A novel viral RNA detection method based on the combined use of trans-acting ribozymes and HCR-FRET analyses.

The diagnoses of retroviruses are essential for controlling the rapid spread of pandemics. However, the real-time Reverse Transcriptase quantitative Polymerase Chain Reaction (RT-qPCR), which has been the gold standard for identifying viruses such as SARS-CoV-2 in the early stages of infection, is associated with high costs and logistical challenges. To innovate in viral RNA detection a novel molecular approach for detecting SARS-CoV-2 viral RNA, as a proof of concept, was developed. This method combines specific viral gene analysis, trans-acting ribozymes, and Fluorescence Resonance Energy Transfer (FRET)-based hybridization of fluorescent DNA hairpins. In this molecular mechanism, SARS-CoV-2 RNA is specifically recognized and cleaved by ribozymes, releasing an initiator fragment that triggers a hybridization chain reaction (HCR) with DNA hairpins containing fluorophores, leading to a FRET process. A consensus SARS-CoV-2 RNA target sequence was identified, and specific ribozymes were designed and transcribed in vitro to cleave the viral RNA into fragments. DNA hairpins labeled with Cy3/Cy5 fluorophores were then designed and synthesized for HCR-FRET assays targeting the RNA fragment sequences resulting from ribozyme cleavage. The results demonstrated that two of the three designed ribozymes effectively cleaved the target RNA within 10 minutes. Additionally, DNA hairpins labeled with Cy3/Cy5 pairs efficiently detected target RNA specifically and triggered detectable HCR-FRET reactions. This method is versatile and can be adapted for use with other viruses. Furthermore, the design and construction of a DIY photo-fluorometer prototype enabled us to explore the development of a simple and cost-effective point-of-care detection method based on digital image analysis.

VarGibbs Usage in the Optimization of Nearest-Neighbor Parameters and Prediction of Melting Temperature of RNA Duplexes.

The nearest-neighbor (NN) model is a general tool for the evaluation for oligonucleotide thermodynamic stability. It is primarily used for the prediction of melting temperatures but has also found use in RNA secondary structure prediction and theoretical models of hybridization kinetics. One of the key problems is to obtain the NN parameters from melting temperatures, and VarGibbs was designed to obtain those parameters directly from melting temperatures. Here we will describe the basic workflow from RNA melting temperatures to NN parameters with the use of VarGibbs. We start by a brief revision of the basic concepts of RNA hybridization and of the NN model and then show how to prepare the data files, run the parameter optimization, and interpret the results.

A fluorometric hybridization assay for detecting and genotyping high-risk human papillomavirus 16 and 18 in archival tissues of cervical specimens.

Early diagnosis and genotyping of high-risk human papillomavirus (HR-HPV) in cervical tissue specimens is significant for cervical cancer prevention. A sensitive microplate fluorometric hybridization assay (MFHA) was designed for the detection of HPV DNA 16 and 18 in cervical tissue. Following optimization and validation of the method, 60 formalin-fixed and paraffin-embedded cervical samples representing different cervical intraepithelial neoplasia grades of HPV-associated lesions were tested to determine the sensitivity and specificity of the assay. Using consensus GP5+/6+ biotin-labeled primers to amplify a conserved region within the L1 gene, the amplicons were added to the microplate wells coated with specific probes for the hybridization of HPV 16 and 18 individually. Final detection was performed with streptavidin-AlexaFluor488 conjugated. The results were then compared with type-specific nested polymerase chain reaction (PCR) and colorimetric microplate assay. While the agreement between the results obtained by the type-specific nested PCR and fluorometric assay for the detection of both HR-HPV types was 100%, this agreement for the detection of HPV type 16 and 18 using microplate colorimetric assay was 94.2% and 85% respectively. Overall, the results of the fluorometric and colorimetric assays are promising for detecting both HR-HPV types in a large number of cervical tissue samples with the higher MFHA assay sensitivity.

Hybridization Capture of Ancient DNA Using RNA Baits.

The majority of DNA recovered from ancient remains is derived from organisms that colonize the remains post-mortem, such as soil microbes, or from contaminants, such as DNA from living humans. Additionally, some ancient DNA research projects aim to target specific genomic regions, such as mitochondrial genomes or variable single nucleotide polymorphisms (SNPs). To overcome the challenge of targeting specific fragments of DNA from within a complex DNA extract, methods have been developed to enrich ancient DNA extracts for target DNA relative to nontarget DNA. This chapter describes a method for target DNA enrichment that uses hybridization to biotinylated RNA baits to capture and amplify specific ancient DNA fragments from within the pool of extracted fragments.

Comparative Molecular Analysis of Gram-Negative Bacteria in Primary Teeth with Irreversible Pulpitis or Periapical Pathology.

Purpose: The purpose of this study was to compare the Gram-negative pathogens identified in the root canals of primary teeth with irreversible inflammatory pulpitis and in teeth showing apical periodontitis. Methods: Samples were collected from 123 root canals of primary teeth from three- to seven-year-old patients. Root canals were assigned to either group one (irreversible inflammatory pulpitis; n equals 63) or group two (pulp necrosis and apical periodontitis; n equals 60). Total number of cells of selected Gram-negative microorganisms was determined by the checkerboard DNA-DNA hybridization technique. Demographic data were compared using either chi-squared or t tests. Total numbers of microorganisms were compared using the Mann-Whitney test (α equals 0.05). Results: There were no significant intergroup differences in gender, age, and tooth group distribution (P>0.05). Among the 123 samples, 17 were discarded due to salivary contamination. The total numbers of Prevotella nigrescens, Treponema denticola, Fusobacterium nucleatum polymorphum, Fusobacterium nucleatum spp nucleatum, Aggregatibacter actinomycetemcomitans serotype a, Aggregatibacter actinomycetemcomitans serotype b, Porphyromonas gingivalis, Prevotella intermedia, and Prevotella melaninogenica were higher in teeth with apical periodontitis compared to those with irreversible inflammatory pulpitis (P<0.05). Conclusion: Higher numbers of Gram-negative bacteria were found in teeth with apical periodontitis compared to teeth with irreversible in- flammatory pulpitis.

Successful and failed mini-implants: microbiological evaluation and quantification of bacterial endotoxin.

OBJECTIVES: Using two groups of mini-implants (successful and failed) the objectives of this in vivo study were: to evaluate the microbial contamination by the checkerboard DNA-DNA hybridization technique and to quantify the bacterial endotoxin by the limulus amebocyte lysate assay. MATERIAL AND METHODS: The 15 successful and 10 failed mini-implants (1.6 mm diameter × 7.0 or 9.0 mm long), placed in the maxilla and/or mandible, were obtained from 15 patients undergoing orthodontic treatment. Data were analyzed statistically by the Wilcoxon rank-sum test using the SAS software (a=0.05). RESULTS: All 40 microbial species were detected in both groups of mini-implants, with different frequencies. No differences were observed between the groups with respect to microbial complexes (blue, purple, yellow, green, orange, red and other species) and endotoxin quantification (p>0.05). CONCLUSION: Neither microbial contamination nor endotoxin quantification was determinant for the early loss of stability of the mini-implants.

Characterization and electrochemical response of DNA functionalized 2nm gold nanoparticles confined in a nanochannel array.

Polyvalent gold nanoparticle oligonucleotide conjugates are subject of intense research. Even though 2nm diameter AuNPs have been previously modified with DNA, little is known about their structure and electrochemical behavior. In this work, we examine the influence of different surface modification strategies on the interplay between the meso-organization and the molecular recognition properties of a 27-mer DNA strand. This DNA strand is functionalized with different sulfur-containing moieties and immobilized on 2nm gold nanoparticles confined on a nanoporous alumina, working the whole system as an electrode array. Surface coverages were determined by EXAFS and the performance as recognition elements for impedance-based sensors is evaluated. Our results prove that low DNA coverages on the confined nanoparticles prompt to a more sensitive response, showing the relevance in avoiding the DNA strand overcrowding. The system was able to determine a concentration as low as 100pM of the complementary strand, thus introducing the foundations for the construction of label-free genosensors at the nanometer scale.

Efficacy of a single session protocol for endodontic treatment in primary teeth: in vivo study.

AIMS: To determine the efficacy of a single session protocol (SSP) in the reduction of septic content of primary teeth root canals and identify the persistence of bacterial species associated with unsuccessful treatment. METHODS: Primary teeth root canals (16) with pulp necrosis and peri-radicular lesions were treated. Samples were collected at baseline (T1), and after chemo-mechanical preparation, before filling (T2). Identification of the microorganisms was determined using checkerboard DNA-DNA hybridisation. STATISTICAL ANALYSIS: Wilcoxon test was applied for comparison of mean number of species, proportion and mean count of each species between the evaluation times. RESULTS: Significant reductions were found in the mean number of bacteria species between T1 and T2 (p < 0.05), but not for the reduction in proportion (p > 0.05). There was a reduction (6.0-4.6) of the mean number of species associated with failure, without statistical significance. CONCLUSION: The SSP was capable of significantly reducing the septic content, even though, many of the bacteria associated with failure persisted at the time of root canal filling.

Infecções endodônticas primária x secundária: perfil microbiano, níveis de endotoxinas e ácido lipoteicóico, sinais e sintomas / Primary x secondary endodontic infections: microbial profile, endotoxins and lipoteichoic acid levels, signs and symptoms

Esta pesquisa clínica teve como objetivo comparar a carga e composição microbiana bem como as concentrações de LPS e LTA encontradas na infecção endodôntica primária (IEP) e na infecção endodôntica secundária (IES). Além disso, a correlação desses achados com características clínicas e tomográficas também foram investigadas. Sessenta dentes de pacientes com IEP (31) e IES (29) foram submetidos à avaliação clínica e tomográfica, seguido do tratamento endodôntico ou retratamento. Amostras foram coletadas de cada canal radicular utilizando cones de papel. Logo após a abertura coronária (IEP) ou após a desobturação dos canais (IES) o conteúdo coletado foi submetido à técnica de cultura microbiológica para determinar a carga microbiana de bactérias anaeróbias e ao método Checkerboard DNA-DNA hybridization para investigação de espécies bacterianas presentes. O teste de Lisado de Amebócito de Limulus e o Ensaio de Imunoabsorção Enzimática foram utilizados para quantificar os níveis de LPS e LTA. Os dados obtidos foram correlacionados com os achados clínicos e tomográficos. Maiores quantidades de bactérias cultiváveis e de LPS foram encontradas na IEP (p < 0,05). Não houve diferença nos níveis de LTA entre IEP e IES (p > 0,05). A mediana de espécies por canal radicular encontrada na IEP foi de 9 espécies e na IES foi de 22 (p < 0,05). As espécies bacterianas mais prevalentes detectadas na IEP foram P. gingivalis (14/31) e S. intermedius (14/31). Na IES, as espécies mais prevalentes foram P. gingivalis (21/29) e C. rectus (20/29). LPS foi correlacionado positivamente com um maior volume da lesão periapical (p < 0,05). Níveis de LTA não foram relacionados a sinais e sintomas ou ao volume da lesão periapical (p > 0,05). Concluiu-se que dentes com IEP tiveram maiores quantidades de carga microbiana e de LPS do que os dentes com IES. Uma maior quantidade de LPS foi correlacionada positivamente com um maior volume de destruição óssea periapical. Uma ampla interação de espécies bacterianas específicas resultou em diferentes características clínicas(AU)

This clinical research aimed to compare the microbial load and composition as well as the LPS and LTA concentrations found in Primary Endodontic Infection (PEI) and Secondary Endodontic Infection (SEI). In addition, the correlation of these findings with clinical and tomographic features was also investigated. Sixty patients' teeth with PEI (31) and SEI (29) were submitted to clinical and tomographic assessment, followed by endodontic treatment or retreatment. Samples were taken from each root canal using paper points. After the coronary opening (PEI) or after the removal of root filling material (SEI), the collected samples were submitted to the microbiological culture technique to determine the microbial load of anaerobic bacteria and to the Checkerboard DNA-DNA hybridization method for investigation of present bacterial species. The Limulus amebocyte lysate assay and enzyme-linked immunosorbent assay were used to quantify LPS and LTA levels. The data obtained were correlated with clinical and tomographic findings. A higher number of cultivable bacteria and LPS was found in PEI (p < 0.05). There was no difference in LTA levels between PEI and SEI (p> 0.05).The median number of species per root canal found in PEI was 9 and 22 in SEI (p < 0.05). The most prevalent bacterial species detected in PEI were P. gingivalis (14/31) and S. intermedius (14/31). In SEI, the most prevalent species were P. gingivalis (21/29) and C. rectus (20/29). LPS was positively correlated with a larger periapical lesion volume (P < 0.05). LTA levels were not related to signs and symptoms or periapical lesion volume (p> 0.05). It was concluded teeth with PEI had higher contents of microbial load and LPS than teeth with SEI. However, a more diverse microbiota was found in SEI than that of PEI. Higher content of LPS was positively correlated with larger periapical bone destruction. A widely interaction of specific microbial species resulted in different clinical features(AU)

Successful and failed mini-implants: microbiological evaluation and quantification of bacterial endotoxin

Abstract Objectives Using two groups of mini-implants (successful and failed) the objectives of this in vivo study were: to evaluate the microbial contamination by the checkerboard DNA-DNA hybridization technique and to quantify the bacterial endotoxin by the limulus amebocyte lysate assay. Material and Methods The 15 successful and 10 failed mini-implants (1.6 mm diameter × 7.0 or 9.0 mm long), placed in the maxilla and/or mandible, were obtained from 15 patients undergoing orthodontic treatment. Data were analyzed statistically by the Wilcoxon rank-sum test using the SAS software (a=0.05). Results All 40 microbial species were detected in both groups of mini-implants, with different frequencies. No differences were observed between the groups with respect to microbial complexes (blue, purple, yellow, green, orange, red and other species) and endotoxin quantification (p>0.05). Conclusion Neither microbial contamination nor endotoxin quantification was determinant for the early loss of stability of the mini-implants.

Nonradioactive Detection of Small RNAs Using Digoxigenin-Labeled Probes.

Small RNAs have been traditionally detected and quantified using small RNA blots, a modified Northern blot technique. The small RNAs are size-fractionated from the rest of the cellular RNA molecules by polyacrylamide gel electrophoresis and transferred by blotting onto a positively charged membrane. A radiolabeled probe was then traditionally used to detect a specific small RNA in the cellular pool. Small RNA blotting is a relatively simple, inexpensive approach to visualize small RNAs without artifacts. However, the radioactive labeling of the probe is sometimes an impediment, especially due to the requirement of specialized facilities. Here we describe a sensitive and simple method to detect and quantify small RNAs using digoxigenin-based nonradioactive RNA blots.

Comparison of DNA Microarray, Loop-Mediated Isothermal Amplification (LAMP) and Real-Time PCR with DNA Sequencing for Identification of Fusarium spp. Obtained from Patients with Hematologic Malignancies.

The performance of three molecular biology techniques, i.e., DNA microarray, loop-mediated isothermal amplification (LAMP), and real-time PCR were compared with DNA sequencing for properly identification of 20 isolates of Fusarium spp. obtained from blood stream as etiologic agent of invasive infections in patients with hematologic malignancies. DNA microarray, LAMP and real-time PCR identified 16 (80%) out of 20 samples as Fusarium solani species complex (FSSC) and four (20%) as Fusarium spp. The agreement among the techniques was 100%. LAMP exhibited 100% specificity, while DNA microarray, LAMP and real-time PCR showed 100% sensitivity. The three techniques had 100% agreement with DNA sequencing. Sixteen isolates were identified as FSSC by sequencing, being five Fusarium keratoplasticum, nine Fusarium petroliphilum and two Fusarium solani. On the other hand, sequencing identified four isolates as Fusarium non-solani species complex (FNSSC), being three isolates as Fusarium napiforme and one isolate as Fusarium oxysporum. Finally, LAMP proved to be faster and more accessible than DNA microarray and real-time PCR, since it does not require a thermocycler. Therefore, LAMP signalizes as emerging and promising methodology to be used in routine identification of Fusarium spp. among cases of invasive fungal infections.

Minicircle classes heterogeneity within the TcIII and TcIV discrete typing units of Trypanosoma cruzi.

The taxon Trypanosoma cruzi, causative agent of Chagas disease, is composed of several discrete typing units (DTUs) named TcI-TcVI, and Tcbat. The history of the taxon T. cruzi is known, even though several controversial aspects remain as the relationships between TcIII and TcIV. We analyzed cloned T. cruzi stocks pertaining to the seven DTUs by filter hybridization tests of PCR amplicons from minicircle variable regions and kinetoplast DNA probes. Minicircle DNA blots from the cloned stocks and filter hybridization with one TcI, one TcII, one TcV, one TcVI, three TcIII, one TcIV from North America and one TcIV kinetoplast DNA probes from South America revealed minicircle variable region cross-reaction in some T. cruzi DTUs probed. TcIII was heterogeneous in minicircle class composition, even though two TcIII probes revealed that a small fraction of minicircles cross-hybridized with the minicircles from the TcIII, TcV and TcVI DTUs. The minicircles of TcIV from North America cross-reacted only with TcIV from North America but not with TcIV stocks from Brazil and Bolivia. The results on minicircle cross-hybridizations are discussed in the context of RNA editing, mitochondrial function in T. cruzi DTUs.

Different sealing materials preventing the microbial leakage into the screw-retained implant restorations: an in vitro analysis by DNA checkerboard hybridization.

OBJECTIVES: The aim of this controlled in vitro study was to identify and quantify up to 38 microbial species penetrating through the screw-retained implant prostheses with different sealing materials. MATERIAL AND METHODS: Sixty morse cone implants were restored with single-unit screw-retained prostheses. All the components were randomly divided into five groups (n = 12) according to the proposed materials: (1) polytetrafluoroethylene tape+composite resin; (2) polytetrafluoroethylene tape+gutta-percha; (3) polytetrafluoroethylene tape+light-polymerized provisional composite; (4) cotton pellet+gutta-percha; and (5) cotton pellet+light-polymerized provisional composite. Human saliva was used as contaminant media, and DNA checkerboard hybridization was used to identify and quantify microbial species. RESULTS: Microbial leakage was observed in all groups: M. salivarium, S. pasteuri, P. nigrescens, and P. melaninogenica were the species presenting the highest values of genome count, prevalence, and proportion within the groups. The total microbial mean counts (×105 , ±SD) were as follows: Group 1 (2.81 ± 0.38), Group 2 (3.41 ± 0.38), Group 3 (6.02 ± 1.48), Group 4 (6.40 ± 1.42), and Group 5 (17.45 ± 1.67). Group 5 showed the higher microbial counts (P < 0.001). CONCLUSIONS: Moderate to high counts of pathogenic/nonpathogenic species were detected in the inner parts of implants from all groups. The lowest values of microbial counts were recorded for polytetrafluoroethylene tape associated with composite resin or gutta-percha; cotton pellet associated with light-polymerized provisional composite presented the highest microbial counts.

A Luminex-based single DNA fragment amplification assay as a practical tool for detecting and serotyping dengue virus.

Dengue is a mosquito-borne viral infection that can evolve from subclinical to severe forms of disease. Early recognition during initial primary and secondary infections correlates with a reduced case-fatality rate in susceptible groups. The aim of this study was to standardize a DNA hybridization assay based on the Luminex technology for detecting and serotyping dengue virus (DENV). Reference DENVs representing the four different serotypes were used as controls to standardize the test. For validation, 16 DENV isolates obtained from a reference laboratory were analyzed in a double-blind manner to validate the test. Sixty blood samples from patients suspected of having dengue fever were used to evaluate the methodology after the validation step, and the results were compared with the reference semi-nested RT-PCR. Additionally, five human samples of each Zika and Chikungunya confirmed patients were used for specificity analysis. The Luminex-based assay correctly identified all 16 DENV isolates. In the evaluation step, the results of the RT-PCR/Luminex assay showed a concordance of 86.7% with those of the semi-nested RT-PCR. None of other virus infection samples was amplified. This is the first description of a hybridization assay that can discriminate the four DENV serotypes using probes against a single DENV sequence. The results indicated that the RT-PCR/Luminex DENV assay designed and evaluated in this study is a valuable additional tool for the early and rapid detection and serotyping of DENV, which could, in the future, be applied to new targets such as the Zika and Chikungunya viruses.

A molecular platform for the diagnosis of multidrug-resistant and pre-extensively drug-resistant tuberculosis based on single nucleotide polymorphism mutations present in Colombian isolates of Mycobacterium tuberculosis.

Developing a fast, inexpensive, and specific test that reflects the mutations present in Mycobacterium tuberculosis isolates according to geographic region is the main challenge for drug-resistant tuberculosis (TB) control. The objective of this study was to develop a molecular platform to make a rapid diagnosis of multidrug-resistant (MDR) and extensively drug-resistant TB based on single nucleotide polymorphism (SNP) mutations present in therpoB, katG, inhA,ahpC, and gyrA genes from Colombian M. tuberculosis isolates. The amplification and sequencing of each target gene was performed. Capture oligonucleotides, which were tested before being used with isolates to assess the performance, were designed for wild type and mutated codons, and the platform was standardised based on the reverse hybridisation principle. This method was tested on DNA samples extracted from clinical isolates from 160 Colombian patients who were previously phenotypically and genotypically characterised as having susceptible or MDR M. tuberculosis. For our method, the kappa index of the sequencing results was 0,966, 0,825, 0,766, 0,740, and 0,625 forrpoB, katG, inhA,ahpC, and gyrA, respectively. Sensitivity and specificity were ranked between 90-100% compared with those of phenotypic drug susceptibility testing. Our assay helps to pave the way for implementation locally and for specifically adapted methods that can simultaneously detect drug resistance mutations to first and second-line drugs within a few hours.

A molecular platform for the diagnosis of multidrug-resistant and pre-extensively drug-resistant tuberculosis based on single nucleotide polymorphism mutations present in Colombian isolates of Mycobacterium tuberculosis

Developing a fast, inexpensive, and specific test that reflects the mutations present in Mycobacterium tuberculosis isolates according to geographic region is the main challenge for drug-resistant tuberculosis (TB) control. The objective of this study was to develop a molecular platform to make a rapid diagnosis of multidrug-resistant (MDR) and extensively drug-resistant TB based on single nucleotide polymorphism (SNP) mutations present in therpoB, katG, inhA,ahpC, and gyrA genes from Colombian M. tuberculosis isolates. The amplification and sequencing of each target gene was performed. Capture oligonucleotides, which were tested before being used with isolates to assess the performance, were designed for wild type and mutated codons, and the platform was standardised based on the reverse hybridisation principle. This method was tested on DNA samples extracted from clinical isolates from 160 Colombian patients who were previously phenotypically and genotypically characterised as having susceptible or MDR M. tuberculosis. For our method, the kappa index of the sequencing results was 0,966, 0,825, 0,766, 0,740, and 0,625 forrpoB, katG, inhA,ahpC, and gyrA, respectively. Sensitivity and specificity were ranked between 90-100% compared with those of phenotypic drug susceptibility testing. Our assay helps to pave the way for implementation locally and for specifically adapted methods that can simultaneously detect drug resistance mutations to first and second-line drugs within a few hours.

Electrosynthesis and characterization of nanostructured polyquinone for use in detection and quantification of naturally occurring dsDNA.

The detection of naturally occurring desoxyribonucleic acid (DNA) has become a subject of study by the projections that would generate to be able to sense the genetic material for the detection of future diseases. Bearing this in mind, to provide new measuring strategies, in the current work the preparation of a low-cost electrode, modified with poly(1-amino-9,10-anthraquinone) nanowires using a SiO2 template, is carried out; the assembly is next modified by covalently attaching ssDNA strands. It must be noted that all this is accomplished by using solely electrochemical techniques, according to methodology developed for this purpose. SEM images of the modified surface show high order and homogeneity in the distribution of modified nanowires over the electrode surface. In turn, after the hybridization with its complementary strand, the voltammetric responses enable corroborating the linear relationship between hybridization at different DNA concentrations and normalized current response, obtaining a limit of detection (LOD) 5.7·10(-12)gL(-1) and limit of quantification (LOQ) 1.9·10(-11)gL(-1). The working dynamic range is between 1.4·10(-7) and 8.5·10(-9)gL(-1) with a correlation coefficient 0.9998. The successful obtaining of the modified electrode allows concluding that the high order reached by the nanostructures, guides the subsequent single strand of DNA (ssDNA) covalent attachment, which after hybridization with its complementary strand brings about a considerable current increase. This result allows foreseeing a guaranteed breakthrough with regard to the use of the biosensor in real samples.

Genetic analysis of enteropathogenic Escherichia coli (EPEC) adherence factor (EAF) plasmid reveals a new deletion within the EAF probe sequence among O119 typical EPEC strains.

BACKGROUND: Enteropathogenic Escherichia coli (EPEC) are classified into typical and atypical strains based on the presence of the E. coli adherence factor (EAF) plasmid. The EAF plasmid contains the bfp (bundle-forming pilus) operon and the perABC (plasmid encoded regulator) gene cluster. A 1-kb cryptic region of EAF plasmid has been widely used as a genetic probe for EPEC detection. However, some EPEC strains may harbor an EAF plasmid lacking the EAF probe sequence, which makes the differentiation between typical and atypical a complex task. In this study, we report the genetic analysis of the EAF plasmid-encoded genes in a collection of EPEC clinical isolates. METHODS: A total of 222 EPEC clinical isolates, which were previously classified as typical (n=70) or atypical (n=152) by EAF probe reactivity, were screened for the presence of different EAF sequences by PCR and DNA hybridization. RESULTS: All typical strains possessed intact bfpA and perA genes, and most of them were positive in the PCR for EAF probe sequence. However, a subset of 30 typical strains, 22 of which belonged to O119 serogroup, presented a 1652 pb deletion in the region between 1093-bp downstream perC and 616-bp of the EAF fragment. The bfpA, bfpG, and per genes were found in all typical strains. In addition, 32 (21%) atypical strains presented the perA gene, and 20 (13.2%) also presented the bfpA gene. Among the 32 strains, 16 belonged to the O119:H2, O119:HND, and ONT:HND serotypes. All 32 atypical strains contained perA mutation frameshifts and possessed an IS1294 element upstream of the per operon as detected by PCR followed by restriction fragment length polymorphism (RFLP) typing and multiplex PCR. Among the 20 bfpA probe-positive strains, eight O119 strains possessed deletion in the bfp operon at the 3'end of bfpA due to an IS66 element. CONCLUSION: Our data show that typical O119 strains may contain a deletion within the EAF probe sequence not previously reported. This new finding suggests that care should be taken when using the previously described EAF PCR assay in epidemiological studies for the detection of typical O119 strains. In addition, we were able to confirm that some atypical strains carry vestiges of the EAF plasmid.