PCR Inhibitors Research Articles

Ganciclovir and Its Hemocompatible More Lipophilic Derivative Can Enhance the Apoptotic Effects of Methotrexate by Inhibiting Breast Cancer Resistance Protein (BCRP)

Efflux transporters, namely ATP-binding cassette (ABC), are one of the primary reasons for cancer chemoresistance and the clinical failure of chemotherapy. Ganciclovir (GCV) is an antiviral agent used in herpes simplex virus thymidine kinase (HSV-TK) gene therapy. In this therapy, HSV-TK gene is delivered together with GCV into cancer cells to activate the phosphorylation process of GCV to active GCV-triphosphate, a DNA polymerase inhibitor. However, GCV interacts with efflux transporters that are responsible for the resistance of HSV-TK/GCV therapy. In the present study, it was explored whether GCV and its more lipophilic derivative (1) could inhibit effluxing of another chemotherapeutic, methotrexate (MTX), out of the human breast cancer cells. Firstly, it was found that the combination of GCV and MTX was more hemocompatible than the corresponding combination with compound 1. Secondly, both GCV and compound 1 enhanced the cellular accumulation of MTX in MCF-7 cells, the MTX exposure being 13–21 times greater compared to the MTX uptake alone. Subsequently, this also reduced the number of viable cells (41–56%) and increased the number of late apoptotic cells (46–55%). Moreover, both GCV and compound 1 were found to interact with breast cancer resistant protein (BCRP) more effectively than multidrug-resistant proteins (MRPs) in these cells. Since the expression of BCRP was higher in MCF-7 cells than in MDA-MB-231 cells, and the cellular uptake of GCV and compound 1 was smaller but increased in the presence of BCRP-selective inhibitor (Fumitremorgin C) in MCF-7 cells, we concluded that the improved apoptotic effects of higher MTX exposure were raised mainly from the inhibition of BCRP-mediated efflux of MTX. However, the effects of GCV and its derivatives on MTX metabolism and the quantitative expression of MTX metabolizing enzymes in various cancer cells need to be studied more thoroughly in the future.

Clinical evaluation of a multiplex real-timeRT-PCR assay for detection of SARS-CoV-2 in individual and pooled upper respiratory tract samples.

The aim of this study was to identify and validate a sensitive, high-throughput, and cost-effective SARS-CoV-2 real-time RT-PCR assay to be used as a surveillance and diagnostic tool for SARS-CoV-2 in a university surveillance program. We conducted a side-by-side clinical evaluation of a newly developed SARS-CoV-2 multiplex assay (EZ-SARS-CoV-2 Real-Time RT-PCR) with the commercial TaqPath COVID-19 Combo Kit, which has an Emergency Use Authorization from the FDA. The EZ-SARS-CoV-2 RT-PCR incorporates two assays targeting the SARS-CoV-2 N gene, an internal control targeting the human RNase P gene, and a PCR inhibition control in a single reaction. Nasopharyngeal (NP) and anterior nares (AN) swabs were tested as individuals and pools with both assays and in the ABI 7500 Fast and the QuantStudio 5 detection platforms. The analytical sensitivity of the EZ-SARS-CoV-2 RT-PCR assay was 250 copies/ml or approximately 1.75 genome copy equivalents per reaction. The clinical performance of the EZ-SARS-CoV-2 assay was evaluated using NP and AN samples tested in other laboratories. The diagnostic sensitivity of the assay ranged between 94 and 96% across the detection platforms, and the diagnostic specificity was 94.06%. The positive predictive value was 94%, and the negative predictive value ranged from 94 to 96%. Pooling five NP or AN specimens yielded 93% diagnostic sensitivity. The overall agreement between these SARS-CoV-2 RT-PCR assays was high, supported by a Cohen’s kappa value of 0.93. The EZ-SARS-CoV-2 RT-PCR assay performance attributes of high sensitivity and specificity with AN sample matrix and pooled upper respiratory samples support its use in a high-throughput surveillance testing program.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00705-021-05148-1.

Protein Domain Specific Covalent Inhibition of Human DNA Polymerase β.

DNA polymerase β (Pol β) is a frequently overexpressed and/or mutated bifunctional repair enzyme. Pol β possesses polymerase and lyase active sites, that are employed in two steps of base excision repair. Pol β is an attractive therapeutic target for which there is a need for inhibitors. Two mechanistically inspired covalent inhibitors (1, IC50=21.0 μM; 9, IC50=18.7 μM) that modify lysine residues in different Pol β active sites are characterized. Despite modifying lysine residues in different active sites, 1 and 9 inactivate the polymerase and lyase activities of Pol β. Fluorescence anisotropy experiments indicate that they do so by preventing DNA binding. Inhibitors 1 and 9 provide the basis for a general approach to preparing domain selective inhibitors of bifunctional polymerases. Such molecules could prove to be useful tools for studying the role of wild type and mutant forms of Pol β and other polymerases in DNA repair.

One size does not fit all: Tuning eDNA protocols for high‐ and low‐turbidity water sampling

AbstractFindings from eDNA metabarcoding are strongly influenced by experimental approach, yet the effect of pre‐PCR sample processing on taxon detection and estimates of biodiversity across different water types is still poorly resolved. To fill this data gap, we investigated the impact of sampling effort, extraction method, and filter pore size on DNA yield, PCR inhibition, and 16S rDNA metabarcoding results for fishes in water samples collected from inshore turbid‐ and offshore clear‐water environments. The turbid‐water samples had high concentrations of suspended organic and/or inorganic material and yielded ~3.2× more DNA and exhibited high levels of PCR inhibition compared with the low‐turbidity, clear‐water samples. Importantly, there were no striking differences in the results of our metabarcoding experiments based on extraction method or filter pore size. While a small number of unique species of relatively low read count were detected in all turbid‐water treatments, most species were consistently detected across samples. Results for the clear‐water samples were strikingly different, with low DNA yield, high levels of variation across replicates, and a high number of non‐overlapping species across treatments. These findings indicate a patchy distribution of eDNA in offshore environments, which means higher volumes of water (≥ 2 L per replicate) must be filtered in habitats where target DNA is likely to be sparse. In semi‐closed systems such as estuaries, higher concentrations of target DNA are expected, and we found that either a 1.0 or 3.0 µm filter pore size was sufficient to capture standing diversity, while decreasing the risk of clogging. For economical DNA extraction and inhibitor removal, we recommend a combination of Omega Bio‐tek E.Z.N.A Tissue DNA kit followed by a PCR inhibitor removal step using the Zymo Kit. Finally, we emphasize that pilot studies should be undertaken whenever sampling in a new environment to identify which protocol is most appropriate.

Fungal Basidiomycete Ceratobasidium theobromae DNA obtained directly from cocoa petioles

Abstract. Junaid M, Purwantara A, Guest G. 2021. Fungal Basidiomycete Ceratobasidium theobromae DNA obtained directly from cocoa petioles. Biodiversitas 22: 2838-2843. Understanding the biology of the fastidious Basidiomycete Ceratobasidium theobromae occupying host-tissue is essential for plant disease management. Direct pathogen DNA extraction from infected plant tissue avoids the need for isolation in artificial media. We report a modified DNA isolation protocol to obtain total plant DNA designed to overcome DNA extraction and isolation problems caused by infected petioles rich in polysaccharides and phenolic substances as a primary source of gummosis. This study examined and compared total plant DNA isolated from petioles high in polysaccharides and polyphenolic compounds using two methods: conventional CTAB lysis buffer and Kits (the standard method), and a new modified CTAB protocol to address these PCR inhibitors. The modified method resulted in higher quality and quantity of C. theobromae crude DNA and amplified PCR product. The modified method produced large quantities of clear, transparent, aqueous DNA-containing lysate (crude DNA) with a clear separation between the upper crude DNA and organic waste layers. Mean DNA absorbance was 1.80, and the lowest DNA yield was 836.6 ng/µl. With the standard method, the blurred, viscous lysate obtained showed signs of gummosis, with poor separation between layers of crude DNA, polysaccharides, protein, and organic waste layers and low yield. Gel electrophoresis indicated poor quality DNA extract. We conclude that this modified method will be valuable for genetic diversity and disease studies in a range of previously challenging plant tissues and their pathogens.

Evaluation of DNA extraction methods for quantifying Helicobacter pylori in water with PCR inhibitors

Abstract DNA extraction methods were evaluated to reduce PCR inhibitors and quantify Helicobacter pylori directly from water samples using real-time PCR. Three nucleic acid extraction methods were evaluated for different types of water samples. While the QIAamp DNA mini kit for tissue was suitable for DNA extraction from treated water, the QIAamp DNA stool mini kit was still efficient in analyzing samples from river water after heavy rain and with high concentration of PCR inhibitors. The FastDNA SPIN Kit for Soil could extract DNA effectively from microbes in river and stream waters without heavy rain. Immunomagnetic separation (IMS) was used prior to DNA extraction and was a useful tool for reducing PCR inhibitors in influent and stream samples. H. pylori in various waters could be quantified directly by real-time PCR while minimizing the effect of PCR inhibitors by an appropriate method through the evaluation of DNA extraction methods considering the characteristics of the matrix water. The findings of the present study suggest that the types or characteristics of water sample by source and precipitation are an important factor in detecting H. pylori and they can be applied when detecting and monitoring of other pathogens in water.

Comparing Quantitative Methods for Analyzing Sediment DNA Records of Cyanobacteria in Experimental and Reference Lakes.

Sediment DNA (sedDNA) analyses are rapidly emerging as powerful tools for the reconstruction of environmental and evolutionary change. While there are an increasing number of studies using molecular genetic approaches to track changes over time, few studies have compared the coherence between quantitative polymerase chain reaction (PCR) methods and metabarcoding techniques. Primer specificity, bioinformatic analyses, and PCR inhibitors in sediments could affect the quantitative data obtained from these approaches. We compared the performance of droplet digital polymerase chain reaction (ddPCR) and high-throughput sequencing (HTS) for the quantification of target genes of cyanobacteria in lake sediments and tested whether the two techniques similarly reveal expected patterns through time. Absolute concentrations of cyanobacterial 16S rRNA genes were compared between ddPCR and HTS using dated sediment cores collected from two experimental (Lake 227, fertilized since 1969 and Lake 223, acidified from 1976 to 1983) and two reference lakes (Lakes 224 and 442) in the Experimental Lakes Area (ELA), Canada. Relative abundances of Microcystis 16S rRNA (MICR) genes were also compared between the two methods. Moderate to strong positive correlations were found between the molecular approaches among all four cores but results from ddPCR were more consistent with the known history of lake manipulations. A 100-fold increase in ddPCR estimates of cyanobacterial gene abundance beginning in ~1968 occurred in Lake 227, in keeping with experimental addition of nutrients and increase in planktonic cyanobacteria. In contrast, no significant rise in cyanobacterial abundance associated with lake fertilization was observed with HTS. Relative abundances of Microcystis between the two techniques showed moderate to strong levels of coherence in top intervals of the sediment cores. Both ddPCR and HTS approaches are suitable for sedDNA analysis, but studies aiming to quantify absolute abundances from complex environments should consider using ddPCR due to its high tolerance to PCR inhibitors.

Influence of ink and smoke ATM security systems on dactyloscopy and subsequent DNA analysis after detonation

Automated Teller Machine bombings are an increasing societal problem that are often committed using Improvised Explosive Devices. The evolution in IEDs and the negative consequences for society require new security measures to prevent these crimes. Ink staining and security smoke devices are added to cash cassettes, in order to protect ATMs and prevent ATM bombings. Traces found at crime scenes, such as fingerprints and DNA, can contribute to the identification of perpetrators. However, the effect of ink staining and security smoke devices on dactyloscopy and DNA profiling is still unknown. In the current study, we demonstrate that procedures using Citrus Cleaner or sulfosalicylic acid were successful in removing ink and security smoke deposited on plastic plates but did result in the massive loss of fingerprint information as only a low number (4%) of good quality fingerprints were recovered after smoke contamination. Secondly, security ink Sun Blue ES2, but not SICPA Green and Sun Blue ES1, had a significant impact on DNA profiling success. DNA concentrations obtained from blood spiked swabs decreased with increasing ink concentration resulting in a complete loss of genotype information with the addition of ≥10 μl Sun Blue ES2. No noticeable PCR inhibition or DNA degradation was detected during quantification, but a decreased efficiency of DNA extraction could not be excluded. Security smoke, on the other hand, does not seem to have a significant influence on DNA analysis. Precautions must therefore be taken in order to avoid contaminating DNA swabs with ink during sampling. Thirdly, only a single negative impression of a glove in ink and a single glove print were able to be visualized with white fingerprint powder on detonated cash cassettes. In conclusion, the detection of glove prints and fingerprints is limited and security ink, contrary to smoke, after detonation can have a negative influence on downstream DNA analysis procedures.

Resource-efficient internally controlled in-house real-time PCR detection of SARS-CoV-2

BackgroundThe reliable detection of SARS-CoV-2 has become one of the most important contributions to COVID-19 crisis management. With the publication of the first sequences of SARS-CoV-2, several diagnostic PCR assays have been developed and published. In addition to in-house assays the market was flooded with numerous commercially available ready-to-use PCR kits, with both approaches showing alarming shortages in reagent supply.AimHere we present a resource-efficient in-house protocol for the PCR detection of SARS-CoV-2 RNA in patient specimens (RKI/ZBS1 SARS-CoV-2 protocol).MethodsTwo duplex one-step real-time RT-PCR assays are run simultaneously and provide information on two different SARS-CoV-2 genomic regions. Each one is duplexed with a control that either indicates potential PCR inhibition or proves the successful extraction of nucleic acid from the clinical specimen.ResultsLimit of RNA detection for both SARS-CoV-2 assays is below 10 genomes per reaction. The protocol enables testing specimens in duplicate across the two different SARS-CoV-2 PCR assays, saving reagents by increasing testing capacity. The protocol can be run on various PCR cyclers with several PCR master mix kits.ConclusionThe presented RKI/ZBS1 SARS-CoV-2 protocol represents a cost-effective alternative in times of shortages when commercially available ready-to-use kits may not be available or affordable.

Effect of disinfection agents and quantification of potentially viable Leptospira in fresh water samples using a highly sensitive integrity-qPCR assay.

Leptospirosis is an emerging worldwide zoonotic disease, but the general biology of the causative agents is still poorly understood. Humans are an occasional host. The main risk factors are water-associated exposure during professional or recreational activities or during outbreaks in endemic areas. Detecting the presence of pathogenic bacteria in aquatic environments and their capacity to resist various inactivation processes are research fields that need to be further developed. In addition, the methods used for detecting and enumerating Leptospira still need to be improved. We aimed to describe a new quantitative polymerase chain reaction coupled to propidium monoazide treatment (PMAqPCR) that targets not only total Leptospira but also discriminates pathogenic from non-pathogenic Leptospira while also addressing PCR inhibitors, a frequently encountered problem when studying environmental water. In a second step, the killing efficiency of Leptospira to different treatments was tested and PMAqPCR compared to culture-based enumeration. This provided information about the effects of temperature, as well as ultraviolet and chlorine disinfection, that are both related to water treatment processes, in particular for the production of drinking water, on the persistence of both saprophytic and pathogenic Leptospira. Finally, PMAqPCR was used for the detection of Leptospira in freshwater samples for a proof-of-concept. In conclusion, our method could be used for routine freshwater monitoring and allows better evaluation of the presence of Leptospira, allowing evaluation of the bacterial dynamics in a designated area or assessment of the efficacy of water disinfection processes.

Selective Inhibition of DNA Polymerase β by a Covalent Inhibitor.

DNA polymerase β (Pol β) plays a vital role in DNA repair and has been closely linked to cancer. Selective inhibitors of this enzyme are lacking. Inspired by DNA lesions produced by antitumor agents that inactivate Pol β, we have undertaken the development of covalent small-molecule inhibitors of this enzyme. Using a two-stage process involving chemically synthesized libraries, we identified a potent irreversible inhibitor (14) of Pol β (KI = 1.8 ± 0.45 μM, kinact = (7.0 ± 1.0) × 10-3 s-1). Inhibitor 14 selectively inactivates Pol β over other DNA polymerases. LC-MS/MS analysis of trypsin digests of Pol β treated with 14 identified two lysines within the polymerase binding site that are covalently modified, one of which was previously determined to play a role in DNA binding. Fluorescence anisotropy experiments show that pretreatment of Pol β with 14 prevents DNA binding. Experiments using a pro-inhibitor (pro-14) in wild type mouse embryonic fibroblasts (MEFs) indicate that the inhibitor (5 μM) is itself not cytotoxic but works synergistically with the DNA alkylating agent, methylmethanesulfonate (MMS), to kill cells. Moreover, experiments in Pol β null MEFs indicate that pro-14 is selective for the target enzyme. Finally, pro-14 also works synergistically with MMS and bleomycin to kill HeLa cells. The results suggest that pro-14 is a potentially useful tool in studies of the role of Pol β in disease.

Extraction of human noroviruses from leafy greens and fresh herbs using magnetic silica beads

Consumption of leafy greens and to a lesser extent fresh herbs has been associated with several foodborne outbreaks including human norovirus (HuNoV). However, the extraction and detection of viruses from these matrices present multiple challenges such as low recovery yields and relatively high PCR inhibition. A new magnetic silica bead based (MSB) extraction protocol was developed and used to recover norovirus from leafy greens and fresh herbs. The performance results were compared to the ISO 15216–1:2017 standard. The HuNoV GII.4 and GI.5 recovery yields from spiked lettuce using the MSB extraction protocol range from 33 to 82%. There was a good correlation between murine norovirus (MNV) and HuNoV recovery yields from fresh herbs and leafy greens. No reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) inhibition was detected from leafy green extracts using the MSB methodology. The selected commercial RT-qPCR detection kit had a major impact on RT-qPCR inhibition levels detected in the ISO 15216–1:2017 RNA extracts. RNase treatment was used to estimate genome recovery from HuNoV with intact capsids. This treatment resulted in similar HuNoV and MNV recovery yields. Between 2019 and 2020, the MSB protocol was used to conduct a survey of HuNoV in domestic and imported leafy greens and fresh herbs sold at retail in Canada. All of the 280 samples tested were negative. Overall, the use of MSB was shown to be an efficient approach to recover HuNoV from leafy greens and certain types of fresh herbs and to conduct surveys.

Optimized protocol for a quantitative SARS-CoV-2 duplex RT-qPCR assay with internal human sample sufficiency control

There is growing evidence that measurement of SARS-CoV-2 viral copy number can inform clinical and public health management of SARS-CoV-2 carriers and COVID-19 patients. Here we show that quantification of SARS-CoV-2 is feasible in a clinical setting, using a duplex RT-qPCR assay which targets both the E gene (Charité assay) and a human RNA transcript, RNase P (CDC assay) as an internal sample sufficiency control.Samples in which RNase P is not amplified indicate that sample degradation has occurred, PCR inhibitors are present, RNA extraction has failed or swabbing technique was insufficient. This important internal control reveals that 2.4 % of nasopharyngeal swabs (15/618 samples) are inadequate for SARS-CoV-2 testing which, if not identified, could result in false negative results.We show that our assay is linear across at least 7 logs and is highly reproducible, enabling the conversion of Cq values to viral copy numbers using a standard curve. Furthermore, the SARS-CoV-2 copy number was independent of the RNase P copy number indicating that the per-swab viral copy number is not dependent on sampling- further allowing comparisons between samples.The ability to quantify SARS-CoV-2 viral copy number will provide an important opportunity for viral burden-guided public health and clinical decision making.

Water pre-filtration methods to improve environmental DNA detection by real-time PCR and metabarcoding.

Environmental DNA (eDNA) analysis is a novel approach for biomonitoring and has been mostly used in clear water. It is difficult to detect eDNA in turbid water as filter clogging occurs, and environmental samples contain various substances that inhibit the polymerase chain reaction (PCR) and affect the accuracy of eDNA analysis. Therefore, we applied a pre-filtration method to better detect the fish species (particularly pale chub, Opsariichthys platypus) present in a water body by measuring eDNA in environmental samples containing PCR inhibitors. Upon conducting 12S rRNA metabarcoding analysis (MiFish), we found that pre-filtration did not affect the number or identities of fish species detected in our samples, but pre-filtration through pore sizes resulted in significantly reduced variance among replicate samples. Additionally, PCR amplification was improved by the pre-filtration of environmental samples containing PCR inhibitors such as humic substances. Although this study may appear to be a conservative and ancillary experiment, pre-filtration is a simple technique that can not only improve the physical properties of water, such as turbidity, but also the quality of eDNA biomonitoring.

Development of duplex TaqMan-based real-time PCR assay for the simultaneous detection of Perkinsus olseni and P. chesapeaki in host Manila clam tissue samples

The aetiological agent Perkinsus olseni is globally recognised as a major threat for shellfish production considering its wide geographical distribution across Asia, Europe, Australia and South America. Another species, Perkinsus chesapeaki, which has never been known to be associated with significant mortality events, was recently detected along French coasts infecting clam populations sporadically in association with P. olseni. Identifying potential cryptic infections affecting Ruditapes philippinarum is essential to develop appropriate host resource management strategies. Here, we developed a molecular method based on duplex real-time quantitative PCR for the simultaneous detection of these two parasites, P. olseni and P. chesapeaki, in the different clam tissues: gills, digestive gland, foot, mantle, adductor muscle and the rest of the soft body. We firstly checked the presence of possible PCR inhibitors in host tissue samples. The qPCR reactions were inhibited depending on the nature of the host organ. The mantle and the rest of the soft body have a high inhibitory effect from threshold of host gDNA concentration of 2 ng.µL−1, the adductor muscle and the foot have an intermediate inhibition of 5 ng.µL−1, and the gills and digestive gland do not show any inhibition of the qPCR reaction even at the highest host gDNA concentration of 20 ng.µL−1. Then, using the gills as a template, the suitability of the molecular technique was checked in comparison with the Ray’s Fluid Thioglycolate Medium methodology recommended by the World Organisation for Animal Health. The duplex qPCR method brought new insights and unveiled cryptic infections as the co-occurrence of P. olseni and P. chesapeaki from in situ tissue samples in contrast to the RFTM diagnosis. The development of this duplex qPCR method is a fundamental work to monitor in situ co-infections that will lead to optimised resource management and conservation strategies to deal with emerging diseases.

MiRNAs detection in equine plasma by quantitative polymerase chain reaction for doping control: Assessment of blood sampling and study of eca-miR-144 as potential erythropoiesis stimulating agent biomarker.

Short half-life doping substances are, quickly eliminated and therefore difficult to control with traditional analytical chemistry methods. Indirect methods targeting biomarkers constitute an alternative to extend detection time frames in doping control analyses. Gene expression analysis (i.e., transcriptomics) has already shown interesting results in both humans and equines for erythropoietin (EPO), growth hormone (GH), and anabolic androgenic steroid (AAS) misuses. In humans, circulating cell-free microRNAs in plasma were described as new potential biomarkers for control of major doping agent (MDA) abuses. The development of a quantitative polymerase chain reaction (qPCR) method allowing the detection of circulating miRNAs was carried out on equine plasma collected on different type of tubes (EDTA, lithium-heparin [LiHep]). Although analyzing plasma collected in EDTA tubes is a standard method in molecular biology, analyzing plasma collected in LiHep tubes is challenging, as heparin is a reverse transcription (RT) and a PCR inhibitor. Different strategies were considered, and attention was paid on both miRNAs extraction quality and detection sensitivity. The detection of endogenous circulating miRNAs was performed and compared between the different types of tubes. In parallel, homologs of human miRNAs characterized as potential biomarkers of doping were sought in equine databases. The miRNA eca-miR-144, described as potential erythropoiesis stimulating agents (ESAs) administration candidate biomarker was retained and assessed in equine post-administration samples. The results about the qPCR method development and optimization are exposed as well as the equine miRNAs detection. To our knowledge, this work is the first study and the proof of concept of circulating miRNAs detection in plasma dedicated to equine doping control.

Monitoring of diverse enteric pathogens across environmental and host reservoirs with TaqMan array cards and standard qPCR: a methodological comparison study

SummaryBackgroundMultiple bacteria, viruses, protists, and helminths cause enteric infections that greatly impact human health and wellbeing. These enteropathogens are transmited via several pathways through human, animal, and environmental reservoirs. Individual qPCR assays have been extensively used to detect enteropathogens within these types of samples, whereas the TaqMan array card (TAC), which allows simultaneous detection of multiple enteropathogens, has only previously been validated in human clinical samples.MethodsIn this methodological comparison study, we compared the performance of a custom 48-singleplex TAC relative to standard qPCR. We established the sensitivity and specificity of each method for the detection of eight enteric targets, by using spiked samples with varying levels of PCR inhibition. We then tested the prevalence and abundance of pathogens in wastewater from Melbourne (Australia), and human, animal, and environmental samples from informal settlements in Suva, Fiji using both TAC and qPCR.FindingsBoth methods exhibited similarly h specificity (TAC 100%, qPCR 94%), sensitivity (TAC 92%, qPCR 100%), and quantitation accuracy (TAC 91%, qPCR 99%) in non-inhibited sample matrices with spiked gene fragments. PCR inhibitors substantially affected detection via TAC, though this issue was alleviated by ten-fold sample dilution. Among samples from informal settlements, the two techniques performed similarly for detection (89% agreement) and quantitation (R2 0·82) for the eight enteropathogen targets. The TAC additionally included 38 other enteric targets, enabling detection of diverse faecal pathogens and extensive environmental contamination that would be prohibitively labour intensive to assay by standard qPCR.InterpretationThe two techniques produced similar results across diverse sample types, with qPCR prioritising greater sensitivity and quantitation accuracy, and TAC trading small reductions in these for a cost-effective larger enteropathogen panel enabling a greater number of enteric pathogens to be analysed concurrently, which is beneficial given the abundance and variety of enteric pathogens in environments such as urban informal settlements. The ability to monitor multiple enteric pathogens across diverse reservoirs could allow better resolution of pathogen exposure pathways, and the design and monitoring of interventions to reduce pathogen load.FundingWellcome Trust Our Planet, Our Health programme.

Development of a 3’:5’ digital PCR assay to determine horse mRNA integrity

Accurate tools to measure RNA integrity are essential to obtain reliable gene expression data. The reverse transcription quantitative PCR (RT-qPCR) based 3’:5′ assay permits a direct determination of messenger RNA (mRNA) integrity. However, the use of standard curves and the possible effect of PCR inhibitors make this method cumbersome and prone to variation, especially in small samples. Here we developed a triplex digital PCR (dPCR) 3’:5’ assay for assessing RNA integrity in equine samples as rapid and simple alternative to RT-qPCR. This dPCR assay not only provides a straight forward analysis of the mRNA integrity, but also of its quantity.

Pathological and Molecular Characterization of Newcastle Disease Virus Isolated from Gallus gallus in Java, Indonesia

Background: Known as the vital avian disease in Indonesia, Newcastle disease (ND) has surged all over Indonesia which cause an immense drop in poultry industry. This investigation intended to examine the pathological and molecular characteristics of the latest Newcastle disease virus (NDV) derived from Gallus gallus in Java, Indonesia.Methods: The samples were inoculated on the embryonic chicken eggs (ECEs), then identified using the hemagglutination test (HA test), insulated isothermal PCR (iiPCR) and hemagglutination inhibition test (HI test). It conducted the reverse transcription-polymerase chain reaction (RT-PCR) using MSF1 and MSF2 primers and followed by nucleotide sequencing for the fusion protein gene. B-cells epitope prediction analysis was applied using the Immune Epitope Database and Analysis Resource (IEDB) and VaxiJen v2.0 to predict its immunogenicity. In addition, it also performed the histopathological examination by applying hematoxylin and eosin staining.Result: The results identified two viruses, NDV-1 and NDV-2, based on the fusion protein gene. Significantly, the cleavage site motif of NDV1 is 112GRQGRL117 (avirulent) and whereas for NDV2 is 112RRRKRF117 (virulent). Furthermore, it also predicted the “CKMGSRPSTKNPAP” peptide from NDV1 could be an immunogenic epitope candidate with a BepiPred linear epitope prediction score of 17.08. In summary, it suggests that NDV1 isolate can be used as a reference for vaccine design in Indonesia.

Генотипування лістерій за допомогою полімеразної ланцюгової реакції (ПЛР) та його епідеміологічне значення

The purpose of the study. The article covers the issues of genotyping of listeria by polymerase chain reaction (PCR) and its epidemiological significance. It is known that molecular genetic methods allow to detect specific microbial pathogens, virulence markers, antimicrobial resistance genes faster and with greater sensitivity than traditional culture methods. Therefore, the development of detection methods and genotyping by polymerase chain reaction (PCR) is relevant. Materials and methods. For the detection and genotyping of Listeria, the technology of DNA chips is becoming increasingly important, which can significantly expand the possibilities of molecular detection. Chip technology can be used to simultaneously identify a whole range of pathogenic microorganisms, to determine genetic virulence markers, the relationship to antibiotics, subtyping, as well as to determine the quality of microorganisms in samples. A simplified version of DNA chip technology is multiplex (numerical) PCR, which is used to detect and genotype listeria. Studies have shown that to detect Listeria spp. using a polymerase chain reaction, it is advisable to use the gene iap (invasive associated protein), known for 6 species of listeria, which encodes a protein P 60 that is common to all species of listeria, including L.murrayi. Computer analysis revealed areas with 100% homology, from which primers were selected for PCR detection of all types of listeria. Areas of genomes characterized by 100% homology were selected for further analysis and labeling of primer sets. The sequences of the constructed primers List 1 and List 2 allowed to identify 6 species of Listeria (L. monocytogenes, L. innocua, L. ivanovii, L. grayi, L. seeligeri, L. welshimeri). Increasing the length of the primer leads to the increasing of specificity of PCR analysis. The greater the length of the primer, the smaller the specific gravity of one error of the unpaired nucleotide. The degree of primer homology is a key parameter that indicates the "quality" of a set of primers. Results and discussion. It is established that a significant disadvantage of the vast majority diagnosed using PCR test systems is the lack of internal control of amplification. The negative result of PCR analysis may be due to the absence in the clinical material of a fragment of the Listeria genome, and the fact that the PCR product was not synthesized for other reasons. They may be as the following ones: operator errors, erroneously determined reaction mixture concentrations and PCR temperature parameters. False-negative results can also be caused by factors that inhibit thermostable DNA polymerase. In its turn, such inhibition of the enzyme responsible for amplification is caused by a very large amount of DNA - template, pre-treatment of clinical samples. It has been shown that 80% of clinical specimens contain a substance that inhibits DNA polymerase. Therefore, it is necessary to use internal control, the positive result of the reaction of which indicates the successful amplification, that is the absence of false positive results. Conclusion. There are several reasons why the accuracy of PCR analysis does not reach 100%. Accuracy depends on the technology (variety) of PCR - the method used (ordinary or fluorescent), detection of amplicons, PCR homogeneous or nested, nested in one test tube or in two test tubes, as well as the level of quality of the survey (primarily on the technical parameters of the amplifier). The test systems used can be used for PCR detection and are recommended as standard primer sets for the detection and cross-species testing of listeria, which is important for the timely implementation of appropriate anti-epidemic measures in listeriosis