Reagent Contamination Research Articles

INVITED REVIEW: Antimicrobial Resistance Genes in Milk: a 10-year-systematic review and critical comment.

The occurrence of antimicrobial resistance genes in milk is eagerly discussed as a public health risk, and frequently investigated. Here, we perform a systematic review on the abundance of antimicrobial resistance genes in milk from primary production over a 10-year-period. We aimed to provide a comprehensive data set on known and emerging antimicrobial resistance genes in major mastitis pathogens, occurring worldwide in milk at primary production, and to critically discuss the relevance and constraints of these findings. We searched Pubmed for peer-reviewed studies published between 2012 and 2022 that fit fixed combinations of keywords and did not meet exclusion criteria such as "mixed with other sources." For synthesis, data on occurrence was extracted from studies and supplements. To address plausibility issues, we performed an NCBI BLAST search. Results & limitations. Our search revealed 2222 publications in total. Of them, 500 studies were eligible for full-text reads and 306 publications were included in data compilation. An overwhelming majority of studies dealt with mecA in Staphylococcus aureus, followed by extended-spectrum β-lactamase-encoding genes such as blaCTXM in Escherichia coli, while other mastitis pathogens such as Streptococcus spp. were scarcely investigated. In most cases, < 5% of milk samples were positive for major pathogens bearing the antimicrobial resistance gene of interest. However, huge study-to-study differences were found between regions, but also on a national level. For instance, the estimate prevalence of Escherichia coli-borne blaCTXM in mastitis milk samples ranged from 0.0% to 55%, with a median value of 8.02%, while in healthy individuals and bulk milk, the prevalence ranged from 0.0% to 20.0%, with a median value of 0.8%. Several studies reported antimicrobial resistance genes for the very first time in a species, but did not stand up to scrutiny. As an example, frequent detection of TEM-genes in streptococci is most likely attributed to contamination of molecular reagents, as reported elsewhere. Despite the huge amount of data, there is a need for more quality control, more representative sampling of milk, more quantitative research, and deeper insights into bacterial genomics, to identify relevant and/or emerging antimicrobial resistance genes in milk. Considering a low percentage of contaminated milk samples, unknown ARG-concentrations and an unproven role in human disease, the risk attributed to ARGs in milk seems to be exaggerated by far. However, the risk of ARG-selection on farm, resulting in low treatment success in cattle, is a real one and should be met by prudent use of antibiotics.

Recent Recall of Iron Reagent-Investigation of Potential Reagent Contamination and Assay Improvement Strategy.

Recently, a major manufacturer recalled several lots of iron assay reagent due to positive bias of roughly 15%-30% and the cause remains unknown. This study investigated the root cause of this positive bias and evaluated a simple practical approach to improve the assay. Performance comparison of recalled and unimpacted iron assay kits was done utilizing calibrators, quality control (QC) materials, and 42 remnant patient samples. Spectral scan and trace elements analysis of R1 and R2 reagents was performed. Copper (Cu) and thiourea (TU) spiking experiments were utilized to elucidate the cause and prevention of positive bias seen with recalled lots. Iron measurements in QC materials and patient samples using recalled reagents generated a positive bias of 17.5% and 21%, respectively. Correspondingly, the recalled R2 reagents, but not R1, showed a rise in basal absorbance along with an unanticipated presence of Cu (22.7 µg/dL) and lead (7.5 µg/L). Cu spiking to recalled and unimpacted R2 reagent intensified the reagent color besides falsely increasing its absorbance, calibration factor, and patient iron measurements. Interestingly, addition of TU (65 mmol/L) to R2 reagent from unimpacted lot prevented the short-term and prolonged Cu-induced spurious rise in calibration factor and patient iron estimations. We conclude that accidental copper contamination of R2 reagent during manufacturing could be a reason underlying the positive bias in the recalled iron reagent lots. Addition of TU in ferene-containing R2 reagent is a simple and effective means to prevent Cu-induced false elevation in iron values.

Determination of Fluorine by Ion-Selective Electrode and High-Resolution Continuum Source Graphite Furnace Molecular Absorption Spectrometry with Respect to Animal Feed Safety.

Fluorine, depending on its concentration and chemical form, is essential or toxic to humans and animals. Therefore, it is crucial to be able to determine it reliably. In this study, fluorine was determined in animal feed after extraction with HCl (gastric juice simulation). The standard potentiometric method with a fluoride-selective electrode (ISE) and newly developed high-resolution continuum source graphite furnace molecular absorption spectrometry (HR-CS GFMAS) method was applied. Feed samples turned out to be a challenge for HR-CS GFMAS. Chemical interferences (formation of competing molecules, CaF, GaCl, and GaP, instead of the target GaF molecule) and spectral effects (including a phosphorous molecule spectrum and atomic lines) were identified. An additional difficulty was caused by reagent contamination with F and memory effects. Difficulties were eliminated/reduced. The quality of ISE analysis was multi-directionally verified (including comprehensive proficiency testing). A risk of inaccuracy at low F concentration, where the calibration relationship is nonlinear, was investigated. The results of both methods were consistent, which confirms the accuracy of the methods and informs that the extracted fluorine is in fluoride form. The results of extensive ISE tests conducted in Poland in 2021-2023 have shown that, in most cases, the fluoride content is significantly lower than the threshold values.

Chemical oxidation co-coagulation-flocculation for the flotation wastewater treatment of lead-zinc oxide ore from Lanping mine

The flotation wastewater produced by "lead preferred flotation-zinc flotation" all-open process with aids of mixed depressants and cationic-anionic collectors has a high turbidity and multitude of reagent contaminants, and fails to meet the discharge standards. This study objective is to remove fine solid particles and flotation reagents in this wastewater by chemical oxidation co-coagulation-flocculation process. Results of chemical oxidation tests indicate peroxymonosulfate (PMS) exhibits superior performance on decreasing COD, and the COD remarkably decreases to 71.8 mg·L&lt;sup&gt;-1&lt;/sup&gt; with 100 mg·L&lt;sup&gt;-1&lt;/sup&gt; PMS addition after 120 min. Moreover, the combined oxidation of radicals (SO&lt;sub&gt;4&lt;/sub&gt;&lt;sup&gt;•-&lt;/sup&gt; and •OH) are responsible for degradation of flotation reagents (Na&lt;sub&gt;2&lt;/sub&gt;S, DCCH, xanthates and amine) in the wastewater. Results of experimental factors confirm that the turbidity of wastewater decreases significantly from 124796 to 71.4 NTU, and the yield of water reaches above 90% with combined usage of lime (500 mg·L&lt;sup&gt;-1&lt;/sup&gt;) and polyacrylamide (PAM, 50 mg·L&lt;sup&gt;-1&lt;/sup&gt;). Besides, the contents of S, P, N, Zn Pb and Fe decrease, and meet the discharge standards. Results of zeta potential analysis suggest lime reduces the electrostatic repulsion between particles, and PAM plays a bridge link role between particles, accelerating the precipitation of suspended particle.

Considerations for performing companion animal skin microbiome studies.

The microbiome field has grown significantly in the past decade, and published studies have provided an overview of the microorganisms inhabiting the skin of companion animals. With the continued growth and interest in this field, concerns have been raised regarding sample collection methods, reagent contamination, data processing and environmental factors that may impair data interpretation (especially as related to low-biomass skin samples). In order to assure transparency, it is important to report all steps from sample collection to data analysis, including use of proper controls, and to make sequence data and sample metadata publicly available. Whilst interstudy variation will continue to exist, efforts to standardise methods will reduce confounding variables, and allow for reproducibility and comparability of results between studies. Companion animal microbiome studies often include clinical cases, and small sample sizes may result in lack of statistical significance within small datasets. The ability to combine results from standardised studies through meta-analyses would mitigate the limitations of these smaller studies, providing for more robust interpretation of results which could then inform clinical decisions. In this narrative review, we aim to present considerations for designing a study to evaluate the skin microbiome of companion animals, from conception to data analysis.

EVALUATION AND IMPLIMENTATION OF A COMMERCIAL QUALITY CONTROL PRODUCT TO MONITOR SERUM INDICES DETECTION IN DAILY ROUTINE USE

Abstract Introduction/Objective Clinical Chemistry analyzers measure serum indices of each sample. Serum indices affect patient result. Yet there is no current common practice of monitoring the Instruments ability to measure serum indices using a commercial QC product. In this study, we focused on such Q.C. product evaluation. Methods/Case Report We evaluated the implementation of daily routine use of serum indices QC. We used a commercially available Bio-Rad (Hercules, California, United States) LiquichekTM Serum Indices QC material on Abbott (Abbott Park, Illinois, United States) Architect c8000 Chemistry analyzers in National Guard Hospital, Jeddah, Saudi Arabia. We run the QC twice a day in three Instruments for Two months. Data transferred to Bio-Rad Unity Real Time, a QC data management software. Mean, standard deviation and coefficient of variation calculated for each Instrument. Results (if a Case Study enter NA) A total of 1,296 data points were collected. The QC performed well with a precision of % CV ≤ 1.97% Hemolysis, ≤ 2.67% Icterus and ≤ 6.80% Lipemia. The three instruments performed with a slightly different mean. No “out of control” situations observed. TAT was not affected. To mimic a possible day-to-day Lab scenario, the saline bottle used as HIL reagent was deliberately contaminated using a few drops of whole blood to study the effect of gross hemolysis reagent contamination on HIL readings. The commercial HIL quality control detected the problem. Its performance showed increase in Hemolysis, slight decrease in Icterus and a slight increase in Lipemia readings. Conclusion Bio-Rad Liquichek Serum Indices QC is easy to use with a long shelf life. It helps to monitor the instrument’s ability to detect the sample integrity. Results can be compared to peer group unlike the home brewed QC. Peer group comparison helps to monitor and troubleshoot any bias or shift from the peer group and brings benefit to the total QC process.

Electrodeposition of UO2 nanoparticles in molten salt with microplasma gaseous cathode

Electrodeposition of UO2 nanoparticles in molten salt with microplasma gaseous cathode

Absence of changes in the milk microbiota during Escherichia coli endotoxin induced experimental bovine mastitis

Changes in the milk microbiota during the course of mastitis are due to the nature of a sporadic occurring disease difficult to study. In this study we experimentally induced mastitis by infusion of Escherichia coli endotoxins in one udder quarter each of nine healthy lactating dairy cows and assessed the bacteriological dynamics and the milk microbiota at four time points before and eight time points after infusion. As control, saline was infused in one udder quarter each of additionally nine healthy cows that followed the same sampling protocol. The milk microbiota was assessed by sequencing of the 16 S rRNA gene and a range of positive and negative controls were included for methodological evaluation. Two different data filtration models were used to identify and cure data from contaminating taxa. Endotoxin infused quarters responded with transient clinical signs of inflammation and increased SCC while no response was observed in the control cows. In the milk microbiota data no response to inflammation was identified. The data analysis of the milk microbiota was largely hampered by laboratory and reagent contamination. Application of the filtration models caused a marked reduction in data but did not reveal any associations with the inflammatory reaction. Our results indicate that the microbiota in milk from healthy cows is unaffected by inflammation.

A Refined, Controlled 16S rRNA Gene Sequencing Approach Reveals Limited Detection of Cerebrospinal Fluid Microbiota in Children with Bacterial Meningitis.

Advances in both laboratory and computational components of high-throughput 16S amplicon sequencing (16S HTS) have markedly increased its sensitivity and specificity. Additionally, these refinements have better delineated the limits of sensitivity, and contributions of contamination to these limits, for 16S HTS that are particularly relevant for samples with low bacterial loads, such as human cerebrospinal fluid (CSF). The objectives of this work were to (i) optimize the performance of 16S HTS in CSF samples with low bacterial loads by defining and addressing potential sources of error, and (ii) perform refined 16S HTS on CSF samples from children diagnosed with bacterial meningitis and compare results with those from microbiological cultures. Several bench and computational approaches were taken to address potential sources of error for low bacterial load samples. We compared DNA yields and sequencing results after applying three different DNA extraction approaches to an artificially constructed mock-bacterial community. We also compared two postsequencing computational contaminant removal strategies, decontam R and full contaminant sequence removal. All three extraction techniques followed by decontam R yielded similar results for the mock community. We then applied these methods to 22 CSF samples from children diagnosed with meningitis, which has low bacterial loads relative to other clinical infection samples. The refined 16S HTS pipelines identified the cultured bacterial genus as the dominant organism for only 3 of these samples. We found that all three DNA extraction techniques followed by decontam R generated similar DNA yields for mock communities at the low bacterial loads representative of CSF samples. However, the limits of detection imposed by reagent contaminants and methodologic bias precluded the accurate detection of bacteria in CSF from children with culture-confirmed meningitis using these approaches, despite rigorous controls and sophisticated computational approaches. Although we did not find current DNA-based diagnostics to be useful for pediatric meningitis samples, the utility of these methods for CSF shunt infection remains undefined. Future advances in sample processing methods to minimize or eliminate contamination will be required to improve the sensitivity and specificity of these methods for pediatric meningitis. IMPORTANCE Advances in both laboratory and computational components of high-throughput 16S amplicon sequencing (16S HTS) have markedly increased its sensitivity and specificity. These refinements have better delineated the limits of sensitivity, and contributions of contamination to these limits, for 16S HTS that are particularly relevant for samples with low bacterial loads such as human cerebrospinal fluid (CSF). The objectives of this work were to (i) optimize the performance of 16S HTS in CSF samples by defining and addressing potential sources of error, and (ii) perform refined 16S HTS on CSF samples from children diagnosed with bacterial meningitis and compare results with those from microbiological cultures. We found that the limits of detection imposed by reagent contaminants and methodologic bias precluded the accurate detection of bacteria in CSF from children with culture-confirmed meningitis using these approaches, despite rigorous controls and sophisticated computational approaches.

Metagenomic Methods for Addressing NASA's Planetary Protection Policy Requirements on Future Missions: A Workshop Report.

Molecular biology methods and technologies have advanced substantially over the past decade. These new molecular methods should be incorporated among the standard tools of planetary protection (PP) and could be validated for incorporation by 2026. To address the feasibility of applying modern molecular techniques to such an application, NASA conducted a technology workshop with private industry partners, academics, and government agency stakeholders, along with NASA staff and contractors. The technical discussions and presentations of the Multi-Mission Metagenomics Technology Development Workshop focused on modernizing and supplementing the current PP assays. The goals of the workshop were to assess the state of metagenomics and other advanced molecular techniques in the context of providing a validated framework to supplement the bacterial endospore-based NASA Standard Assay and to identify knowledge and technology gaps. In particular, workshop participants were tasked with discussing metagenomics as a stand-alone technology to provide rapid and comprehensive analysis of total nucleic acids and viable microorganisms on spacecraft surfaces, thereby allowing for the development of tailored and cost-effective microbial reduction plans for each hardware item on a spacecraft. Workshop participants recommended metagenomics approaches as the only data source that can adequately feed into quantitative microbial risk assessment models for evaluating the risk of forward (exploring extraterrestrial planet) and back (Earth harmful biological) contamination. Participants were unanimous that a metagenomics workflow, in tandem with rapid targeted quantitative (digital) PCR, represents a revolutionary advance over existing methods for the assessment of microbial bioburden on spacecraft surfaces. The workshop highlighted low biomass sampling, reagent contamination, and inconsistent bioinformatics data analysis as key areas for technology development. Finally, it was concluded that implementing metagenomics as an additional workflow for addressing concerns of NASA's robotic mission will represent a dramatic improvement in technology advancement for PP and will benefit future missions where mission success is affected by backward and forward contamination.

Electrolysis of Bacteria Based on Microfluidic Technology.

Cell lysis is a key step for studying the structure and function of proteins in cells and an important intermediate step in drug screening, cancer diagnosis, and genome analysis. The current cell lysis methods still suffer from limitations, such as the need for large instruments, a long and time-consuming process, a large sample volume, chemical reagent contamination, and their unsuitability for the small amount of bacteria lysis required for point-of-care testing (POCT) devices. Therefore, a fast, chemical-free, portable, and non-invasive device needs to be developed. In the present study, we designed an integrated microfluidic chip to achieve E. coli lysis by applying an alternating current (AC) electric field and investigated the effects of voltage, frequency, and flow rate on the lysis. The results showed that the lysis efficiency of the bacteria was increased with a higher voltage, lower frequency, and lower flow rate. When the voltage was at 10 Vp-p, the lysis efficiency was close to 100%. The study provided a simple, rapid, reagent-free, and high-efficiency cleavage method for biology and biomedical applications involving bacteria lysis.

324. Detection of Antibiotic Resistance Genes and Associated Genera Direct from Blood Samples by Multiplex PCR and Next Generation Sequencing

Abstract Background While blood culture and genotypic or phenotypic testing is standard procedure for identification of sepsis pathogens and resistance, blood culture negative sepsis occurs in 40-60% of patients. A culture independent diagnostic can identify pathogens that do not grow in culture and may provide a more sensitive indicator to blood stream infections. Here we discuss an assay that combines multiplex PCR with next generation sequencing (NGS) to detect resistance genes and pathogenic genera directly from whole blood. These genes include mecA, mecC, vanA, vanB, blaKPC, blaOXA-48 family, blaNDM, blaVIM, blaIMP, blaCMY-2, blaDHA, and blaCTX-M-14 and blaCTX-M-15 groups. Methods K2EDTA whole blood samples were spiked with low titers of antimicrobial resistant bacterial isolates. Sample processing involved chemical lysis of red blood cells, concentration and mechanical lysis of pathogen cells, and multiplex amplification of resistance genes and 16S rRNA gene. A synthetic DNA oligo was added at a fixed concentration as a read control. Samples were sequenced on Illumina platforms. NGS data was analyzed using a bioinformatics pipeline to process and match sequences to curated databases with BLAST. The number of reads per sequence was normalized by the number of reads for the read control, resulting in a read-control-normalized (RCN) value that could be used to establish positivity cutoffs. Results Resistance genes in bacterial pathogen spiked samples were identified at titers ≤ 10 CFU/mL. Owing to a unique dual indexing strategy, cross-reactivity was absent or low enough that cutoffs could be set using RCN values without reducing sensitivity. The expected genus was also detected at ≤ 10 CFU/mL for all samples; however, genera that may be representative of reagent or environmental contamination were observed at levels higher than a universal RCN cutoff in some samples. Conclusion We have shown that this NGS assay can detect 13 antimicrobial resistance genes with high sensitivity and specificity direct from blood. Bacterial genera associated with these genes were detected by this assay. As there is a risk of detection of contaminating species with a 16S target, further research into low DNA reagents or environmental controls may be required. Disclosures Daniel Gamero, BS, T2 Biosystems, inc: WO2018213641A8, EP3861013A1, WO2020055887A1, WO2020252084A1, WO2020257691A1|T2 Biosystems, inc: Stocks/Bonds Kelly Dabrowski, M.S., T2 Biosystems, Inc: Stocks/Bonds Analisa DeVito, B.S., T2 Biosystems, Inc: Stocks/Bonds Roger Smith, Ph.D., T2 Biosystems, Inc: US20180188195A1, EP3849938A1,WO2020257691A1, WO2020252084A1|T2 Biosystems, Inc: Stocks/Bonds Jessica L. Snyder, Ph.D., T2 Biosystems, Inc: CA3062882A1,US20180171388A1,WO2017180745A1 , EP3861013A1 , AU2020296183A1, WO2020055887A1, WO2020252084A1|T2 Biosystems, Inc: Stocks/Bonds.

Vapor Generation of Rare-Earth Elements via Reaction with Tetrahydroborate: Chemical Identities and Mechanisms of Formation.

This technical note reports for the first time the simultaneous vapor generation of 16 rare-earth elements (REEs) via reaction with sodium tetrahydroborate (THB). Significant improvement in REE vapor generation efficiency was discovered in the presence of arsenazo III (ARS). Subsequent to a critical evaluation on the impacts of various experimental variables, including the concentrations of acids, THB, and ARS, blank-limited (reagent contamination and impurity from ARS) detection limits (LODs) in the range of 0.004-0.15 μg L-1 were achieved based on three times the standard deviation of the method blank by the proposed vapor generation inductively coupled plasma mass spectrometry (ICP-MS). The nature of released REE species was studied by various approaches and identified to be REE nanoparticles and REE-ARS chelates; a dual-route mechanism for the vapor generation of REEs was thereby proposed.

Human Clinical Isolates of Pathogenic Fungi Are Host to Diverse Mycoviruses.

ABSTRACTFungi host viruses from many families, and next-generation sequencing can be used to discover previously unknown genomes. Some fungus-infecting viruses (mycoviruses) confer hypovirulence on their pathogenic hosts, raising the possibility of therapeutic application in the treatment of fungal diseases. Though all fungi probably host mycoviruses, many human pathogens have none documented, implying the mycoviral catalogue remains at an early stage. Here, we carried out virus discovery on 61 cultures of pathogenic fungi covering 27 genera and at least 56 species. Using next-generation sequencing of total nucleic acids, we found no DNA viruses but did find a surprising RNA virus diversity of 11 genomes from six classified families and two unclassified lineages, including eight genomes likely representing new species. Among these was the first jivivirus detected in a fungal host (Aspergillus lentulus). We separately utilized rolling circle amplification and next-generation sequencing to identify ssDNA viruses specifically. We identified 13 new cressdnaviruses across all libraries, but unlike the RNA viruses, they could not be confirmed by PCR in either the original unamplified samples or freshly amplified nucleic acids. Their distributions among sequencing libraries and inconsistent detection suggest low-level contamination of reagents. This highlights both the importance of validation assays and the risks of viral host prediction on the basis of highly amplified sequencing libraries. Meanwhile, the detected RNA viruses provide a basis for experimentation to characterize possible hypovirulent effects, and hint at a wealth of uncharted viral diversity currently frozen in biobanks.IMPORTANCE Fungal pathogens of humans are a growing global health burden. Viruses of fungi may represent future therapeutic tools, but for many fungal pathogens there are no known viruses. Our study examined the viral content of diverse human-pathogenic fungi in a clinical biobank, identifying numerous viral genomes, including one lineage previously not known to infect fungi.

Air contamination of therapeutic drug monitoring assay reagents results in falsely high plasma ammonia levels.

Accumulating evidence shows that contamination of blood samples by atmospheric ammonia affects blood ammonia test levels; however, reports on the effect of ammonia contamination of assay reagents are limited. Here, we aimed to clarify the effect of ammonia contamination of assay reagents, particularly the therapeutic drug monitoring (TDM) reagents, on the detection levels of blood ammonia using enzymatic assays. Ammonia gas was measured in the refrigerator compartment of the automatic analyser and the reaction tank water, probe wash water and drain outlets connected to the automatic analyser. At different time points following the closure of the cold storage, ammonia levels in quality control plasma samples were measured using three commercial assay reagents to evaluate the effect of air contamination. The distribution of evaporated ammonia in the reagent was measured using the CicaLiquid NH3 assay kit containing the assay reagent most affected by air contamination. It was confirmed that ammonia gas was generated in the cold storage of the automatic analyser. More than half of the reagents detected >0.25 ppm ammonia, and the highest concentration was detected in the TDM reagent. The ammonia levels obtained using all three reagents increased significantly after 3h of air contamination. The effect was resolved by measuring a 'dummy' sample or mixing the reagents by inversion. We demonstrated that air contamination by TDM reagents placed in cold storage could result in significantly falsely high ammonia measurements. Preventing this effect would improve the accuracy of ammonia measurements.

Metagenomic Identification of Viral Sequences in Laboratory Reagents.

Metagenomic next-generation sequencing has transformed the discovery and diagnosis of infectious disease, with the power to characterise the complete ‘infectome’ (bacteria, viruses, fungi, parasites) of an individual host organism. However, the identification of novel pathogens has been complicated by widespread microbial contamination in commonly used laboratory reagents. Using total RNA sequencing (“metatranscriptomics”) we documented the presence of contaminant viral sequences in multiple ‘blank’ negative control sequencing libraries that comprise a sterile water and reagent mix. Accordingly, we identified 14 viral sequences in 7 negative control sequencing libraries. As in previous studies, several circular replication-associated protein encoding (CRESS) DNA virus-like sequences were recovered in the blank control libraries, as well as contaminating sequences from the Totiviridae, Tombusviridae and Lentiviridae families of RNA virus. These data suggest that viral contamination of common laboratory reagents is likely commonplace and can comprise a wide variety of viruses.

Contamination Issue in Viral Metagenomics: Problems, Solutions, and Clinical Perspectives

We describe the most common internal and external sources and types of contamination encountered in viral metagenomic studies and discuss their negative impact on sequencing results, particularly for low-biomass samples and clinical applications. We also propose some basic recommendations for reducing the background noise in viral shotgun metagenomic (SM) studies, which would limit the bias introduced by various classes of contaminants. Regardless of the specific viral SM protocol, contamination cannot be totally avoided; in particular, the issue of reagent contamination should always be addressed with high priority. There is an urgent need for the development and validation of standards for viral metagenomic studies especially if viral SM protocols will be more widely applied in diagnostics.

Contamination-resistant, rapid emulsion-based isothermal nucleic acid amplification with Mie-scatter inspired light scatter analysis for bacterial identification

An emulsion loop-mediated isothermal amplification (eLAMP) platform was developed to reduce the impact that contamination has on assay performance. Ongoing LAMP reactions within the emulsion droplets cause a decrease in interfacial tension, causing a decrease in droplet size, which results in decreased light scatter intensity due to Mie theory. Light scatter intensity was monitored via spectrophotometers and fiber optic cables placed at 30° and 60°. Light scatter intensities collected at 3 min, 30° were able to statistically differentiate 103 and 106 CFU/µL initial Escherichia coli O157:H7 concentrations compared to NTC (0 CFU/µL), while the intensity at 60° were able to statistically differentiate 106 CFU/µL initial concentrations and NTC. Control experiments were conducted to validate nucleic acid detection versus bacterial adsorption, finding that the light scatter intensities change is due specifically to ongoing LAMP amplification. After inducing contamination of bulk LAMP reagents, specificity lowered to 0% with conventional LAMP, while the eLAMP platform showed 87.5% specificity. We have demonstrated the use of angle-dependent light scatter intensity as a means of real-time monitoring of an emulsion LAMP platform and fabricated a smartphone-based monitoring system that showed similar trends as spectrophotometer light scatter data, validating the technology for a field deployable platform.

Detection of cell-free microbial DNA using a contaminant-controlled analysis framework

BackgroundThe human microbiome plays an important role in cancer. Accumulating evidence indicates that commensal microbiome-derived DNA may be represented in minute quantities in the cell-free DNA of human blood and could possibly be harnessed as a new cancer biomarker. However, there has been limited use of rigorous experimental controls to account for contamination, which invariably affects low-biomass microbiome studies.ResultsWe apply a combination of 16S-rRNA-gene sequencing and droplet digital PCR to determine if the specific detection of cell-free microbial DNA (cfmDNA) is possible in metastatic melanoma patients. Compared to matched stool and saliva samples, the absolute concentration of cfmDNA is low but significantly above the levels detected from negative controls. The microbial community of plasma is strongly influenced by laboratory and reagent contaminants introduced during the DNA extraction and sequencing processes. Through the application of an in silico decontamination strategy including the filtering of amplicon sequence variants (ASVs) with batch dependent abundances and those with a higher prevalence in negative controls, we identify known gut commensal bacteria, such as Faecalibacterium, Bacteroides and Ruminococcus, and also other uncharacterised ASVs. We analyse additional plasma samples, highlighting the potential of this framework to identify differences in cfmDNA between healthy and cancer patients.ConclusionsTogether, these observations indicate that plasma can harbour a low yet detectable level of cfmDNA. The results highlight the importance of accounting for contamination and provide an analytical decontamination framework to allow the accurate detection of cfmDNA for future biomarker studies in cancer and other diseases.

Choice of Commercial DNA Extraction Method Does Not Affect 16S Sequencing Outcomes in Cloacal Swabs.

Simple SummaryThe cloacal anatomy is unique because the fecal, urinary, and reproductive tracts converge into one orifice. Therefore, sampling for microbiome research can be difficult in birds, especially in agricultural production settings where it may not be feasible to sample the intestines, and cloacal swabs are often used. There is a need to evaluate laboratory methods for 16S rRNA sequencing in cloacal swab samples to ensure reproducible and trustworthy downstream results. We compared four DNA extraction methods from two commercially available magnetic-based DNA extraction kits. Mock communities and negative controls were included for each method and subjected to 16S rRNA sequencing. While extraction quality and yield differed between each extraction method, overall sequencing results were not affected, including alpha and beta diversity. Positive and negative controls are an important aspect of microbiome science and our findings lend guidance to future microbiome research in poultry.As the applications of microbiome science in agriculture expand, laboratory methods should be constantly evaluated to ensure optimization and reliability of downstream results. Most animal microbiome research uses fecal samples or rectal swabs for profiling the gut bacterial community; however, in birds, this is difficult given the unique anatomy of the cloaca where the fecal, urinary, and reproductive tracts converge into one orifice. Therefore, avian gut microbiomes are usually sampled from cloacal swabs, creating a need to evaluate sample preparation methods to optimize 16S sequencing. We compared four different DNA extraction methods from two commercially available kits on cloacal swabs from 10 adult commercial laying hens and included mock communities and negative controls, which were then subjected to 16S rRNA amplicon sequencing. Extracted DNA yield and quality, diversity analyses, and contaminants were assessed. Differences in DNA quality and quantity were observed, and all methods needed further purification for optimal sequencing, suggesting contaminants due to cloacal contents, method reagents, and/or environmental factors. However, no differences were observed in alpha or beta diversity between methods. Importantly, multiple bacterial contaminants were detected in each mock community and negative control, indicating the prevalence of laboratory and handling contamination as well as method-specific reagent contamination. We found that although the extraction methods resulted in different extraction quality and yield, overall sequencing results were not affected, and we did not identify any method that would be an inappropriate choice in extracting DNA from cloacal swabs for 16S rRNA sequencing. Overall, our results highlight the need for careful consideration of positive and negative controls in addition to DNA isolation method and lend guidance to future microbiome research in poultry.