pylori in healthy subjects have not been studied sufficiently. Therefore, we tested a noninvasive method for the characterization of H. pylori using stool specimens. This method involved H. pylori antigen detection in stool specimens by immunochromatography; confirmation of H. pylori DNA by real-time PCR that involved the detection of its 16S rRNA gene in the DNA extracted from stool specimens; and nested PCR with genotype-specific primer pairs. A total of 80 samples obtained from asymptomatic subjects were assessed using this method. The results showed that the prevalence of H. pylori in asymptomatic Japanese individuals was 37.5%. The detection rate of the virulence factor gene cagA was 18.8%. Furthermore, all the detected cagA belonged to the highly virulent East-Asian type. These data suggest that the method used in this study is valuable for studying the molecular epidemiology of H. pylori infection in asymptomatic people.     

123例腹泻患儿粪便中艰难梭菌毒素基因特征及感染危险因素分析

探讨腹泻患儿粪便中艰难梭菌毒素基因特征, 并分析产毒艰难梭菌感染的危险因素。

采集2019年1月至2021年3月嘉兴市第二医院儿科收治的腹泻患儿的粪便标本共123份, 其中社区获得性腹泻患儿60例, 医院获得性腹泻患儿63例。标本进行厌氧培养, 采用实时荧光PCR鉴定艰难梭菌tpi基因, 并检测tcd A、tcd B毒素基因。收集患儿临床资料, 采用Logistic回归分析产毒艰难梭菌感染的危险因素。

123例粪便标本共检出艰难梭菌tpi基因阳性35例(28.46%), 毒素基因中tcd A⁺ B⁺为19例(15.45%), tcd A⁺B^-为3例(2.44%), tcd A^-B⁺为2例(1.63%), tcd A^-B^-为11例(8.94%)。社区获得性腹泻患儿中tcd A/B产毒艰难梭菌感染率为28.33%, 高于医院获得性腹泻患儿的11.11%(P < 0.05)。产毒艰难梭菌感染腹泻患儿与非产毒艰难梭菌感染腹泻患儿的性别、年龄、居住地、是否早产、有无先天性疾病、既往有无胃肠道手术、抗生素应用种类、近1个月内有无机械通气、临床症状、血液白细胞计数、血红蛋白、粪便白细胞计数、血清C反应蛋白、血清白细胞介素-6、血清白蛋白、血肌酐、血清总胆红素水平比较差异无统计学意义(均P > 0.05)。产毒艰难梭菌感染腹泻患儿中的非母乳喂养、近1个月内抗生素用药史、抗生素持续用药时间 > 7 d、近1个月内糖皮质激素用药史、近1个月内抑酸药用药史、近1个月内肠内营养者的比例显著高于非产毒艰难梭菌感染腹泻患儿(均P < 0.05)。非母乳喂养(OR=2.433)、近1个月内抗生素用药史(OR=3.040)、近1个月内肠内营养(OR=2.330)是腹泻患儿产毒艰难梭菌感染的独立危险因素(均P < 0.05)。

嘉兴地区儿童艰难梭菌毒素基因主要为tcd A/B基因, 患儿以社区获得性腹泻为主, 非母乳喂养、抗生素用药史、肠内营养的儿童更容易感染艰难梭菌。

     

A simple and rapid method for preparing genomic DNA from gram-positive bacteria

Molecular epidemiologic and other studies may require preparation of genomic DNA from large numbers of bacteria in sufficiently pure form for restriction endonuclease digestion, cloning, RAPD-PCR, Southern hybridization, and so on.Staphylococcus and other Gram-positive bacteria have a rigid cell wall and can be difficult to lyse. Here, a simple and rapid method for the preparation of genomic DNA from multiple samples is reported. This method produces clean DNA for use in most molecular biology methods in <90 min.     

A DNA microarray facilitates the diagnosis of Bacillus anthracis in environmental samples

Aims:  In order to improve the diagnosis of Bacillus anthracis in environmental samples, we established a DNA microarray based on the ArrayTube technology of Clondiag.
Methods and Results:  Total DNA of a bacterial colony is randomly biotinylated and hybridized to the array. The probes on the array target the virulence genes, the genomic marker gene rpoB , as well as the selective 16S rDNA sequence regions of B. anthracis , of the Bacillus cereus group and of Bacillus subtilis . Eight B. anthracis reference strains were tested and correctly identified. Among the analysed environmental Bacillus isolates, no virulent B. anthracis strain was detected.
Conclusions:  This array clearly differentiates B. anthracis from members of the B. cereus group and other Bacillus species in environmental samples by chromosomal ( rpoB ) and plasmid markers. Additionally, recognition of B. cereus strains harbouring the toxin genes or atypical B. anthracis strains that have lost the virulence plasmids is feasible.
Significance and Impact of the Study:  The array is applicable to the complex diagnostics for B. anthracis detection in environmental samples. Because of low costs, high security and easy handling, the microarray is applicable to routine diagnostics.     

A rapid procedure for the isolation of plasmid DNA from environmental bacteria.

The INSTA-MINI-PREP method, a rapid protocol for plasmid DNA extraction, was originally developed to prepare plasmid DNA from 1 to 3 ml miniprep Escherichia coli cultures. Direct extraction of plasmid DNA is achieved by a two-phase solution which is separated by centrifugation in the presence of the INSTA-PREP gel barrier material. This method has been successfully tested on various environmental Salmonella strains, although it was not suitable for Pseudomonas aeruginosa and enterococci strains. The INSTA-MINI-PREP method is a new alternative procedure to screen plasmid contents of Salmonella and E. coli strains rapidly and easily.     

DNA extraction protocol for rapid PCR detection of pathogenic bacteria

Twelve reagents were evaluated to develop a direct DNA extraction method suitable for PCR detection of foodborne bacterial pathogens. Many reagents exhibited strong PCR inhibition, requiring significant dilution of the extract with a corresponding reduction in sensitivity. Most reagents also exhibited much lower recovery of DNA from the gram-positive test organism (Listeria monocytogenes) than from the gram-negative organism (Escherichia coli O157:H7), preventing unbiased detection and quantitation of both organisms. The 5× HotSHOT + Tween reagent exhibited minimal inhibition and high extraction efficiency for both test organisms, providing a 15-min single-tube DNA-extraction protocol suitable for highly sensitive quantitative PCR assays.     

Multicenter comparison of rapid lateral flow stool antigen immunoassay and stool antigen enzyme immunoassay for the diagnosis of Helicobacter pylori infection in children

BACKGROUND AND AIM: The stool antigen enzyme immunoassay (EIA) methods are widely used for diagnosing Helicobacter pylori infection. Recently, a novel, rapid stool antigen test, the lateral flow immunoassay (LFI) method, has been developed. The primary purpose of this study was to compare the EIA method with the LFI method for the diagnosis of H. pylori infection in children. MATERIALS AND METHODS: Stool specimens from children being evaluated for H. pylori infection were also examined using the LFI (ImmunoCard STAT! HpSA) and EIA methods (Premier Platinum HpSA). The sensitivity, specificity and accuracy of the test were based on the 13C-labeled urea breath test. RESULTS: One hundred and eighty-two children and adolescents, 3-17 years of age (mean 9.2 years), were studied. In addition, 29 patients who received eradication therapy were re-evaluated 2 or 3 months post-treatment. The 13C-labeled urea breath test was positive in 64 patients (35.2%). The sensitivity, specificity and accuracy of the LFI method were 90.6% (95% CI = 80.7-96.5%), 95.8% (92.1-99.4%), and 94.0% (90.5-97.4%), respectively and for the EIA method, sensitivity, specificity and accuracy were 96.8% (95% CI, 89.0-99.6%) and 99.2% (97.5-100%), and 98.3% (96.5-100%), respectively. There were no significant differences in results among the age groups 3-5, 6-10 and 11-17 years. As for the assessment of H. pylori eradication, the results of the LFI and EIA methods agreed with those of 13C-urea breath test in 27/29 and 29/29 patients, respectively. CONCLUSIONS: The LFI stool antigen method showed a good sensitivity, specificity and accuracy for diagnosing H. pylori infection in children. This novel method may be useful in clinical practice as an office-based test because it is rapid, reliable and easy to perform.     

A novel Mixture Model Method for identification of differentially expressed genes from DNA microarray data

Background  

The main goal in analyzing microarray data is to determine the genes that are differentially expressed across two types of tissue samples or samples obtained under two experimental conditions. Mixture model method (MMM hereafter) is a nonparametric statistical method often used for microarray processing applications, but is known to over-fit the data if the number of replicates is small. In addition, the results of the MMM may not be repeatable when dealing with a small number of replicates. In this paper, we propose a new version of MMM to ensure the repeatability of the results in different runs, and reduce the sensitivity of the results on the parameters.