MC-PRPA-HLFIA Cascade Detection System for Point-of-Care Testing Pan-Drug-Resistant Genes in Urinary Tract Infection Samples.

Recently, urinary tract infection (UTI) triggered by bacteria carrying pan-drug-resistant genes, including carbapenem resistance gene blaNDM and blaKPC, colistin resistance gene mcr-1, and tet(X) for tigecycline resistance, have been reported, posing a serious challenge to the treatment of clinical UTI. Therefore, point-of-care (POC) detection of these genes in UTI samples without the need for pre-culturing is urgently needed. Based on PEG 200-enhanced recombinase polymerase amplification (RPA) and a refined Chelex-100 lysis method with HRP-catalyzed lateral flow immunoassay (LFIA), we developed an MCL-PRPA-HLFIA cascade assay system for detecting these genes in UTI samples. The refined Chelex-100 lysis method extracts target DNA from UTI samples in 20 min without high-speed centrifugation or pre-incubation of urine samples. Following optimization, the cascade detection system achieved an LOD of 102 CFU/mL with satisfactory specificity and could detect these genes in both simulated and actual UTI samples. It takes less than an hour to complete the process without the use of high-speed centrifuges or other specialized equipment, such as PCR amplifiers. The MCL-PRPA-HLFIA cascade assay system provides new ideas for the construction of rapid detection methods for pan-drug-resistant genes in clinical UTI samples and provides the necessary medication guidance for UTI treatment.

Rapid Nucleic Acid Extraction for Aquatic Animal DNA Virus Determination Using Chelex 100 Resin via Conventional PCR and Digital Droplet PCR Detection.

Molecular diagnostic testing for viral pathogens is crucial in aquaculture. The efficient and convenient preparation of pathogenic microbial nucleic acids is the basis of molecular diagnosis. Here, we developed a simplified deoxyribonucleic acid (DNA) extraction method from aquatic animal DNA viruses using the Chelex 100 resin. The nucleic acid was extracted from infected tissues and cell culture for the detection of three common aquatic viral pathogens (CEV, CyHV-2, and GSIV). We compared the extraction effects of a current commercial kit extraction method and the Chelex 100 resin extraction method according to nucleic acid concentration, conventional polymerase chain reaction (PCR), and digital droplet PCR (ddPCR). The results indicated that both extraction procedures could obtain high-quality nucleotide samples. Extracting DNA using the Chelex 100 resin led to better detective efficiency for ddPCR molecular diagnostic testing. The whole process took less than 20 min, and only Chelex 100 resin solution was added to the tissues or cells without multiple tubes being transferred several times. The extracted DNA concentration and the detection sensitivity were high. These results indicated that the Chelex 100 resin solution has the advantages of speed, efficiency, and economy compared to the commercial kit. In addition, the higher pH value (10-11) of the Chelex 100 resin solution markedly improved the detection sensitivity compared to a lower pH value (9-10). In conclusion, the comparison of the Chelex 100 Resin and commercial viral DNA extraction kits revealed the good performance of the Chelex 100 resin solution at pH 10-11 in DNA extraction for PCR amplification from aquatic animal viral samples of tissues and cells in molecular diagnostic testing. It is both rapid and cost-effective.

Preservation and Extraction of Malaria Parasite DNA from Dried Blood Spots.

Molecular studies related to diagnosis and research rely on collection of blood samples and extraction of high-quality DNA. In Africa, where the populations carried 94% of the total burden of cases and deaths due to malaria in 2019, collection of samples is often challenged by remote study areas and lack of a cold chain to transport and store samples. Collection of blood on filter paper is a technique that is less invasive and has simpler requirements regarding training of staff, storage, and transport of samples than collection of venous blood samples. Dried blood spots (DBS) are therefore commonly used in many research projects. However, DNA quality can be affected by duration and conditions of storage. The quality of the DNA for molecular analyses also depends on a DNA extraction methodology that provides high-quality DNA with high purity and yield. Several protocols for DNA extraction have been described, and many comparative studies have analyzed and optimized the different methodologies to find an alternative to the more costly commercial extraction kits. This chapter describes recommendations for storage and preservation of DBS, and a Chelex-based protocol for extraction of DNA from DBS.

Development of a Simple DNA Extraction Method and Candida Pan Loop-Mediated Isothermal Amplification Assay for Diagnosis of Candidemia.

To reduce the morbidity and mortality of candidemia patients through rapid treatment, the development of a simple, rapid molecular diagnostic method that is based on nucleic acid extraction and is superior to conventional methods for detecting Candida in the blood is necessary. We developed a multiplex Candida Pan/internal control (IC) loop-mediated isothermal amplification (LAMP) assay and a simple DNA extraction boiling protocol using Chelex-100 that could extract yeast DNA in blood within 20 min. The Chelex-100/boiling method for DNA extraction showed comparable efficiency to that of the commercial QIAamp UCP Pathogen Mini Kit using Candida albicans qPCR. In addition, the Candida Pan/IC LAMP assay showed superior sensitivity to that of general Candida Pan and species qPCRs against clinical DNA samples extracted with the QIAamp UCP Pathogen Mini Kit and Chelex-100/boiling method. The Candida Pan/IC LAMP assay followed by Chelex-100/boiling-mediated DNA extraction showed high sensitivity (100%) and specificity (100%) against clinical samples infected with Candida. These results suggest that the Candida Pan/IC LAMP assay could be used as a rapid molecular diagnostic test for candidemia.

Use of Chelex-100 for the molecular diagnosis of five animal pathogens / Uso de Chelex-100 para el diagnóstico molecular de cinco patógenos de animales

Abstract Molecular tools have improved conventional veterinary diagnosis. Acid nucleic extraction is a key step for downstream applications. This work aimed to compare the DNA extraction method Chelex-100 resin (M1) with Whatman® cards (M2), phenol-chloroform (M3), or commercial kits (M4), and to determine the most sensitive and inexpensive one for its diagnosis of animal pathogens that, despite their economic or zoonotic relevance, receive little attention. DNA was isolated from urine, organs, semen, blood and intestinal mucous, from the bacteria Leptospira interrogans serovar Pomona Pomona (by M1 and M2), Brucella melitensis (by M1, M3 and M4), and Salmonella ser. Abortusequi (by M1 and M4), and the parasites Leishmania spp. (by M1, M3 and M4), and Eimeria spp. (by M1 and M3), respectively. The sensitivity of each method was assayed by Polymerase Chain Reaction (PCR). The M1 showed similar sensitivity for Salmonella ser. Abortusequi, Leishmania spp., and Eimeria spp., being better for L. interrogans serovar Pomona Pomona and slightly lower for B. melitensis. For the first time, a simple and economic method was successfully employed for extracting DNA from these animal pathogens, especially important in low-resource settings, contributing to the diagnosis of leptospirosis, brucellosis, leishmaniasis, and coccidiosis; as well as to the molecular epidemiology of salmonellosis in stallion from semen samples.

Resumen Las técnicas moleculares han contribuido a mejorar el diagnóstico veterinario tradicional y la extracción de ácidos nucleicos es determinante. El objetivo de este trabajo fue comparar el método de extracción de ADN Chelex-100 (M1) con papel Whatman (M2), fenol-cloroformo (M3) o kits comerciales (M4), y determinar un método sensible y de bajo costo para el diagnóstico de patógenos de animales económica o zoonóticamente relevantes y que reciben poca atención. A partir de orina, órganos, semen, sangre y mucosa intestinal se extrajo el ADN de las bacterias Leptospira interrogans serovar Pomona Pomona (con M1 y M2), Brucella melitensis (con M1, M3 y M4), Salmonella ser. Abortusequi (M1 y M4), y de los parásitos Leishmania spp. (M1, M3 y M4) y Eimeria spp. (M1 y M3), respectivamente. La sensibilidad de los protocolos fue analizada por PCR. El método M1 demostró una sensibilidad similar para S. Abortusequi, Leishmania spp. y Eimeria spp., siendo mejor para L. interrogans y levemente menor para B. melitensis. Por primera vez se usó exitosamente en estos patógenos veterinarios un método simple y económico para extraer ADN, especialmente importante en laboratorios de bajos recursos económicos, contribuyendo al diagnóstico de leptospirosis, brucelosis, leishmaniasis y coccidiosis, así como también a la epidemiología molecular de salmonelosis en muestras de semen de caballos.

Two simple and inexpensive methods for preparing DNA suitable for digital PCR from a small number of cells in 96-well plates.

BACKGROUND: Although DNA of high quality can be easily prepared from cultured cells with commercially available kits, many studies involve a large number of samples which increases the cost drastically. We optimized two simple and inexpensive methods for preparing DNA suitable for digital PCR from a small number of cells directly from wells of 96-well plates. METHODS: Cells (number: 103 -104 ) were lysed with a Direct PCR® lysis buffer or a 10% Chelex100® solution. The lysates were further purified and concentrated by means of DNA precipitation with a blue-colored glycogen as a carrier. PCR and digital PCR were used to evaluate the efficiency of the two methods. RESULTS: For 1000 cells from one primary culture and two tumor cell lines, DNA was reproducible and obtained with recovery rate (obtained/expected amount of DNA) in the range of 50%-90% as measured by the fluorometer dyes instrument Qubit. Using 8 out of a total of 10 µL DNA solution for 1000 cells, both conventional PCR and digital PCR were successful. For digital PCR, more than 1600 positive droplets were obtained for DNA from 1000 cells using the Direct PCR® method, corresponding to a yield efficiency of approximately 80%. Further reducing the number of cells down to 100 would be possible with 160 positive droplets expected. Both reagents are inexpensive (0.08€/sample). CONCLUSIONS: Two methods are efficient, especially the Direct PCR® reagent-based method provides a simple and inexpensive method for preparing DNA suitable for digital PCR from small number of cells.

Use of a Polymer Inclusion Membrane and a Chelating Resin for the Flow-Based Sequential Determination of Copper(II) and Zinc(II) in Natural Waters and Soil Leachates.

A bi-parametric sequential injection method for the determination of copper(II) and zinc(II) when present together in aqueous samples was developed. This was achieved by using a non-specific colorimetric reagent (4-(2-pyridylazo)resorcinol, PAR) together with two ion-exchange polymeric materials to discriminate between the two metal ions. A polymer inclusion membrane (PIM) and a chelating resin (Chelex 100) were the chosen materials to retain zinc(II) and copper(II), respectively. The influence of the flow system parameters, such as composition of the reagent solutions, flow rates and standard/sample volume, on the method sensitivity were studied. The interference of several common metal ions was assessed, and no significant interferences were observed (<10% signal deviation). The limits of detection were 3.1 and 5.6 µg L-1 for copper(II) and zinc(II), respectively; the dynamic working range was from 10 to 40 µg L-1 for both analytes. The newly developed sequential injection analysis (SIA) system was applied to natural waters and soil leachates, and the results were in agreement with those obtained with the reference procedure.

Use of embedded Chelex chelating resin and sediment toxicity bioassays with medaka embryos to determine the bioavailability and toxicity of lead-contaminated sediment.

The aquatic sediment acts as a reservoir for multiple sources of pollutants including toxic metals. Most analytical methods used for estimating the bioavailability of sediment heavy metals have not been biologically validated by correlation with an aquatic organism's response. A reliable whole-sediment contacting toxicity assay using vertebrate species is lacking and the exposure routes for sediment metals are unclear. This study established a novel bio-analytical approach involving the Chelex-100 resin detection system and sediment toxicity assessment with embryo-larval stages of medaka fish (Oryzias latipes) to evaluate the bioavailability and toxicity of lead (Pb) contamination in sediment to fish. Treated fish exposed to the Pb-spiked artificial sediment with whole-sediment exposure showed more dose-dependent toxic responses than those from pore- or overlying-water exposure extracted from the same sediment. The Chelex-100 resin-extractable Pb content was highly correlated with mortality, total malformation and Pb bioaccumulation in medaka embryos or hatchlings from Pb-spiked sediment at environmentally relevant concentrations. The environmental sediment with higher contents of clay or organic carbon showed lower potency of releasing Pb from sediment to overlying water, as compared to those observed with artificial sediment. Our results suggest that the bio-analytical method can be practically applied in situ to evaluate the adverse effect of heavy metal-contaminated sediment on the aquatic ecosystem.

Isolation and next generation sequencing of archival formalin-fixed DNA.

DNA from archived organs is presumed unsuitable for genomic studies because of excessive formalin-fixation. As next generation sequencing (NGS) requires short DNA fragments, and Uracil-N-glycosylase (UNG) can be used to overcome deamination, there has been renewed interest in the possibility of genomic studies using these collections. We describe a novel method of DNA extraction capable of providing PCR amplicons of at least 400 bp length from such excessively formalin-fixed human tissues. When compared with a leading commercial formalin-fixed DNA extraction kit, our method produced greater yields of DNA and reduced sequence variations. Analysis of PCR products using bacterial sub-cloning and Sanger sequencing from UNG-treated DNA unexpectedly revealed increased sequence variations, compared with untreated samples. Finally, whole exome NGS was performed on a myocardial sample fixed in formalin for 2 years and compared with lymphocyte-derived DNA (as a gold standard) from the same patient. Despite the reduction in the number and quality of reads in the formalin-fixed DNA, we were able to show that bioinformatic processing by joint calling and variant quality score recalibration (VQSR) increased the sensitivity four-fold to 56% and doubled specificity to 68% when compared with a standard hard-filtering approach. Thus, high-quality DNA can be extracted from excessively formalin-fixed tissues and bioinformatic processing can optimise sensitivity and specificity of results. Sequencing of several sub-cloned amplicons is an important methodological step in assessing DNA quality.

Comparison between different methods of DNA isolation from dried blood spots for determination of malaria to determine specificity and cost effectiveness.

DNA extraction from filter paper by using different methods was compiled through a thorough review of many research articles published in various journals. When performing malaria epidemiological surveys in remote area, it is difficult to collect blood samples and transport it. In field particularly in remote area where facilities for storing and processing of samples does not exist, there surveillance and diagnosis of malaria is very difficult. In this review we are focused upon four simple methods of DNA isolation from the field collected blood and mosquito abdomen blood meal spotted on Whatman No. 1 or No. 3 filter paper. The main DNA isolation methods are Chelex-100, Tris-EDTA (TE) buffer; Methanol based DNA extraction and Phosphate buffer saline (PBS) using Lysis buffer and Phenol-Chloroform method. Efforts have been taken to identify the methods which are cost-effective and take less time to extract DNA from dried blood spots (DBS) and whole mosquitoes. The purpose of this paper is to update the knowledge and find a method to extract DNA from DBS which will be specific, rapid, cost-effective, less time consuming and feasible for epidemiological survey in remote area.

Improved Method for DNA Extraction and Purification from Tetrahymena pyriformis.

Tetrahymena pyriformis (protozoa) is intensely investigated as a model organism, offering numerous advantages in comprehensive and multidisciplinary studies using morphologic or molecular methods. Since DNA extraction is a vital step of any molecular experiment, here a new mixed surfactant (Sodium dodecyl sulfate (SDS) 20%/Triton X-100) was adopted for effective DNA extraction from Tetrahymena pyriformis under an easy, fast protocol. The efficiency of this technique was then compared with three widely-used alternative techniques, namely the Chelex 100 matrix, Ammonium pyrrolidine dithiocarbamate (APD) complex and SDS-chloroform methods. DNA extraction was analyzed by pulsed-field gel electrophoresis, spectral measurement, fluorometry (Qubit), restriction enzyme digestion, and polymerase chain reaction. Data analysis revealed that the quantity and quality of the recovered DNA varied depending on the applied DNA extraction method. The new method (SDS 20%/Triton X-100) was the most efficient for extracting DNA from Tetrahymena pyriformis with high integrity and purity, affordable cost, less time, and suitability for molecular applications.

Preparation of Zirconium-89 Solutions for Radiopharmaceutical Purposes: Interrelation Between Formulation, Radiochemical Purity, Stability and Biodistribution.

Zirconium-89 is a promising radionuclide for nuclear medicine. The aim of the present work was to find a suitable method for obtaining zirconium-89 solutions for radiopharmaceutical purposes. For this purpose, the ion exchange behavior of zirconium-89 solutions was studied. Radio-TLC (thin layer chromatography) and biodistribution studies were carried out to understand speciation of zirconium-89 complexes and their role in the development of new radiopharmaceuticals. Three methods of zirconium-89 isolation were studied using ZR (hydroxamate) and Chelex-100 resins. It was found that ZR-resin alone is not enough to obtain stable zirconium-89 formulations. An easy and effective method of reconstitution of [89Zr]Zr-oxalate to [89Zr]Zr-citrate using Chelex-100 resin was developed. Developed procedures allow obtaining [89Zr]Zr-oxalate (in 0.1 M sodium oxalate solution) and [89Zr]Zr-citrate (in 0.1-1.0 M sodium citrate solution). These solutions are perfectly suitable and convenient for radiopharmaceutical purposes. Our results prove [89Zr]Zr-citrate to be advantageous over [89Zr]Zr-oxalate. During evaluation of speciation of zirconium-89 complexes, a new TLC method was developed, since it was proved that there is no comprehensive method for analysis or zirconium-89 preparations. The new method provides valuable insights about the content of "active" ionic form of zirconium-89. The interrelation of the chromatographic behavior of zirconium-89 preparations and their biodistribution was studied.

Comparison of four DNA extraction methods to extract DNA from cigarette butts collected in Lebanon.

Cigarette butts collected from crime scenes represent valuable sources of DNA. However the extraction of the genetic material may deem challenging especially when different contaminants may compromise the integrity, quality, and quantity of DNA obtained. This study aims at comparing four extraction methods (Chelex-100, soaking + Chelex-100, Chelex-100 + PK, and DNA IQ™ System) with the intention of identifying the one with maximal recovery rate and profiling success. DNA was extracted using aforementioned four methods from 70 cigarette butts collected from sites across Lebanon. DNA was quantified by qPCR using TaqMan Quantifiler Kit on an Applied Biosystems 7300 SDS instrument and genotypes were obtained using the PowerPlex® 21 kit on an Applied Biosystems 3130 Genetic Analyser. The findings of this work showed that DNA extraction with Chelex-100 + PK is preferred to the other three methods when seeking both, a high yield and the generation of maximal numbers of full profiles. The Chelex-100 + PK method is simple, cost effective, and therefore suitable for routine cigarette butts case studies.

Comparative analysis of three methods from dried blood spots for expeditious DNA extraction from mosquitoes; suitable for PCR based techniques.

The objective of this work was to compare the quality, purity and quantity of DNA isolated from dried blood spots (DBS) by three methods (Chelex-100, QIAamp DNA mini kit, and TE (Tris EDTA)-Buffer). Sample collection was performed in six districts in Odisha, India and screened for cases of clinical malaria and dengue and vector density. Mosquito abdomens were spotted on Whatman 3MM (MERCK) Filter paper and dried for 10 min at room temperature. DNA was isolated from DBS using three methods (Chelex-100, QIAamp DNA mini kit, and TE-Buffer), and PCR was used to determine the feeding behaviours of vector mosquitoes. DNA was quantified using a UV-spectrophotometer, and q-PCR was used to determine the target gene copy number to compare the methods. The QIAamp DNA mini kit method was used as the reference method. The yield and purity of DNA extracted with Chelex-100 and TE were 14-72 ng/µl and 1.51-1.85 and 9-50 ng/µl and 1.68-2.1, respectively. DNA extracted using the Chelex-100 method was stored for over 1 month at - 20 °C and was suitable for later use. The Chelex-100 method had a sensitivity of 99.5% and specificity of 78%. A Bland-Altman plot suggested that the Chelex-100 method was similar to the QIAamp DNA mini kit method for determining the feeding behaviours of vector mosquitoes. The Chelex-100 method is simple, cost-effective, and safe and requires minimal time for DNA extraction from dried blood spots. In malaria and dengue research, detecting the feeding behaviours from mosquito DNA from dried blood spots on filter paper by PCR is an easy, minimally invasive and inexpensive molecular technique that can be performed in remote areas.

A simple and efficient method to recover isotopically enriched Ni-64 from electrolytic solutions.

Production of 64Cu via the proton irradiation of 64Ni, electrodeposited on a suitable backing substrate, remains the most common method to produce this emerging radionuclide. Some unforeseen cases arise when the electrodeposition does not work and the electrolytic solution needs to be reprocessed, but the presence of salt buffers makes it difficult to recover the nickel to prepare a fresh solution. The aim of this work was to develop a simple and efficient method to recover 64Ni from bath solutions.

Simultaneous measurement of fifteen rare earth elements using diffusive gradients in thin films.

A new method for simultaneous measurement of fifteen rare earth elements (REEs) [La (Ⅲ), Ce (Ⅲ), Pr (Ⅲ), Nd (Ⅲ), Sm (Ⅲ), Eu (Ⅲ), Gd (Ⅲ), Tb (Ⅲ), Dy (Ⅲ), Ho (Ⅲ), Er (Ⅲ), Tm (Ⅲ), Yb (Ⅲ), Lu (Ⅲ), and Y (Ⅲ)] was established in this study using the diffusive gradients in thin films (DGT) technique with Chelex®100 binding gel. Five different types of ion exchange resins (Chelex®100, D418, D001-cc, 001 × 7, and HSTY®-SS) were selected for the initial investigation of their adsorption performance for REEs. The Chelex®100 binding gel had the greatest uptake efficiencies of >95% for the fifteen REE ions, which was used for all subsequent experiments. The binding gel exhibited rapid binding dynamics to REEs in mixed solution of the fifteen REE ions. Elution efficiencies ranging from 86.5% to 93.8% for these REEs were obtained based on extraction using 2.0 M HCl. The Chelex®100 DGT uptake of the fifteen REE ions increased linearly with the deployment time and found to be independent of pH (3-9) and ionic strength (3 mM-100 mM). The capacities of Chelex®100 DGT for measurement of the mixed elements were determined at a range of 5.39-6.75 µg cm-2. Application of the DGT for soil analysis showed that Chelex®100 DGT was a useful tool in simultaneous measurement of the fifteen REE ions, even in a soil with high concentrations of REEs. This study demonstrated the advantage of Chelex®100 DGT in simultaneous measurement of the fifteen REE ions due to high uptake efficiencies and a wide tolerance to environmental interference.

Method for improving the quality of genomic DNA obtained from minute quantities of tissue and blood samples using Chelex 100 resin.

BACKGROUND: Although genomic DNA isolation using the Chelex 100 resin is rapid and inexpensive, the DNA obtained by this method has a low concentration in solution and contains suspended impurities. The presence of debris in the DNA solution may result in degradation of DNA on long term storage and inhibition of the polymerase chain reaction. In order to remove impurities and concentrate the DNA in solution, we have introduced modifications in the existing DNA isolation protocol using Chelex-100. We used ammonium acetate to precipitate proteins and a sodium acetate- isopropanol mixture to pellet out DNA which was washed with ethanol. RESULTS: A pure DNA pellet that can be dissolved in water or Tris-EDTA buffer and stored for a long time at - 80 °C was obtained. We also observed a 20-fold change in the DNA concentration following precipitation and re-dissolution. CONCLUSION: Our method is different from other extraction methods since it uses non-toxic, easily available and inexpensive reagents as well as minimal amounts of blood or tissue samples for the DNA extraction process. Besides its use in sex determination and genotyping in lab animals as described in this paper, it may also have applications in forensic science and diagnostics such as the easy detection of pathogenic DNA in blood.

Comparison of Chelex based resins in diffusive gradients in thin-film for high resolution assessment of metals.

The passive sampling technique of diffusive gradients in thin-film (DGT) is widely used to determine 1D profiles (using Chelex-100 resin) and 2D images (using suspended particulate reagent-iminodiacetate resin, abbreviated as SPR-IDA resin) of metals in sediment pore waters and in oxic/anoxic soils. However, when deployed in anoxic sediments with high metal concentrations, Fe and Mn concentrations determined with the Chelex-100 resin gel were ~ 5 times higher than concentrations measured with the SPR-IDA resin gel. This discrepancy suggests that the SPR-IDA resin gel is saturated faster than the Chelex-100 resin gel. Here, we tested the adsorption capacity of the SPR-IDA resin gel and compared it to the Chelex-100 resin gel. Fe and Mn binding capacities on a SPR-IDA gel disc are less than 0.1 µmoles, which means that they are far below those on a Chelex-100 gel disc (around 3.2 µmoles), while competition with stronger binding metals such as Cu and Cd further lowers Fe and Mn capacities. This restricts the SPR-IDA resin gel to be used in contaminated marine sediments. We propose the use of a ground Chelex-100 resin, which is prepared by grinding Chelex-100 resin in a ball-mill prior to gel preparation. The capacities of Fe and Mn on a ground Chelex-100 resin gel disc are around 1.6 µmoles, more than 16 times higher than the capacity on SPR-IDA gel disc. In addition, the bead size of the ground Chelex-100 resin is small enough (~ 10 µm) to allow high resolution LA-ICP-MS imaging of Fe, Mn and trace metals in sediment pore waters as well as soils.

[Comparison of MPure-12 Automatic Nucleic Acid Purification and Chelex-100 Method].

OBJECTIVES: To explore the forensic application value of MPure-12 automatic nucleic acid purification （MPure-12 Method） for DNA extraction by extracting and typing DNA from bloodstains and various kinds of biological samples with different DNA contents. METHODS: Nine types of biological samples, such as bloodstains, semen stains, and saliva were collected. DNA were extracted using MPure-12 method and Chelex-100 method, followed by PCR amplification and electrophoresis for obtaining STR-profiles. RESULTS: The samples such as hair root, chutty, butt, muscular tissue, saliva stain, bloodstain and semen stain were typed successfully by MPure-12 method. Partial alleles were lacked in the samples of saliva, and the genotyping of contact swabs was unsatisfactory. Additional, all of the bloodstains （20 µL, 15 µL, 10 µL, 5 µL, 1 µL） showed good typing results using Chelex-100 method. But the loss of alleles occurred in 1 µL blood volume by MPure-12 method. CONCLUSIONS: MPure-12 method is suitable for DNA extraction of a certain concentration blood samples．Chelex-100 method may be better for the extraction of trace blood samples．This instrument used in nucleic acid extraction has the advantages of simplicity of operator, rapidity, high extraction efficiency, high rate of reportable STR-profiles and lower man-made pollution.

In Vitro Activity of the Siderophore Cephalosporin, Cefiderocol, against a Recent Collection of Clinically Relevant Gram-Negative Bacilli from North America and Europe, Including Carbapenem-Nonsusceptible Isolates (SIDERO-WT-2014 Study).

Cefiderocol (formerly S-649266) is an investigational siderophore cephalosporin. Iron-depleted cation-adjusted Mueller-Hinton broth (ID-CAMHB) was prepared according to the Clinical and Laboratory Standards Institute (CLSI) protocol and used to perform broth microdilution testing of cefiderocol against a 2014-2015 collection of clinical isolates of Gram-negative bacilli from North America (n = 4,239) and Europe (n = 4,966). The concentrations of cefiderocol inhibiting 90% of isolates tested (MIC90s) were 0.5 µg/ml (North America; n = 3,007) and 1 µg/ml (Europe; n = 3,080) for all isolates of Enterobacteriaceae; 1 µg/ml (North America; n = 30) and 4 µg/ml (Europe; n = 139) for meropenem-nonsusceptible (MIC ≥ 2 µg/ml) isolates of Enterobacteriaceae; 0.5 µg/ml for both North American (n = 765) and European (n = 765) isolates of Pseudomonas aeruginosa; 0.5 µg/ml (North America; n = 151) and 1 µg/ml (Europe; n = 202) for meropenem-nonsusceptible (MIC ≥ 4 µg/ml) isolates of P. aeruginosa; 1 µg/ml for both North American (n = 309) and European (n = 839) isolates of all Acinetobacter baumannii strains as well as for both North American (n = 173) and European (n = 595) isolates of meropenem-nonsusceptible A. baumannii; and 0.5µg/ml (North America; n = 152) and 0.25 µg/ml (Europe; n = 276) for isolates of Stenotrophomonas maltophilia MICs of cefiderocol were ≤4 µg/ml for 99.9% (6,078/6,087) of all Enterobacteriaceae, 97.0% (164/169) of meropenem-nonsusceptible Enterobacteriaceae, 99.9% (1,529/1,530) of all P. aeruginosa isolates, 100% (353/353) of meropenem-nonsusceptible P. aeruginosa isolates, 97.6% (1,120/1,148) of all A. baumannii isolates, 96.9% (744/768) of meropenem-nonsusceptible A. baumannii isolates, 100% of isolates of S. maltophilia (428/428) and 93.8% of isolates of Burkholderia cepecia (11/12). We conclude that cefiderocol demonstrated potent in vitro activity against a recent collection of clinical isolates of commonly encountered Gram-negative bacilli, including carbapenem-nonsusceptible isolates.