PCR Inhibitors Research Articles

Molecular Beacons - Loop-Mediated Amplification (MB-LAMP).

High specificity has been demonstrated in polymerase chain reaction (PCR) with the use of molecular beacons (MBs) to detect amplified sequences containing mutations or single-nucleotide polymorphisms (SNPs). MBs have been adapted for use with the isothermal nucleic acid amplification technology loop-mediated amplification (LAMP) by targeting single-stranded loop sequences under optimized conditions to enable applications such as plant genotyping. LAMP has several benefits over PCR, such as rapid amplification, single-temperature reaction conditions enabling low-cost equipment, and robustness to certain PCR inhibitors. However, and despite the increased number of primers required, the specificity of LAMP is limited, and false positive results can be problematic. In this chapter, design considerations for molecular beacons in LAMP assays are described, as well as a method for MB-LAMP amplification and detection, with an example of gene sequences in genetically modified (GM) maize samples.

Энхансеры ПЦР. I. Общие сведения

PCR is the most popular method for nucleic acids amplification, which characterized by high specificity and sensitivity. PCR is widely used in research and in practice. However, when nucleotide sequences, called “difficult” templates, are amplified, false-negative results can be obtained for samples containing a target in the reaction mixture. The same result can be caused by impurities that are co-extracted with nucleic acids and inhibit DNA polymerase. To eliminate this, substances called PCR enhancers are added to the reaction mixture, which can stabilize the DNA polymerase and/or, on the contrary, destabilize hydrogen bonds in GC-rich regions. The use of proteins, amino acids, carbohydrates, polyhydric alcohols, amides, sulfones, sulfoxides, nonionic detergents, zwitterionic compounds, various nanoparticles, as well as analogues of dGTP and dCTP, which form two hydrogen bonds instead of three when paired with complementary nucleobases, is described.

β-Like DNA polymerases and prospects for their use as targets in chemotherapy of tumors.

DNA polymerases β are enzymes that perform repair of damaged DNA. In the cells of malignant tumors, there is a change in the production and properties of these enzymes, which is accompanied by altered viability of tumor cells. Analysis of the publications available in Russian and international databases (Pubmed, Elsevier) on the structure and properties of DNA polymerases β and their role in cell growth and proliferation, published over the past 20 years, has shown overexpression of genes encoding β-like DNA polymerases in many types of malignant tumors cells. This explains the maintenance of their viability and proliferative activity. Targeted inhibition of β-like DNA polymerases is accompanied by antiproliferative and antitumor effects. Stable paramagnetic isotopes of magnesium (25Mg2+) or other divalent metals (43Ca2+ and 67Zn2+) with uncompensated nuclear spin isotopes, as well as short single-stranded polydeoxyribonucleotides, can be used as promising antitumor pharmacophores.

Selenium-ruthenium complex blocks H1N1 influenza virus-induced cell damage by activating GPx1/TrxR1.

Background: Influenza A (H1N1) virus is an acute respiratory infectious disease that causes massive morbidity and mortality worldwide. As an essential trace element, selenium is widely applied in the treatment of various diseases because of its functions of enhancing immune response, antioxidant and antiviral mutation. In this study, we constructed the selenium-containing metal complex drug delivery system Ru(biim)(PhenSe)2 (RuSe), and investigated the anti-influenza virus efficacy and the potential antiviral mechanism for RuSe. Methods: The inhibitory effect of RuSe on influenza-mediated apoptosis was examined by cell count assay, cell cycle assay, Annenxin-V assay, TUNEL-DAPI assay and reactive oxygen species level determination. Virulence assay, PCR and neuraminidase inhibition assay revealed the inhibition of RuSe on influenza virus. At the level of animal experiments, two animal models were used to clarify the role of RuSe through HE staining, immunohistochemical staining, cytokine determination, selenium metabolism determination and selenium protein expression level determination. Results: The results of this study confirm that RuSe enhances the expression levels of selenium proteins GPx1 and TrxR1 by regulating selenium metabolism, thereby inhibiting viral replication and assembly and regulating virus-mediated mitochondria-related apoptosis. On the other hand, animal experiments show that RuSe can reduce lung tissue inflammation and inhibit lung tissue cell apoptosis in mice, and improve the survival state of mice. In addition, RuSe significantly improves the low immune response of Se-deficient mice by regulating selenium metabolism, and effectively alleviated lung fibrosis and lung tissue apoptosis in Se-deficient mice. Conclusions: This study suggests that RuSe provides a promising new approach for the clinical treatment of influenza virus.

Bacteria and Yeast Colony PCR.

The bacteria Escherichia coli and the yeast Saccharomyces cerevisiae are currently the two most important organisms in synthetic biology. E. coli is almost always used for fundamental DNA manipulation, while yeast is the simplest host system for studying eukaryotic gene expression and performing large-scale DNA assembly. Yeast expression studies may also require altering the chromosomal DNA by homologous recombination. All these studies require the verification of the expected DNA sequence, and the fastest method of screening is colony PCR, which is direct PCR of DNA in cells without prior DNA purification. Colony PCR is hampered by the difficulty of releasing DNA into the PCR mix and by the presence of PCR inhibitors. We hereby present one protocol for E. coli and two protocols for S. cerevisiae differing in efficiency and complexity as well as an overview of past and possible future developments of efficient S. cerevisiae colony PCR protocols.

Evaluation of the clinical performance of OncoPredict HPV® SCR assay within the VALGENT-2 framework.

Human papillomavirus (HPV) assays used in cervical cancer screening should be clinically validated according to international criteria. OncoPredict HPV® Screening (SCR) is a partial genotyping multiplex real-time PCR assay targeting E6/E7 genes of 13 high-risk (hr) HPVs. OncoPredict HPV® SCR (index assay) identifies HPV-16 and HPV-18 separately, 11 other hrHPV in aggregate and includes quality controls for sample adequacy, DNA extraction efficiency and PCR inhibition. 1300 VALGENT-2 study samples (from women aged 20-60 attending the Scottish cervical cancer screening program) were tested with the index assay and the GP5+/6+ PCR enzyme immunoassay (standard comparator assay). Non-inferior accuracy detecting cervical intraepithelial neoplasia of grade 2 or worse (CIN2+) of the index versus comparator was verified. Intra- and interlaboratory reproducibility of the index was evaluated by overall concordance and Cohen's kappa, using a sub-population (n = 526). Relative sensitivity and specificity for CIN2+of the index versus comparator were 1.01 (95% confidence interval [CI]: 0.99-1.03) and 1.02 (95%CI: 1.0-1.04), respectively. Noninferiority pvalues were all ≤0.05, except for CIN3+in patients ≥30 years. Excellent intra- and interlaboratory reproducibility was shown with concordance >98% and kappas >0.95. OncoPredict HPV® SCR fulfills the three international validation criteria for hrHPV DNA tests in cervical cancer screening.

Identification of Pseudomonas fuscovaginae, Pseudomonas syringae and Xanthomonas translucens in wheat seeds using PCR

The causative agents of grain crops bacteriosis viz. Pseudomonas fuscovaginae , Pseudomonas syringae and Xanthomonas translucens are regulated by phytosanitary requirements of the largest importers of Russian grain - Egypt, Turkey, Bangladesh, Nigeria and Pakistan. Therefore, it requires the development of rapid methods for their diagnosis. The PCR method, which is the fastest and most reliable in testing laboratories, needs optimal preparation of the test material. The aim of the study was to optimize the process of preparing seed samples for subsequent detection and identification of P. fuscovaginae, P. syringae and X. translucens by PCR. Wheat grain samples were soaked in phosphate-buffered saline (PBS) for 2 hours and infected with suspensions of P. fuscovaginae, P. syringae pv. coronafaciens and X. translucens at various concentrations. Then, the infected grain samples were crushed and subjected to two-stage centrifugation. DNA was isolated from the obtained analytical samples and species-specific PCR was performed for each bacterial species. It was found that a two-hour soaking of the seeds and their treatment with a homogenizer is sufficient to effectively destroy each grain in the sample and ensure the release of bacteria into the liquid part of the sample. The first low-speed centrifugation allowed the crushed grain to settle efficiently and remove excess starch from the supernatant. High-speed centrifugation of the supernatant made it possible to obtain a concentrated microbiota contained in the grain sample. To obtain DNA of sufficient quality for PCR test, the kit Proba-GS (AgroDiagnostika, Russia) was used for DNA extraction. Using Pseudomonas fuscovaginae-RT kit (Syntol, Russia) and PsyF/PsyR and 4F1/4R 1 primers, DNA of P. fuscovaginae P. syringae and X. translucens , respectively, was successfully detected in each of the samples infected with these bacteria at concentrations of 103 CFU/ml. The absence of PCR inhibition was noted. The method of removing starch from samples for molecular diagnostics of phytopathogens was used for the first time. Application of these methods will allow diagnosing pathogens of bacterioses within one day.

Cost-Effective PCR-Based Identification of Tunga penetrans (Siphonaptera) Larvae Extracted from Soil Samples Containing PCR Inhibitor-Rich Material

Tungiasis is a neglected tropical disease caused by skin-penetrating female Tunga penetrans fleas. Although tungiasis causes severe health problems, its ecology is poorly understood and morphological descriptions of the larvae are unavailable. To identify T. penetrans immature stages and sites where they develop, diagnostic PCRs are required. However, flea larvae feed on soil organic matter rich in PCR inhibitors. Here, three DNA preparation methods, including a soil DNA kit that removes inhibitors, a simple ammonium acetate precipitation approach (AmAcet) and a crude lysate of larvae (CL), were combined with amplification by the highly processive FIREPol® Taq or the inhibitor-resistant Phusion® polymerase. Independent of the polymerase used, the frequency of successful amplification, Cq values and PCR efficacies for the low-cost CL and AmAcet methods were superior to the commercial kit for amplification of a 278 bp partial internal transcribed spacer-2 (ITS-2) and a 730 bp pan-Siphonaptera cytochrome oxidase II PCR. For the CL method combined with Phusion® polymerase, the costs were approximately 20-fold lower than for the methods based on the soil DNA kit, which is a considerable advantage in resource-poor settings. The ITS-2 PCR did not amplify Ctenocephalides felis genomic or Tunga trimammilata ITS-2 plasmid DNA, meaning it can be used to specifically identify T. penetrans.

Assessment of cetyl-trimethyl-ammonium bromide (CTAB) based method for the extraction of soil-transmitted helminth DNAs from stools for molecular dagnostic of soil-transmitted helminthiasis

Although several protocols were developed to extract DNA for soil-transmitted helminthiasis diagnostic, amplifying these extracts remains challenging due to DNA polymerase inhibitors. This study aimed to assess a DNA extraction method for efficient detection of soil-transmitted helminth species by determining stool mass and the type of DNA polymerase that can be used for this extraction method. For this study, 141 stool samples harbouring soil-transmitted eggs and 50 samples without egg were obtained from school-aged children of Makenene in the Centre region of Cameroon. DNA was extracted from 10, 20, 40 and 80 mg of stool using commercial kit and/or cetyltrimethylammonium bromide (CTAB)-based method. The amount of stool for molecular diagnostic of soil-transmitted helminthiasis was determined by amplifying Ascaris lumbricoides DNA. The performances of three DNA polymerases and CTAB-based method were assessed by amplifying DNA of different soil-transmitted helminth species. For this study, 94 stools with A. lumbricoides eggs, 39 with Trichuris trichuria and 15 with hookworm were analyzed. DNA of A. lumbricoides, T. trichuria, Necator americanus and Ancylostoma duodenale were detected in 97.9% of extracts from stools harbouring soil-transmitted helminth eggs. Soil-transmitted helminth DNAs were significantly (X2 = 17.66; df = 3; p 〈00001) more amplified in extracts from 10 and 20 mg than those from 40 and 80 mg. The amplification rate with “Q5 high fidelity DNA polymerase” was significantly (X2 = 30.54; df = 2; p < 0.00001) higher than that of other DNA polymerases. Multiplex-PCR confirmed co-infections of A. lumbricoides with either T. trichuria or N. americanus. The extraction cost for the CTAB-based method was $1.45. This method appearedis reliable and 3 times cost effective than commercial kit. Its combination with the “Q5 high fidelity DNA polymerase” may improve soil-transmitted helminthiasis diagnostic.

Replication stress drives chromosomal instability in fibroblasts of childhood cancer survivors with second primary neoplasms

New development and optimization of oncologic strategies are steadily increasing the number of long-term cancer survivors being at risk of developing second primary neoplasms (SPNs) as a late consequence of genotoxic cancer therapies with the highest risk among former childhood cancer patients. Since risk factors and predictive biomarkers for therapy-associated SPN remain unknown, we examined the sensitivity to mild replication stress as a driver of genomic instability and carcinogenesis in fibroblasts from 23 long-term survivors of a pediatric first primary neoplasm (FPN), 22 patients with the same FPN and a subsequent SPN, and 22 controls with no neoplasm (NN) using the cytokinesis-block micronucleus (CBMN) assay. Mild replication stress was induced with the DNA-polymerase inhibitor aphidicolin (APH). Fibroblasts from patients with the DNA repair deficiency syndromes Bloom, Seckel, and Fanconi anemia served as positive controls and for validation of the CBMN assay supplemented by analysis of chromosomal aberrations, DNA repair foci (γH2AX/53BP1), and cell cycle regulation. APH treatment resulted in G2/M arrest and underestimation of cytogenetic damage beyond G2, which could be overcome by inhibition of Chk1. Basal micronuclei were significantly increased in DNA repair deficiency syndromes but comparable between NN, FPN, and SPN donors. After APH-induced replication stress, the average yield of micronuclei was significantly elevated in SPN donors compared to FPN (p = 0.013) as well as NN (p = 0.03) donors but substantially lower than for DNA repair deficiency syndromes. Our findings suggest that mild impairment of the response to replication stress induced by genotoxic impacts of DNA-damaging cancer therapies promotes genomic instability in a subset of long-term cancer survivors and may drive the development of an SPN. Our study provides a basis for detailed mechanistic studies as well as predictive bioassays for clinical surveillance, to identify cancer patients at high risk for SPNs at first diagnosis.

Characterization of Seed Mycobiota Using Culture-Dependent and Culture-Independent Approaches.

Seed fungi are potentially important for their roles in seedling microbiome assembly and seedling health, but surveys of full seed fungal communities remain limited. While culture-dependent methods have been used to characterize some members of the seed mycobiota, recent culture-independent studies have improved the ease in identifying and characterizing full seed fungal communities. In this chapter, we describe how to survey seed fungi using both traditional culture-based methods and culture-free metabarcoding. We first describe protocols for the isolation and long-term preservation of fungal strains from individual seeds and for the extraction and amplification of DNA from such fungal isolates for identification with Sanger sequencing. We also detail how to extract, amplify, and sequence fungal DNA directly from individual seeds. Finally, we provide suggestions for troubleshooting media choices, PCR inhibition by isolates and plant tissue, and PCR limitation by low fungal DNA.

RIAM: A Universal Accessible Protocol for the Isolation of High Purity DNA from Various Soils and Other Humic Substances.

A single universal open protocol RIAM (named after Research Institute for Agricultural Microbiology) for the isolation of high purity DNA from different types of soils and other substrates (high and low in humic, clay content, organic fertilizer, etc.) is proposed. The main features of the RIAM protocol are the absence of the sorption-desorption stage on silica columns, the use of high concentrations of phosphate in buffers, which prevents DNA sorption on minerals, and DNA precipitation using CTAB. The performance of RIAM was compared with a reference commercial kit and showed very good results in relation to the purity and quantity of DNA, as well as the absence of inhibitory activity on PCR. In all cases, the RIAM ensured the isolation of DNA in quantities much greater than the commercial kit without the effect of PCR inhibition up to 50 ng DNA per reaction in a volume of 15 µL. The latter circumstance along with the ability of the protocol to extract low molecular weight DNA fractions makes the method especially suitable for those cases where quantitative assessments, detection of minor components of soil microbiota, and completeness of isolation of all DNA fractions are required.

Heat Shock Protein Family A Member 1 Promotes Intracellular Amplification of Hepatitis B Virus Covalently Closed Circular DNA.

Hepatitis B virus (HBV) contains a partially double-stranded relaxed circular DNA (rcDNA) genome that is converted into a covalently closed circular DNA (cccDNA) in the nucleus of the infected hepatocyte by cellular DNA repair machinery. cccDNA associates with nucleosomes to form a minichromosome that transcribes RNA to support the expression of viral proteins and reverse transcriptional replication of viral DNA. In addition to the de novo synthesis from incoming virion rcDNA, cccDNA can also be synthesized from rcDNA in the progeny nucleocapsids within the cytoplasm of infected hepatocytes via the intracellular amplification pathway. In our efforts to identify cellular DNA repair proteins required for cccDNA synthesis using a chemogenetic screen, we found that B02, a small-molecule inhibitor of DNA homologous recombination repair protein RAD51, significantly enhanced the synthesis of cccDNA via the intracellular amplification pathway in human hepatoma cells. Ironically, neither small interfering RNA (siRNA) knockdown of RAD51 expression nor treatment with another structurally distinct RAD51 inhibitor or activator altered cccDNA amplification. Instead, it was found that B02 treatment significantly elevated the levels of multiple heat shock protein mRNA, and siRNA knockdown of HSPA1 expression or treatment with HSPA1 inhibitors significantly attenuated B02 enhancement of cccDNA amplification. Moreover, B02-enhanced cccDNA amplification was efficiently inhibited by compounds that selectively inhibit DNA polymerase α or topoisomerase II, the enzymes required for cccDNA intracellular amplification. Our results thus indicate that B02 treatment induces a heat shock protein-mediated cellular response that positively regulates the conversion of rcDNA into cccDNA via the authentic intracellular amplification pathway. IMPORTANCE Elimination or functional inactivation of cccDNA minichromosomes in HBV-infected hepatocytes is essential for the cure of chronic hepatitis B virus (HBV) infection. However, lack of knowledge of the molecular mechanisms of cccDNA metabolism and regulation hampers the development of antiviral drugs to achieve this therapeutic goal. Our findings reported here imply that enhanced cccDNA amplification may occur under selected pathobiological conditions, such as cellular stress, to subvert the dilution or elimination of cccDNA and maintain the persistence of HBV infection. Therapeutic inhibition of HSPA1-enhanced cccDNA amplification under these pathobiological conditions should facilitate the elimination of cccDNA and cure of chronic hepatitis B.

Combination prophylaxis in CMV high risk heart transplant recipients: A single center experience.

Cytomegalovirus (CMV) is the most clinically relevant infectious agent following heart transplantation (HTX). Data on the beneficial effects of prophylactic use of CMV immunoglobulins (CMVIG) are scarce. In this single-center, retrospective study, we reported patient outcomes following cardiac transplantation using prophylactic CMV treatment, including CMVIG. Distinct clinically relevant outcomes were compared across different CMV risk groups (CMV D-/R-, CMV D-/R+, CMV D+/R+, and CMV D+/R- or CMV high risk group). We included 272 heart transplant procedures, performed between 1/1/2009 and 1/11/2020. Sixty-one (22%) procedures belonged to the CMV high risk group, while 96 (35%), 50 (18%), and 65 (24%) were CMV D-/R-, CMV D-/R+, and CMV D+/R+, respectively. Baseline donor and recipient characteristics (sex, age, body mass index, cause of death, indication for HTX), ischemia times and baseline immunosuppressive regimens were similar across the different CMV risk groups, yet fewer patients were bridged with a mechanical circulatory support in the CMV D+/R- group. CMV disease following cardiac transplantation was more common in the CMV D+/R- risk group (n=40 or 66.7%; p<.001), yet mortality and re-transplantation rates, cardiac allograft vasculopathy (CAV) severity, rejection episodes, and development of donor-specific antibodies (DSA), post-transplant lymphoproliferative diseases (PTLD), and EBV infections were similar across all four CMV risk groups. High risk CMV D+/R- patients had a similar survival compared to low and intermediate CMV risk groups using a prophylactic strategy combining CMVIG and viral DNA polymerase inhibitors. This may be related to a number of factors unrelated to prophylaxis strategy as two out of three CMV D+/R- recipients developed CMV primary infection after prophylaxis was discontinued.

DNA extraction procedures and validation parameters of a real-time PCR method to control milk containing only A2 β-casein

Bovine milk mainly contains two types of β-casein: A1 and A2 variants. In recent years, a new variety of cows’ milk has emerged in dairy sector called “A2 milk”. This novel product is characterised by the absence of A1 β-casein, which has been associated with possible gastrointestinal discomfort due to β-casomorphin-7 (BCM-7) release during gastrointestinal digestion. In this context, methods to verify A1 allele absence in A2 milk are required as a quality control in the A2 milk commercialisation. Therefore, the aim of the present study was to develop a locked nucleic acid (LNA) probe-based duplex real-time PCR (qPCR) assay for A1 allele detection in A2 milk samples. Firstly, four DNA isolation methods from milk somatic cells were optimised and evaluated. Results suggests that the commercial kit NucleoSpin Tissue was the most suitable method in terms of DNA quality and amplificability for downstream applications. Then, optimisation and validation of the qPCR assay were carried out. For both A1 and A2 alleles, the absolute limits of detection of this qPCR assay were 7.3 DNA copies/reaction (2 x 10 −5 ng DNA) and 30.4 DNA copies/reaction (0.1 ng DNA) at a 95% confidence level with synthetic reference DNA samples and heterozygous genotyped DNA sample, respectively. The relative limits of detection were 2% (15 copies) and 5% (152 copies) for A1 allele in A2 samples at a 95% confidence with synthetic reference and genotyped DNA samples, respectively. The qPCR assay was robust, with intra- and inter-assay variability below 4.3%, and specific, differentiating between A1 and A2 alleles with 100% genotyping accuracy. In conclusion, this cost-effective and fast method could be used to discriminate A1 allele in A2 samples and, consequently, to verify A1 allele absence in “A2 milk” by screening commercial product on the market. • The LNA duplex qPCR assay developed could be used to verify A1 allele absence in “A2 milk”. • LNA probes reliably discriminated between A1 and A2 alleles. • The relative limits of detection reach up to 2% (15 copies) for A1 allele in A2 samples at a 95% confidence. • Milk is a challenging matrix for DNA isolation due to the presence of PCR inhibitors that leads to low DNA yield.

Solvent-sensitive nanoparticle-enhanced PCR assay for the detection of enterotoxigenic Escherichia coli

Stimulus-responsive nanoparticles are among the most utilized nanoscale materials in biomedical applications. As these nanoparticles exhibit a manipulable response to a particular stimulus, such as pH, heat, and organic solvent, they are potential signalling units in diagnostic assays. This study aims to enhance the limit of detection and reduce the turnaround time of magnetic nanoparticle polymerase chain reaction (PCR) enzyme-linked gene assay (MELGA), an advanced PCR-based technique termed the solvent-sensitive nanoparticle (SSNP)-enhanced PCR assay. This technique was proposed to detect pathogenic enterotoxigenic Escherichia coli (ETEC) through applying stimulus-responsive nanoparticles. The SSNPs were elaborated with three main components, including mesoporous silica nanoparticles as a structural unit, organic dye (Nile red) as a payload, and the corresponding organic solvent-sensitive polymer shell as “gatekeeper” (poly(maleic anhydride-alt-methyl vinyl ether, PMAMVE). A suitable organic solvent capable of inducing polymer swelling and dye dissolution was investigated by considering a solubility parameter. Using ethanol, the encapsulated Nile red can diffuse out of the SSNPs faster than other solvents and reach a constant concentration within 15 min. For the PCR inhibition study, various SSNPs concentrations (10–30 μg/reaction) were mixed with the ETEC gene and PCR reagent. The results showed that the particles in this concentration range did not inhibit PCR. By comparing the efficacy of conventional PCR, MELGA, and SSNP-enhanced PCR assay, the proposed technique showed a better detection limit than that of PCR, whereas that of MELGA was the lowest. Moreover, compared to MELGA or conventional PCR, this technique provided remarkably faster results in the postamplification process.

3D chromatin connectivity underlies replication origin efficiency in mouse embryonic stem cells.

In mammalian cells, chromosomal replication starts at thousands of origins at which replisomes are assembled. Replicative stress triggers additional initiation events from 'dormant' origins whose genomic distribution and regulation are not well understood. In this study, we have analyzed origin activity in mouse embryonic stem cells in the absence or presence of mild replicative stress induced by aphidicolin, a DNA polymerase inhibitor, or by deregulation of origin licensing factor CDC6. In both cases, we observe that the majority of stress-responsive origins are also active in a small fraction of the cell population in a normal S phase, and stress increases their frequency of activation. In a search for the molecular determinants of origin efficiency, we compared the genetic and epigenetic features of origins displaying different levels of activation, and integrated their genomic positions in three-dimensional chromatin interaction networks derived from high-depth Hi-C and promoter-capture Hi-C data. We report that origin efficiency is directly proportional to the proximity to transcriptional start sites and to the number of contacts established between origin-containing chromatin fragments, supporting the organization of origins in higher-level DNA replication factories.

Epigenomic signatures associated with spontaneous and replication stress-induced DNA double strand breaks.

Common fragile sites (CFSs) are specific regions of all individuals' genome that are predisposed to DNA double strand breaks (DSBs) and undergo subsequent rearrangements. CFS formation can be induced in vitro by mild level of DNA replication stress, such as DNA polymerase inhibition or nucleotide pool disturbance. The mechanisms of CFS formation have been linked to DNA replication timing control, transcription activities, as well as chromatin organization. However, it is unclear what specific cis- or trans-factors regulate the interplay between replication and transcription that determine CFS formation. We recently reported genome-wide mapping of DNA DSBs under replication stress induced by aphidicolin in human lymphoblastoids for the first time. Here, we systematically compared these DSBs with regards to nearby epigenomic features mapped in the same cell line from published studies. We demonstrate that aphidicolin-induced DSBs are strongly correlated with histone 3 lysine 36 trimethylation, a marker for active transcription. We further demonstrate that this DSB signature is a composite effect by the dual treatment of aphidicolin and its solvent, dimethylsulfoxide, the latter of which potently induces transcription on its own. We also present complementing evidence for the association between DSBs and 3D chromosome architectural domains with high density gene cluster and active transcription. Additionally, we show that while DSBs were detected at all but one of the fourteen finely mapped CFSs, they were not enriched in the CFS core sequences and rather demarcated the CFS core region. Related to this point, DSB density was not higher in large genes of greater than 300kb, contrary to reported enrichment of CFS sites at these large genes. Finally, replication timing analyses demonstrate that the CFS core region contain initiation events, suggesting that altered replication dynamics are responsible for CFS formation in relatively higher level of replication stress.

Establishment of a digital PCR method for detection of Borrelia burgdorferi sensu lato complex DNA in cerebrospinal fluid

Direct detection of Borrelia burgdorferi sensu lato bacteria in patient samples for diagnosis of Lyme neuroborreliosis (LNB) is hampered by low diagnostic sensitivity, due to few bacteria in cerebrospinal fluids (CSF) samples. Evaluation of novel molecular methods, including digital PCR (dPCR), as future tools in diagnostics of LNB is desirable. This study aimed to establish a dPCR assay and validate pre-PCR procedures for detection of Borrelia in CSF. Synthetic DNA fragments and cultured Borrelia reference strains were used during optimisation experiments. In addition, 59 CSF specimens from patients examined for LNB were included for clinical validation. The results showed that the pre-PCR parameters with the highest impact on Borrelia-specific dPCR method performance were incubation of the PCR-plate at 4 °C for stabilization of droplets, centrifugation for target concentration, quick-spin for dPCR rain reduction, and PCR inhibition by matrix components. Borrelia DNA in CSF was detected in one out of nine patients with LNB. Diagnostic sensitivity was determined to be 11.1% and specificity 100%. In conclusion, this study reports an optimized Borrelia-specific dPCR method for direct detection of Borrelia in CSF samples. The present study does not support the use of Borrelia-specific dPCR as a routine method for diagnosing LNB.

Getting rid of 'rain' and 'stars': Mitigating inhibition effects on ddPCR data analysis, the case study of the invasive crayfish Pacifastacus leniusculus in the streams of Luxembourg.

ddPCR is becoming one of the most widely used tool in the field of eDNA-based aquatic monitoring. Although emulsion PCR used in ddPCR confers a partial mitigation to inhibition due to the high number of reactions for a single sample (between 10K and 20K), it is not impervious to it. Our results showed that inhibition impacts the amplitude of fluorescence in positive droplets with a different intensity among rivers. This signal fluctuation could jeopardize the use of a shared threshold among samples from different origin, and thus the accurate assignment of the positive droplets which is particularly important for low concentration samples such as eDNA ones: amplification events are scarce, thus their objective discrimination as positive is crucial. Another issue, related to target low concentration, is the artifactual generation of high fluorescence droplets ('stars'). Indeed, these could be counted as positive with a single threshold solution, which in turn could produce false positive and incorrect target concentration assessments. Approximating the positive and negative droplets distribution as normal, we proposed here a double threshold method accounting for both high fluorescence droplets ('stars') and PCR inhibition impact in delineating positive droplets clouds. In the context of low concentration template recovered from environmental samples, the application of this method of double threshold establishment could allow for a consistent sorting of the positive and negative droplets throughout ddPCR data generated from samples with varying levels of inhibitor contents. Due to low concentrations template and inhibition effects, Quantasoft software produced an important number of false negatives and positive comparatively to the double threshold method developed here. This case study allowed the detection of the invasive crayfish P. leniusculus in 32 out of 34 sampled sites from two main rivers (Alzette and Sûre) and five of their tributaries (Eisch, Attert, Mamer, Wiltz and Clerve).