Efficiency of Using Commercial DNA Extraction Kits and the Organic Extraction Method in Removal of Polymerase Chain Reaction Inhibitors

The first step in the determination of a perpetrator of a crime using DNA profiling is obtaining good quality DNA. The substrate on which the body fluid is located may contain substances that can co-extract with DNA and may inhibit subsequent PCR analysis. In this study, the efficiency of the removal of such contaminants were tested using three different methods, namely the PrepFiler DNA extraction kit, DNA IQ DNA extraction system, and the organic DNA extraction protocol. One, two, five, and ten μl of whole blood were deposited on soil, wood chips, and cotton swatches treated with bleach. DNA was extracted from all samples and controls using three extraction procedures and quantitated. The AmpflSTR Identifiler kit was used to develop genetic profiles to assess the quality of the extracted DNA. The overall recovery of DNA was reduced in all the treated samples compared to untreated ones. No DNA was recovered using the organic extraction on the soil and wood treated samples for all the sample volumes tested. Several fold reduction in the DNA recovery was noted for the PrepFiler kit and the DNA IQ system. Allelic drop-outs or complete absence of a genetic profile were identified for the soil and wood treated samples that were extracted using organic and PrepFiler extraction methods. The DNA IQ system was found to remove all the inhibitors from bleach, wood, and soil treated samples, yielding a complete genetic profile

Molecular detection and study of Campylobacter and related microorganisms

Philosophiae Doctor - PhDSpecies of Campylobacter, Arcobacter and Helicobacter have been associated with various diseases in humans and animals; and chickens have been identified as a reservoir of these microorganisms. Two published techniques and a new technique, developed in this dissertation, were evaluated to test its efficiency in removing PCR inhibitors from chicken samples. All of the techniques were based on agarose/DNA slants and were evaluated using multiplex PCR and an Internal Amplification Control. The new technique was found to be most effective and consequently used further in the study. A novel study was done to evaluate the survival of Campylobacter, Arcobacter and Helicobacter strains in chicken blood at -20, 4, 37 and 42ºC as well as at ambient room temperature (±222ºC). It was found that all strains could survive at all temperatures, albeit at different duration times. Most notably, an A. butzleri strain was able to survive at 4ºC for up to 297 days.South Afric

PCR detection of Burkholderia multivorans in water and soil samples

Background: Although semi-selective growth media have been developed for the isolation of Burkholderia cepacia complex bacteria from the environment, thus far Burkholderia multivorans has rarely been isolated from such samples. Because environmental B. multivorans isolates mainly originate from water samples, we hypothesized that water rather than soil is its most likely environmental niche. The aim of the present study was to assess the occurrence of B. multivorans in water samples from Flanders (Belgium) using a fast, culture-independent PCR assay. Results: A nested PCR approach was used to achieve high sensitivity, and specificity was confirmed by sequencing the resulting amplicons. B. multivorans was detected in 11 % of the water samples (n = 112) and 92 % of the soil samples (n = 25) tested. The percentage of false positives was higher for water samples compared to soil samples, showing that the presently available B. multivorans recA primers lack specificity when applied to the analysis of water samples. Conclusions: The results of the present study demonstrate that B. multivorans DNA is commonly present in soil samples and to a lesser extent in water samples in Flanders (Belgium)

PCR detection of Burkholderia multivorans in water and soil samples

Background: Although semi-selective growth media have been developed for the isolation of Burkholderia cepacia complex bacteria from the environment, thus far Burkholderia multivorans has rarely been isolated from such samples. Because environmental B. multivorans isolates mainly originate from water samples, we hypothesized that water rather than soil is its most likely environmental niche. The aim of the present study was to assess the occurrence of B. multivorans in water samples from Flanders (Belgium) using a fast, culture-independent PCR assay. Results: A nested PCR approach was used to achieve high sensitivity, and specificity was confirmed by sequencing the resulting amplicons. B. multivorans was detected in 11 % of the water samples (n = 112) and 92 % of the soil samples (n = 25) tested. The percentage of false positives was higher for water samples compared to soil samples, showing that the presently available B. multivorans recA primers lack specificity when applied to the analysis of water samples. Conclusions: The results of the present study demonstrate that B. multivorans DNA is commonly present in soil samples and to a lesser extent in water samples in Flanders (Belgium)

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

Development of an optimized method for the detection of airborne viruses with real-time PCR analysis

<p>Abstract</p> <p>Background</p> <p>Airborne viruses remain one of the major public health issues worldwide. Detection and quantification of airborne viruses is essential in order to provide information regarding public health risk assessment.</p> <p>Findings</p> <p>In this study, an optimized new, simple, low cost method for sampling of airborne viruses using Low Melting Agarose (LMA) plates and a conventional microbial air sampling device has been developed. The use of LMA plates permits the direct nucleic acids extraction of the captured viruses without the need of any preliminary elution step. Molecular detection and quantification of airborne viruses is performed using real-time quantitative (RT-)PCR (Q(RT-)PCR) technique. The method has been tested using Adenoviruses (AdVs) and Noroviruses (NoVs) GII, as representative DNA and RNA viruses, respectively. Moreover, the method has been tested successfully in outdoor experiments, by detecting and quantifying human adenoviruses (HAdVs) in the airborne environment of a wastewater treatment plant.</p> <p>Conclusions</p> <p>The great advantage of LMA is that nucleic acids extraction is performed directly on the LMA plates, while the eluted nucleic acids are totally free of inhibitory substances. Coupled with QPCR the whole procedure can be completed in less than three (3) hours.</p

Potential Panacea to the Complexities of Polymerase Chain Reaction (PCR)

The polymerase chain reaction (PCR) allows the exponential amplification of target DNA sequences, and has greatly impacted the world of scientific research. Although the reagents, equipment needed and the process (denaturation, annealing and elongation) appears simple and straightforward, PCR is adorned by complexities. Hence this paper seeks to discuss the complexities often encountered whilst performing PCR. Findings showed that primers, annealing temperature, wholesomeness and purity of template DNA, PCR contaminants and inhibitory substances, choice of polymerase, Magnessium chloride concentration, and number of cycles influence the outcome of a PCR. These factors sometimes enhance quality but compromise yield and vice versa. A high amount of Magnesium chloride or number of cycles in a PCR would result to a good yield but compromises quality and vice versa. In contrast, a high quality but lower yield of PCR is attained where the annealing temperature is either close to or higher than the melting temperatures of the primers.  Thus performing PCR is more or less choosing between quality and yield, and this paper would enable researchers strike the needed balance to overcome some of the associated complexities. Keywords: PCR, DNA, Taq polymerase, PCR inhibitory substances, primer

Characterization of the Hemagglutinin Cleaving Transmembrane Serine Proteases Matriptase and TMPRSS2

Influenza is one of the commonest infectious diseases affecting millions of people every year including 290,000 – 650,000 heavy casualties. Influenza viruses undergo constant genetic changes and every 10 – 50 years new influenza virus strains emerge that potentially cause a severe pandemic. In this modern interconnected world, experts believe the next influenza pandemic will be a “devastating global health event with far-reaching consequences” [1]. Novel effective anti-influenza drugs are in need. One strategy of influenza research is to focus on host-specific proteases that are essential for virus activation and spread. Trypsin-like serine proteases are crucial for influenza activation by mediating the cleavage of the viral surface glycoprotein HA and hence promoting the fusion potential of the virus. Therefore, their inhibition provides a promising therapeutic approach. The present work focused on the characterization of two relevant HA cleaving type-II transmembrane serine proteases matriptase and TMPRSS2. Chapter 3 and chapter 4 of this thesis engaged with the recombinant production of matriptase (chapter 3) in order to obtain a pure functional enzyme of high quality for a SAR study with novel monobasic (hence potentially bioavailable) matriptase inhibitors of the 3-amidinophenylalanine type (chapter 4). Adequate amounts of high-quality matriptase enzymes were isolated using a new expression system and in total 5 matriptase crystals were available at the end of this thesis for structural analysis. The matriptase inhibitor design in this thesis focused on matriptase-affine compounds with a fair selectivity profile against the blood coagulation enzymes thrombin and fXa. In total, 18 new monobasic and potentially bioavailable, as well as four new dibasic compounds of the 3-amidinophenylalanine types were tested. Based on the last published crystal structure of this inhibitor type in complex with matriptase from 2006 (PDB code 2GV6) docking was used as a structure-based virtual screening method for lead optimization of the compounds N-terminus. Selected compounds were suggested to interact with the carbonyl side chain of Gln175 of matriptase to achieve a higher affinity of matriptase compared to fXa. The 4-tert-butylureido-piperidine could be identified as suitable C-terminus in combination with 3-fluoro-4-hydroxymethyl biphenylsulphonyl N-terminally in order to obtain excellent selectivity over thrombin. The binding mode of this compound (compound 55) was crystallographically determined in complex with matriptase as well as trypsin. Trypsin proved as a suitable alternative to matriptase for detailed binding mode analysis of the compounds N-terminus. However, different preferences were detected for the C-terminus. Dibasic compounds showed higher matriptase affinity and selectivity in comparison with the monobasic analogues. However, the tested monobasic compounds were still decent matriptase inhibitors that are additionally suitable for cell culture and animal studies in their benzamidine prodrug forms, which are well established from related inhibitors of thrombin. In addition, selected monobasic as well as dibasic compounds demonstrated strong suppression of the replication of certain H9N2 influenza viruses in a matriptase-expressing MDCK II cell model. These matriptase inhibitors could be potential lead structures for the development of new drugs against H9 strains for influenza. TMPRSS2 is widely discussed for its role in influenza activation. With a TMPRSS2 dependancy of HA-activation of certain subtypes, the characterization of this protease is an important prerequisite for being available as a target for influenza drug design. However, only little is known about the physiological function of TMPRSS2 and no experimental structure data are available at the moment to enable a structure-based drug development. Therefore, chapter 5 of this thesis focused on the characterization of TMPRSS2 in order to develop a strategy for the isolation of proteolytically active TMPRSS2 from cell culture. Even though, no functional TMPRSS2 could be recovered at the end of this work some new structural characteristics of TMPRSS2 were identified as crucial for functionality insight the cell. In general, TMPRSS2 without the cytosolic part, the transmembrane domain and the LDLRA domain is able to undergo autocatalytically activation if an artificial signal peptide was added N-terminal to enable entry into the endoplasmic reticulum. The presence of the cysteine-rich SRCR domain and the presence of the disulfide chain that connects the SPD and the stem region after activation cleavage have been identified as crucial for activity. N-terminal truncation of TMPRSS2 did not result in obvious dislocation within the cell: as the full-length positive control truncated TMPRSS2 was exclusively found in cell compartments surrounding the nucleus in immunofluorescence experiments. However, a reduced proteolytic cleavage activity towards H3-HA in co-expression experiments has been observed and might be a result of dislocation, since truncated TMPRSS2 is not bound to the biomembrane anymore. In addition, TMPRSS2 has been identified as a potential substrate of matriptase in vitro, which suggests possible participation in several zymogen cascades

Optimized protocol for DNA extraction from ancient skeletal human remains using Chelex-100

©2021 The Author. Published by AkiNik Publications. This is an open access article available under a Creative Commons licence. The published version can be accessed at the following link on the publisher’s website: https://www.forensicpaper.com/archives/2021.v3.i1.A.35PCR-based analysis of skeletonized human remains is a common aspect in both forensic human identification as well as Ancient DNA research. In this, both areas not merely utilize very similar methodology, but also share the same problems regarding quantity and quality of recovered DNA and presence of inhibitory substances in samples from excavated remains. To enable amplification based analysis of the remains, development of optimized DNA extraction procedures is thus a critical factor in both areas. The study here presents an optimized protocol for DNA extraction from ancient skeletonized remains using Chelex-100, which proved to be effective in yielding amplifiable extracts from sample material excavated after centuries in a soil environment, which consequently have high inhibitor content and overall limited DNA preservation. Success of the optimization strategies utilized is shown in significantly improved amplification outcomes compared to the predecessor method