RESUMO
The use of direct nucleic acid amplification of pathogens from food matrices has the potential to reduce time to results over DNA extraction-based approaches as well as traditional culture-based approaches. Here we describe protocols for assay design and experiments for direct amplification of foodborne pathogens in food sample matrices using loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR). The examples provided include the detection of Escherichia coli in milk samples and Salmonella in pork meat samples. This protocol includes relevant reagents and methods including obtaining target sequences, assay design, sample processing, and amplification. These methods, though used for specific example matrices, could be applied to many other foodborne pathogens and sample types.
Assuntos
DNA Bacteriano , Microbiologia de Alimentos , Leite , Técnicas de Amplificação de Ácido Nucleico , Reação em Cadeia da Polimerase , Salmonella , Técnicas de Amplificação de Ácido Nucleico/métodos , Microbiologia de Alimentos/métodos , Animais , Leite/microbiologia , Salmonella/genética , Salmonella/isolamento & purificação , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Doenças Transmitidas por Alimentos/microbiologia , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Técnicas de Diagnóstico Molecular/métodos , SuínosRESUMO
Oral infections can activate local and systemic inflammation. The inflammatory response plays a main role in atherosclerosis. several studies have reported a relation between oral pathogen infection and Atherosclerosis. Recently it was indicated that some oral microbiome has a significant role in triggering atherosclerosis. Denaturing Gradient Gel Electrophoresis (DGGE) is an acceptable assay for identification of uncultivable bacteria. Therefore, we compared the bacterial population diversity in the oral microbiota between atherosclerosis patients and healthy people. Oral microbiota profiling was performed for 139 individuals including 89 patients with CAD and 50 healthy individuals. After DNA extracted from saliva, PCR products were examined and evaluated using DGGE assay. We found that significant relationship between the increased risk of atherosclerosis and the presence of Actinomyces oris, Enterococcus faecalis, Bacterium strain sulresv, Bacterium Culaenoe, NC4, NC7, and NC5 in atherosclerosis patients and healthy individuals. There was also a significant relationship between reducing the risk of atherosclerosis in the presence of NC3 and Entreococcus munotii in atherosclerosis patients and healthy individuals. In conclusion, presence of some oral microbiota increases the risk of atherosclerosis and the presence of some oral microbiota reduces the risk, so the oral microbiota should be further examined to determine its potential as a biomarker for atherosclerosis.
Assuntos
Aterosclerose , Eletroforese em Gel de Gradiente Desnaturante , Microbiota , Boca , Humanos , Aterosclerose/microbiologia , Microbiota/genética , Feminino , Masculino , Pessoa de Meia-Idade , Boca/microbiologia , Estudos de Casos e Controles , Saliva/microbiologia , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Idoso , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , AdultoRESUMO
Pathogenic bacteria in food or environment, can pose threats to public health, highlighting the requirement of tools for rapid and accurate detection of viable pathogenic bacteria. Herein, we report a sequential endoprotein RNase H2-activating DNAzyme assay (termed epDNAzyme) that enables nucleic acid extraction- and amplification-free detection of viable Salmonella enterica (S. enterica). The direct detection allows for a rapid detection of viable S. enterica within 25 min. Besides, the assay, based on sequential reporting strategy, circumvents internal modifications in the DNAzyme's active domain and improve its catalytic activity. The multiple-turnover DNAzyme cutting and the enhanced catalytic activity of DNAzyme render the epDNAzyme assay to be highly sensitive, and enables the detection of 190 CFU/mL and 0.1% viable S. enterica. The assay has been utilized to detect S. enterica contamination in food and clinical samples, indicating its potential as a promising tool for monitoring pathogen-associated biosafety.
Assuntos
Técnicas Biossensoriais , DNA Catalítico , Salmonella enterica , DNA Catalítico/química , Técnicas Biossensoriais/métodos , Salmonella enterica/isolamento & purificação , Salmonella enterica/patogenicidade , Salmonella enterica/genética , Humanos , Ribonuclease H/metabolismo , Ribonuclease H/química , Microbiologia de Alimentos , Limite de Detecção , Infecções por Salmonella/microbiologia , DNA Bacteriano/análise , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/genéticaRESUMO
The isolation and identification of pathogenic bacteria from a variety of samples are critical for controlling bacterial infection-related health problems. The conventional methods, such as plate counting and polymerase chain reaction-based approaches, tend to be time-consuming and reliant on specific instruments, severely limiting the effective identification of these pathogens. In this study, we employed the specificity of the cell wall-binding (CBD) domain of the Staphylococcus aureus bacteriophage 80 alpha (80α) endolysin towards the host bacteria for isolation. Amidase 3-CBD conjugated magnetic beads successfully isolated as few as 1 × 102 CFU/mL of S. aureus cells from milk, blood, and saliva. The cell wall hydrolyzing activity of 80α endolysin promoted the genomic DNA extraction efficiency by 12.7 folds on average, compared to the commercial bacterial genomic DNA extraction kit. Then, recombinase polymerase amplification (RPA) was exploited to amplify the nuc gene of S. aureus from the extracted DNA at 37 °C for 30 min. The RPA product activated Cas12a endonuclease activity to cleave fluorescently labeled ssDNA probes. We then converted the generated signal into a fluorescent readout, detectable by either the naked eye or a portable, self-assembled instrument with ultrasensitivity. The entire procedure, from isolation to identification, can be completed within 2 h. The simplicity and sensitivity of the method developed in this study make it of great application value in S. aureus detection, especially in areas with limited resource supply.
Assuntos
Técnicas Biossensoriais , Endopeptidases , Staphylococcus aureus , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/virologia , Técnicas Biossensoriais/métodos , Endopeptidases/química , Endopeptidases/isolamento & purificação , Endopeptidases/genética , Bacteriófagos/química , Bacteriófagos/genética , Bacteriófagos/isolamento & purificação , Humanos , Fagos de Staphylococcus/genética , Fagos de Staphylococcus/química , Fagos de Staphylococcus/isolamento & purificação , Animais , Técnicas de Amplificação de Ácido Nucleico/métodos , Infecções Estafilocócicas/microbiologia , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Nuclease do Micrococo/química , Nuclease do Micrococo/metabolismo , Nuclease do Micrococo/genética , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
Obtaining reliable and informative DNA data from soil samples is challenging due to the presence of interfering substances and typically low DNA yields. In this work, we prepared poly(ethylene glycol)-modified magnetic particles (PEG@Fe3O4) for DNA purification. The particles leverage the facilitative effect of calcium ions (Ca2+), which act as bridges between DNA and PEG@Fe3O4 by coordinating with the phosphate groups of DNA and the hydroxyl groups on the particles. The addition of 2-propanol further enhances this Ca2+-mediated DNA adsorption by inducing a conformational change from the B-form to the more compact A-form of DNA. PEG@Fe3O4 demonstrates a DNA adsorption capacity of 144.6 mg g-1. When applied to the extraction of genomic DNA from soil samples, PEG@Fe3O4 outperforms commercial kits and traditional phenol-chloroform extraction methods in terms of DNA yield and purity. Furthermore, we developed a 16-channel automated DNA extraction device to streamline the process and reduce the extraction time. The successful amplification of target bacterial and fungal amplicons underscores the potential of this automated, device-assisted method for studying soil microbial diversity.
Assuntos
2-Propanol , Cálcio , Polietilenoglicóis , Polietilenoglicóis/química , 2-Propanol/química , Cálcio/química , Microbiologia do Solo , DNA/química , DNA/isolamento & purificação , Solo/química , Adsorção , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/genéticaRESUMO
Targeted high-throughput sequencing (HTS) has revolutionized the way we look at bacterial communities. It can be used for the species-specific detection of bacteria as well as for the determination of the microbiome and resistome and can be applied to samples from almost any environment. However, the results of targeted HTS can be influenced by many factors, which poses a major challenge for its use in clinical diagnostics. In this study, we investigated the impact of the DNA extraction method on the determination of the bacterial microbiome and resistome by targeted HTS using principles from metrology and diagnostics such as repeatability and analytical sensitivity. Sputum samples spiked with Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa at three different concentrations (103-106 cells/mL) were used. DNA was extracted from each sample on 2 separate days in three replicates each using three different extraction methods based on cetrimonium bromide, magnetic beads, and silica membranes. All three spiked bacteria were detected in sputum, and the DNA extraction method had no significant effect on detection. However, the DNA extraction method had significant effects on the composition of the microbiome and the resistome. The sequencing results were repeatable in the majority of cases. The silica membrane-based DNA extraction kit provided the most repeatable results and the highest diversity of the microbiome and resistome. Targeted HTS has been shown to be a reliable tool for determining the microbiome and resistome; however, the method of DNA extraction should be carefully selected to minimize its impact on the results. IMPORTANCE: High-throughput sequencing (HTS) is one of the crucial new technologies that gives us insights into previously hidden parts of microbial communities. The DNA extraction method is an important step that can have a major impact on the results, and understanding this impact is of paramount importance for their reliable interpretation. Our results are of great value for the interpretation of sputum microbiome and resistome results obtained by targeted HTS. Our findings allow for a more rational design of future microbiome studies, which would lead to higher repeatability of results and easier comparison between different laboratories. This could also facilitate the introduction of targeted HTS in clinical microbiology for reliable identification of pathogenic bacteria and testing for antimicrobial resistance (AMR). As AMR is a major threat to public health, the improved methods for determining AMR would bring great benefits to both the healthcare system and society as a whole.
Assuntos
DNA Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala , Microbiota , Escarro , Escarro/microbiologia , Humanos , Microbiota/genética , DNA Bacteriano/genética , DNA Bacteriano/análise , DNA Bacteriano/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/efeitos dos fármacos , Bactérias/genética , Bactérias/isolamento & purificação , Bactérias/efeitos dos fármacosRESUMO
OBJECTIVE: The aim of this study is to systematically examine and compare the characteristics distinguishing colorectal adenomatous polyps from normal mucosal intestinal microbiota. METHODS: A total of 30 specimens were obtained from patients diagnosed with colorectal adenomatous polyps (adenoma group) who underwent endoscopic removal at Wenzhou People's Hospital between September 2021 and November 2021. Concurrently, 30 normal mucosal specimens were collected from patients without adenomatous polyps (control group). Subsequently, microbiome total DNA extraction was carried out, followed by PCR amplification targeting the V3-V4 region of the 16S rDNA. High-throughput sequencing was conducted using the Illumina MiSeq platform. Subsequent to sequencing, bioinformatics analysis was used to assess the diversity, composition, and functional aspects of the intestinal microbiota in both study groups. RESULTS: A notable dissimilarity in the microbiota structure was identified, specifically within the transverse colon, between these two groups ( P â <â 0.05). Species composition analysis revealed that Escherichia , Fusobacterium , and Bacteroides were predominant bacteria in both groups, with Escherichia and Enterobacter displaying significant differences at the genera level between the control group and the adenoma group ( P â <â 0.05). Correlation analysis and functional prediction demonstrated substantial disparities in interactions among dominant intestinal microbial genera within patients from both groups. Additionally, it was discovered that the intestinal microbiomes in patients in the adenoma group exhibited a significantly higher pathogenic potential. CONCLUSION: Upon conducting a comprehensive analysis, it was discerned that the microbiota present in the transverse colon of the control group exhibited distinctive characteristics that may contribute to the maintenance of intestinal health.
Assuntos
Pólipos Adenomatosos , Neoplasias Colorretais , Microbioma Gastrointestinal , Mucosa Intestinal , Humanos , Masculino , Feminino , Pessoa de Meia-Idade , Pólipos Adenomatosos/microbiologia , Pólipos Adenomatosos/patologia , Mucosa Intestinal/microbiologia , Neoplasias Colorretais/microbiologia , Idoso , Adulto , Estudos de Casos e Controles , RNA Ribossômico 16S/genética , Fusobacterium/isolamento & purificação , Fusobacterium/genética , Sequenciamento de Nucleotídeos em Larga Escala , Bacteroides/isolamento & purificação , Bacteroides/genética , Enterobacter/isolamento & purificação , Enterobacter/genética , Pólipos do Colo/microbiologia , Bactérias/isolamento & purificação , Bactérias/genética , Bactérias/classificação , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/análiseRESUMO
Environmental surveillance is recognized as an important tool for assessing public health in the post-pandemic era. Water, in particular wastewater, has emerged as the source of choice to sample pathogen burdens in the environment. Wastewater from open drains and community water treatment plants is a reservoir of both pathogens and antimicrobial resistance (AMR) genes, and frequently comes in contact with humans. While there are many methods of tracking AMR from water, isolating good-quality DNA at high yields from heterogeneous samples remains a challenge. To compensate, sample volumes often need to be high, creating practical constraints. Additionally, environmental DNA is frequently fragmented, and the sources of AMR (plasmids, phages, linear DNA) consist of low-molecular-weight DNA. Yet, few extraction processes have focused on methods for high-yield extraction of linear and low-molecular-weight DNA. Here, a simple method for high-yield linear DNA extraction from small volumes of wastewater using the precipitation properties of polyethylene glycol (PEG) is reported. This study makes a case for increasing overall DNA yields from water samples collected for metagenomic analyses by enriching the proportion of linear DNA. In addition, enhancing low-molecular-weight DNA overcomes the current problem of under-sampling environmental AMR due to a focus on high-molecular-weight and intracellular DNA. This method is expected to be particularly useful when extracellular DNA exists but at low concentrations, such as with effluents from treatment plants. It should also enhance the environmental sampling of AMR gene fragments that spread through horizontal gene transfer.
Assuntos
Águas Residuárias , Águas Residuárias/microbiologia , Águas Residuárias/química , Polietilenoglicóis/química , Peso Molecular , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Resistência Microbiana a Medicamentos/genética , Farmacorresistência Bacteriana/genéticaRESUMO
BACKGROUND: Developing an alternative and benign method for DNA extraction is imperative due to the high cost and potential harms associated with conventional techniques. Investigation of Ionic liquid (IL) as a solvent for DNA storage and stability revealed the ability of IL to assist DNA processes. IL-based aqueous biphasic system emerges as a comprehensive extraction platform capitalizing on the task-specificity of ILs and the wide applicability of ABS for biomolecule extractions. Therefore, it is beneficial to optimize an IL-based ABS specifically for DNA extraction, taking into account the fundamental interactions between the IL and DNA. RESULTS: The primary objective was to design ABS consisting of Ammonium based ILs, and Potassium phosphate buffer as the salting-out agent for the partitioning of salmon sperm DNA. The analysis focused on optimizing biocompatible anions for the extraction. Moreover, the stability of the DNA in the IL rich phases was analysed to validate the method. The proposed process was then employed for extracting plasmid DNA from bacteria, demonstrating results comparable to those obtained with a commercially available kit. Further validation using agarose gel electrophoresis and transformation of the extracted DNA into E.coli were conducted, producing promising outcomes. Although there is room for improvement in terms of recovery of DNA and reusability of ABS, the described approach is comparable with the conventional one while being cost-effective, and showcases a noticeable and convincing link to eco-friendly processes. SIGNIFICANCE: There is limited literature on IL-based ABS for DNA extraction, and the existing studies predominantly concentrate on systems derived from Cholinium ILs. However, their high hydrophilicity limits the choice of the second-phase forming component to polymers for the formation of ABS. Ammonium ILs efficiently form biphasic systems with various available salting-out agents, and biocompatible anions are introduced to mitigate the toxicity of the ILs.
Assuntos
DNA Bacteriano , Líquidos Iônicos , Líquidos Iônicos/química , DNA Bacteriano/isolamento & purificação , Salmão , Animais , Escherichia coli/genética , Escherichia coli/química , Água/químicaRESUMO
Tongue swabs hold promise as a non-invasive sample for diagnosing tuberculosis (TB). However, their utility as replacements for sputum has been limited by their varied diagnostic performance in PCR assays compared to sputum. The use of silica-based DNA extraction methods may limit sensitivity due to incomplete lysis of Mycobacterium tuberculosis (MTB) cells and co-extraction of non-target nucleic acid, which may inhibit PCR. Specificity may also be compromised because these methods are labor-intensive and prone to cross-contamination. To address these limitations, we developed a sample preparation method that combines sonication for MTB lysis and a sequence-specific MTB DNA capture method using hybridization probes immobilized on magnetic beads. In spiked tongue swabs, our hybridization capture method demonstrated a 100-fold increase in MTB DNA yield over silica-based Qiagen DNA extraction and ethanol precipitation. In a study conducted on clinical samples from South Africa, our protocol had 74% (70/94) sensitivity and 98% (41/42) specificity for detecting active pulmonary TB with sputum Xpert MTB/RIF Ultra as the reference standard. While hybridization capture did not show improved sensitivity over Qiagen DNA extraction and ethanol precipitation, it demonstrated better specificity than previously reported methods and was easier to perform. With integration into point-of-care platforms, these strategies have the potential to help enable rapid non-sputum-based TB diagnosis across key underserved patient populations.
Assuntos
DNA Bacteriano , Mycobacterium tuberculosis , Hibridização de Ácido Nucleico , Sonicação , Língua , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Humanos , Hibridização de Ácido Nucleico/métodos , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/análise , Língua/microbiologia , Sensibilidade e Especificidade , Escarro/microbiologia , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/microbiologia , Tuberculose/diagnóstico , Tuberculose/microbiologiaRESUMO
INTRODUCTION: Buruli ulcer (BU) caused by Mycobacterium ulcerans (MU) is a devastating necrotic skin disease. PCR, recommended for confirmation of BU by WHO, requires an adequately equipped laboratory, therefore often delaying timely diagnosis and treatment of BU patients in remote settings. Loop-mediated isothermal amplification (LAMP) is a PCR-based protocol for isothermal amplification of DNA that has been suggested for diagnosis of BU in low-resource settings. STUDY AIMS AND METHODS: This is an exploratory diagnostic test evaluation study, with an embedded qualitative sub-study. Its aims are two-fold: First, to evaluate a simple rapid syringe-based DNA extraction method (SM) in comparison with a more elaborate conventional DNA extraction method (CM), followed by a LAMP assay targeting IS2404 for the detection of MU, either using a commercially available pocket warmer (pw) or a heat block (hb) for incubation. Second, to complement this by exploring the diagnostic workflow for BU at a community-based health centre in an endemic area in rural Ghana as an example of a potential target setting, using interviews with researchers and health care workers (HCWs). Diagnostic test evaluation results are discussed in relation to the requirements of a target product profile (TPP) for BU diagnosis and the target setting. RESULTS: A protocol using SM for DNA extraction followed by IS2404 PCR (IS2404 PCRSM) was able to identify MU DNA in 73 out of 83 BU clinical specimens submitted for diagnosis. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of IS2404 PCRSM were 90.12%, 100%, 100% and 65.21% respectively, as compared to the reference standard IS2404 PCR in combination with a standard extraction protocol for mycobacterial DNA. Evaluation of the LAMP assay on 64 SM DNA extracts showed a sensitivity, specificity, PPV and NPV of 83.6%, 100%, 100% and 50%, respectively, using either pocket warmer (pwLAMPSM) or heat block (hbLAMPSM) for incubation of the reaction, as compared to the same reference standard. The limit of detection of pwLAMPSM was found to be 30 copies of the IS2404 target. Interview findings explored barriers to BU diagnosis and treatment, including perceptions of the disease, costs, and availability of transport. Participants confirmed that a diagnosis at the PoC, in addition to screening based on clinical criteria, would be advantageous in order to prevent delays and loss to follow-up. DISCUSSION AND CONCLUSIONS: The high diagnostic and analytic accuracy of the pwLAMP, evaluated by us in combination with a syringe-based DNA extraction method, supports its potential use for the rapid detection of MU in suspected BU samples at the community or primary health care level without reliable electricity supply. Further optimization needs include a lysis buffer, evaluation directly at the PoC and/or other sites, assessing staff training requirements and quality control.
Assuntos
Úlcera de Buruli , DNA Bacteriano , Técnicas de Diagnóstico Molecular , Mycobacterium ulcerans , Técnicas de Amplificação de Ácido Nucleico , Sensibilidade e Especificidade , Humanos , Úlcera de Buruli/diagnóstico , Úlcera de Buruli/microbiologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Mycobacterium ulcerans/isolamento & purificação , Mycobacterium ulcerans/genética , Técnicas de Diagnóstico Molecular/métodos , Feminino , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Masculino , Adulto , Criança , Adolescente , Adulto Jovem , Gana , Pré-Escolar , Pessoa de Meia-Idade , Elementos de DNA Transponíveis , Idoso , Região de Recursos LimitadosRESUMO
Developing an effective method for isolating bacterial genetic material from plants is a relatively challenging task and often does not yield adequately prepared material for further analyses. Previous studies often overlook connections, primarily focusing on laboratory investigations. With advancements in high-throughput sequencing techniques, we can now revisit and delve deeper into these interactions. Our study focuses on the initial phase of these investigations: genetic material isolation. Extracting bacterial DNA from aboveground plant parts, known as the phyllosphere, poses a significant challenge due to plant-derived contaminants. Existing isolation protocols frequently yield inconsistent results, necessitating continuous refinement and optimization. In our study, we developed an effective isolation protocol employing mechanical-chemical lysis, sonication, and membrane filtration. This approach yielded high-quality DNA at a concentration of 38.08 ng/µL, suitable for advanced sequencing applications. Our results underscore the effectiveness and necessity of these methods for conducting comprehensive microbiological analyses. Furthermore, our research not only lays the groundwork for further studies on lettuce microbiota, but also highlights the potential for utilizing our developed protocol in investigating other plants and their microbiomes.
Assuntos
DNA Bacteriano , Lactuca , Lactuca/microbiologia , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Sonicação , Bactérias/genética , Bactérias/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Microbiota/genéticaRESUMO
Isolation is the first and crucial step to study possible roles of lactic acid bacteria (LAB) in environment, especially in food fermentation. It is also important to use the organisms for further application. LABs are diverse bacterial group and have diverse growth characteristics. Culture condition of LAB is thus varied, and selection of a suitable culture medium is essential for the purposes. Identification is also important step, since certain desirable and undesirable characteristics are shared within species. Identification was classically carried out by phenotypic characteristics but is usually performed for DNA sequence-based approaches. 16S rRNA gene sequencing is generally used for the identification, and sequencing of housekeeping genes is used when needed. In addition, identification based on whole-genome sequence similarities was well established during the last decade. Here, we describe methods for isolation and identification of LAB briefly.
Assuntos
Lactobacillales , RNA Ribossômico 16S , Lactobacillales/genética , Lactobacillales/isolamento & purificação , Lactobacillales/classificação , RNA Ribossômico 16S/genética , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Microbiologia Ambiental , Análise de Sequência de DNA/métodos , Meios de Cultura/químicaRESUMO
Fecal immunochemical test (FIT) followed by colonoscopy in positive cases is commonly used for population-based colorectal cancer screening. However, specificity of FIT for colorectal cancer is not ideal and has poor performance for advanced adenoma detection. Fecal Fusobacterium nucleatum (Fn) detection has been proposed as a potential noninvasive biomarker for colorectal cancer and advanced adenoma detection. We aimed to evaluate the diagnostic performance of Fn detection using droplet digital PCR (ddPCR) in FIT samples from individuals enrolled in a colorectal cancer screening program with colorectal adenoma or cancer. We evaluated Fn presence in DNA isolated from FIT leftover material of 300 participants in a colorectal cancer screening program using ddPCR. The Fn DNA amount was classified as Fn-low/negative and Fn-high, and the association with patients' clinicopathological features and accuracy measurements was calculated. Fn-high levels were more prevalent in FIT-positive (47.2%, n = 34 of 72) than FIT-negative samples (28.9%, n = 66 of 228; P < 0.04). Among FIT-positive samples, high Fn levels were significantly more frequent in patients with cancer (CA, n = 8) when compared to normal (NT, n = 16; P = 0.02), non-advanced adenomas (NAA, n = 36; P = 0.01), and advanced adenomas (AA, n = 12; P = 0.01). Performance analysis of Fn in FIT-positive samples for colorectal cancer detection yielded an AUC of 0.8203 [confidence interval (CI), 0.6464-0.9942], with high sensitivity (100%) and specificity of 50%. Concluding, we showed the feasibility of detecting Fn in FIT leftovers using the ultrasensitive ddPCR technique. Furthermore, we highlighted the potential use of Fn levels in fecal samples to ameliorate colorectal cancer detection. Prevention Relevance: Fusobacterium nucleatum detection by droplet digital PCR could prioritize the selection of fecal immunochemical test-positive individuals who might benefit the most from the colonoscopy procedure.
Assuntos
Neoplasias Colorretais , Detecção Precoce de Câncer , Fezes , Fusobacterium nucleatum , Reação em Cadeia da Polimerase , Humanos , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/microbiologia , Fusobacterium nucleatum/isolamento & purificação , Fusobacterium nucleatum/genética , Feminino , Masculino , Pessoa de Meia-Idade , Detecção Precoce de Câncer/métodos , Fezes/microbiologia , Fezes/química , Reação em Cadeia da Polimerase/métodos , Idoso , Adenoma/diagnóstico , Adenoma/microbiologia , Sangue Oculto , Infecções por Fusobacterium/diagnóstico , Infecções por Fusobacterium/microbiologia , DNA Bacteriano/análise , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/genética , Colonoscopia/métodos , Sensibilidade e Especificidade , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genéticaRESUMO
Molecular methods have improved the sensitivity of the detection of pneumococcal carriage in saliva. However, they typically require sample culture enrichment and nucleic acid extraction prior to performing the detection assay and may limit scalability for extensive surveillance of pneumococcus, particularly in low-resource settings. We evaluated the performance of a DNA-extraction-free method for the detection of pneumococcus in saliva. We developed a streamlined qPCR-based protocol for the detection of pneumococcus, omitting culture enrichment and DNA extraction. Using saliva samples collected from children attending childcare centers (New Haven, CT, USA), we evaluated the detection of pneumococcus using saliva lysates as compared to purified DNA extracted from culture-enriched aliquots of the paired samples using qPCR targeting the pneumococcal piaB gene. Of the 759 saliva samples tested from 92 children [median age 3.65 years; IQR (2.46-4.78)], pneumococcus was detected in 358 (47.2%) saliva lysates prepared using the extraction-free protocol and in 369 (48.6%) DNA extracted from culture-enriched samples. We observed near-perfect agreement between the two protocols (Cohen's kappa: 0.92; 95% CI: 0.90-0.95). Despite a high correlation between CT values generated by the two methods (r = 0.93, P < 0.0001), the CT values generated from saliva lysates were higher (lower concentration) than those from culture-enriched samples (ΔCT = 6.69, P < 0.00001). The cost of detecting pneumococcus using saliva lysates was at least fivefold lower (US$2.53) compared to the cost of the culture-enriched method (range: US$13.60-US$19.46). For pneumococcal carriage surveillance in children, our findings suggest that a DNA extraction-free approach may offer a cost-effective alternative to the resource-intensive culture-enrichment method.IMPORTANCESurveillance for carriage of pneumococcus is a key component of evaluating the performance of pneumococcal vaccines and informing new vaccination strategies. To improve the scalability of pneumococcal carriage surveillance, we show that molecular detection of pneumococcus in saliva from children can be performed without culture enrichment and DNA extraction. Our findings show that using the extraction-free method can improve surveillance efforts for pneumococcal carriage in children, overcoming the resource-intensive hurdle that comes with the use of molecular methods, particularly in low-resource settings.
Assuntos
Portador Sadio , DNA Bacteriano , Infecções Pneumocócicas , Saliva , Streptococcus pneumoniae , Humanos , Saliva/microbiologia , Streptococcus pneumoniae/isolamento & purificação , Streptococcus pneumoniae/genética , Pré-Escolar , Infecções Pneumocócicas/diagnóstico , Infecções Pneumocócicas/microbiologia , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/genética , Feminino , Masculino , Portador Sadio/diagnóstico , Portador Sadio/microbiologia , Criança , Reação em Cadeia da Polimerase em Tempo Real/métodos , Lactente , Sensibilidade e EspecificidadeRESUMO
Monitoring the levels of opportunistic pathogens in drinking water is important to plan interventions and understand the ecological niches that allow them to proliferate. Quantitative PCR is an established alternative to culture methods that can provide a faster, higher-throughput, and more precise enumeration of the bacteria in water samples. However, PCR-based methods are still not routinely applied for Legionella monitoring, and techniques, such as DNA extraction, differ notably between laboratories. Here, we quantify the impact that DNA extraction methods had on downstream PCR quantification and community sequencing. Through a community science campaign, we collected 50 water samples and corresponding shower hoses, and compared two commonly used DNA extraction methodologies to the same biofilm and water phase samples. The two methods showed clearly different extraction efficacies, which were reflected in both the quantity of DNA extracted and the concentrations of Legionella enumerated in both the matrices. Notably, one method resulted in higher enumeration in nearly all samples by about one order of magnitude and detected Legionella in 21 samples that remained undetected by the other method. 16S rRNA amplicon sequencing revealed that the relative abundance of individual taxa, including sequence variants of Legionella, significantly varied depending on the extraction method employed. Given the implications of these findings, we advocate for improvement in documentation of the performance of DNA extraction methods used in drinking water to detect and quantify Legionella, and characterize the associated microbial community.IMPORTANCEMonitoring for the presence of the waterborne opportunistic pathogen Legionella is important to assess the risk of infection and plan remediation actions. While monitoring is traditionally carried on through cultivation, there is an ever-increasing demand for rapid and high-throughput molecular-based approaches for Legionella detection. This paper provides valuable insights on how DNA extraction affects downstream molecular analysis such as the quantification of Legionella through droplet digital PCR and the characterization of natural microbial communities through sequencing analysis. We analyze the results from a risk-assessment, legislative, and ecological perspective, showing how initial DNA processing is an important step to take into account when shifting to molecular-based routine monitoring and discuss the central role of consistent and detailed reporting of the methods used.
Assuntos
DNA Bacteriano , Água Potável , Legionella , RNA Ribossômico 16S , Microbiologia da Água , Legionella/isolamento & purificação , Legionella/genética , Legionella/classificação , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , RNA Ribossômico 16S/genética , Água Potável/microbiologia , Reação em Cadeia da Polimerase/métodosRESUMO
The success of polymerase chain reaction (PCR) depends on the quality of deoxyribonucleic acid (DNA) templates. This study developed a cost-effective and eco-friendly DNA extraction system utilizing poly(3,4-dihydroxyphenylalanine)-modified cellulose paper (polyDOPA@paper). PolyDOPA@paper was prepared by oxidatively self-polymerizing DOPA under weak alkaline conditions and utilizing the adhesive property of polyDOPA on different materials. Compared to the uncoated cellulose paper, polyDOPA coating significantly enhances DNA adsorption owing to its abundant amino, carboxyl, and hydroxyl moieties. The DNA extraction mechanism using polyDOPA@paper was discussed. The maximum adsorption capacity of polyDOPA@paper for DNA was 20.7 µg cm-2. Moreover, an automated extraction system was designed and fabricated using 3D printing technology. The device simplifies the operation and ensures the reproducibility and consistency of the results. More importantly, it eliminates the need for specialized training of operators. The feasibility of the polyDOPA@paper-based automated extraction system was evaluated by quantitatively detecting Escherichia coli in spiked milk samples via a real-time PCR. The detection limit was 102 cfu mL-1. The results suggest that the system would have significant potential in detecting pathogens.
Assuntos
Celulose , Di-Hidroxifenilalanina , Limite de Detecção , Leite , Papel , Polímeros , Celulose/química , Celulose/análogos & derivados , Adsorção , Di-Hidroxifenilalanina/química , Di-Hidroxifenilalanina/isolamento & purificação , Di-Hidroxifenilalanina/análogos & derivados , Polímeros/química , Leite/química , Escherichia coli , Animais , Reprodutibilidade dos Testes , DNA/isolamento & purificação , DNA/química , Impressão Tridimensional , Reação em Cadeia da Polimerase em Tempo Real , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/análiseRESUMO
OBJECTIVE: Bartonella are emerging bacterial zoonotic pathogens. Utilization of clotted blood samples for surveillance of these bacteria in wildlife has begun to supersede the use of tissues; however, the efficacy of these samples has not been fully investigated. Our objective was to compare the efficacy of spleen and blood samples for DNA extraction and direct detection of Bartonella spp. via qPCR. In addition, we present a protocol for improved DNA extraction from clotted, pelleted (i.e., centrifuged) blood samples obtained from wild small mammals. RESULTS: DNA concentrations from kit-extracted blood clot samples were low and A260/A280 absorbance ratios indicated high impurity. Kit-based DNA extraction of spleen samples was efficient and produced ample DNA concentrations of good quality. We developed an in-house extraction method for the blood clots which resulted in apposite DNA quality when compared to spleen samples extracted via MagMAX DNA Ultra 2.0 kit. We detected Bartonella in 9/30 (30.0%) kit-extracted spleen DNA samples and 11/30 (36.7%) in-house-extracted blood clot samples using PCR. Our results suggest that kit-based methods may be less suitable for DNA extraction from blood clots, and that blood clot samples may be superior to tissues for Bartonella detection.
Assuntos
Animais Selvagens , Infecções por Bartonella , Bartonella , DNA Bacteriano , Baço , Animais , Bartonella/isolamento & purificação , Bartonella/genética , DNA Bacteriano/sangue , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Baço/microbiologia , Infecções por Bartonella/diagnóstico , Infecções por Bartonella/sangue , Infecções por Bartonella/microbiologia , Animais Selvagens/microbiologia , Reação em Cadeia da Polimerase em Tempo Real/métodosRESUMO
The development of high molecular weight (HMW) genomic DNA (gDNA) extraction protocols for non-model species is essential to fully exploit long-read sequencing technologies in order to generate genome assemblies that can help answer complex questions about these organisms. Obtaining enough high-quality HMW gDNA can be challenging for these species, especially for tissues rich in polysaccharides such as biomass from species within the Botryococcus genus. The existing protocols based on column-based DNA extraction and biochemical lysis kits can be inefficient and may not be useful due to variations in biomass polysaccharide content. We developed an optimized protocol for the efficient extraction of HMW gDNA from Botryococcus biomass for use in long-read sequencing technologies. The protocol utilized an initial wash step with sorbitol to remove polysaccharides and yielded HMW gDNA concentrations up to 220 ng/µL with high purity. We then demonstrated the suitability of the HMW gDNA isolated from this protocol for long-read sequencing on the Oxford Nanopore PromethION platform for three Botryococcus species. Our protocol can be used as a standard for efficient HMW gDNA extraction in microalgae rich in polysaccharides and may be adapted for other challenging species.
Assuntos
Biomassa , Peso Molecular , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/genética , Clorofíceas/genética , Análise de Sequência de DNA/métodos , Genoma Bacteriano , Genômica/métodosRESUMO
BACKGROUND: Polymerase chain reaction is a method to detect bacterial DNA and is widely used because it delivers results within a few hours with the potential to guide postoperative antibiotic treatment. This study aims to determine if polymerase chain reaction can accurately detect bacteria in the peritoneal fluid compared with conventional culture from patients operated for acute appendicitis. METHODS: This prospective cohort study included patients above the age of 18 years who underwent laparoscopic surgery for acute appendicitis. Peritoneal samples were collected before the appendectomy procedure for conventional culture and polymerase chain reaction using the BioFire Blood Culture Identification 2 Panel for comparison. During surgery, the surgeon assessed the appendicitis as either complicated or noncomplicated. RESULTS: Samples from 102 patients were eligible for analysis. Twelve samples were polymerase chain reaction positive, and 14 samples were culture positive. The concordance of positive results when comparing these 2 methods was 71.4%. The most commonly found bacteria were Escherichia coli and Bacteroides fragilis. Of the 36 patients with complicated appendicitis, no bacteria were detected by either conventional culture or polymerase chain reaction in 21 (58%) of the patients. In patients with uncomplicated appendicitis, bacteria were demonstrated in 1 out of 66 (2%) patients. CONCLUSION: This study suggests that polymerase chain reaction can be used to detect bacteria in the peritoneal fluid and has the potential to guide postoperative antibiotic treatment.