A validation study of the Qiagen Investigator DIPplex? kit; an INDEL-based assay for human identification

Marker sets that are based on small insertion/deletion (INDEL) alleles can serve as useful supplementary or stand-alone assays for human identification. A validation study has been performed on a human identification assay based on a panel of 30 INDELs and amelogenin using the Investigator DIPplex? kit (Qiagen). The assay was able to type DNA from a number of forensically relevant sample types and obtain full profiles with 62?pg of template DNA and partial profiles with as little as 16?pg of template DNA. The assay is reproducible, precise, and non-overlapping alleles from minor contributors were detectable in mixture analysis ranging from 6:1 to 19:1 mixtures. Population studies were performed on the 30 indels, and there were no significant departures from Hardy-Weinberg equilibrium or significant linkage disequilibrium between the markers (after correction for sampling). In all populations, the random match probability was 1.43?×?10(-11) or less, and the power of exclusion was greater than .999999999. We also discovered several microvariant alleles in our population samples. The data support that the Investigator DIPplex? kit provides a powerful supplement or stand-alone capability for human identity testing.     

Population genetics of 30 insertion–deletion polymorphisms in two Chinese populations using Qiagen Investigator® DIPplex kit

Insertion–deletion polymorphisms (INDELs) are short length diallelic polymorphisms caused by the insertion or deletion of several bases. INDEL markers can serve as useful supplementary or stand-alone assays for human identification. The Qiagen Investigator^® DIPplex kit multiplexes 30 autosomal INDELs plus amelogenin for forensic use. The objective of this study was to estimate genetic diversity of 30 INDEL markers in the Han (the largest ethnic group of China, n = 565) and She population (almost the smallest ethnic group of China, n = 119), and to evaluate their usefulness in forensic genetics. In the Han and She, the mean observed heterozygosity values were 0.4133 and 0.3896, and the combined matching probability values were 1.80 × 10⁻¹¹ and 3.17 × 10⁻¹¹, respectively. Furthermore, the allele frequencies for each locus were compared with those in other reported Chinese subpopulations, and the forensic efficacy was compared between this kit and in-house developed INDEL assay. This study demonstrates that the Investigator^® DIPplex kit can be used as a supplementary tool for human identity testing in China.     

Compatibility of DNA IQ™, QIAamp® DNA Investigator,and QIAsymphony® DNA Investigator® with various fingerprint treatments

Latent fingerprint and touch DNA are the two most important contact evidence for individualization in forensic science which provide complementary information that can lead to direct and unequivocal identification of the culprit. In order to retrieve useful information from both fingerprints and DNA, which are usually mingled together, one strategy is to perform fingerprint examination prior to DNA analysis since common DNA sampling technique such as swabbing could disturb or even destroy fingerprint details. Here, we describe the compatibility of three automatic DNA extraction systems, namely, DNA IQ™, QIAamp^® DNA Investigator, and QIAsymphony^® DNA Investigator^®, with respective to the effects of various fingerprint detection techniques. Our results demonstrate that Super Glue fingerprint treatment followed by DNA IQ™ extraction shows better effectiveness in DNA profiling. Aluminum powder dusting offers the least interference to the three DNA extraction systems above. Magnetic powder dusting, on the other hand, strongly impedes DNA recovery. Physical Developer is the most intrusive, which yields profiles with poor quality, including lower peak heights, poor peak height ratios, and poor intra-color balance. In terms of the choice of extraction method, DNA IQ™ system is recommended for sampling after fingerprint treatments, but not the two DNA Investigator systems.

     

Synovial fluid as an alternative specimen for quantification of drugs of abuse by GC–MS

Forensic Toxicology -     

The DNA‐Buster: The evaluation of an alternative DNA recovery approach

Touch DNA recovery techniques can have limitations, as their effectiveness depends on the substrate on which the DNA of a person of interest can be found. In this study, an in-house dry-vacuuming device, the DNA-Buster, was compared to traditional methods for its DNA recovery performance from items typically examined in forensic casework. The aim was to evaluate whether this dry-vacuuming approach can recover DNA efficiently, potentially complementing the well-established recovery strategies. For this, the performances of swabbing, taping, wet- (M-Vac®) and dry-vacuuming (DNA-Buster) were investigated quantitatively and qualitatively for touch DNA deposited on carpet, cotton sweater, stone, tile and wood. For the sweater, both vacuuming methods outperformed the other collection tools quantitatively. While the highest DNA amounts for the carpet were yielded by swabbing and taping, dry-vacuuming was equally good in reaching full DNA profiles, whereas less complete profiles were observed for the M-Vac®. For stone and tile, swabbing was optimal, whereas dry-vacuuming clearly underperformed for these substrates. Taping was the best recovery method for wood. Despite applying single donor DNA after thoroughly cleaning the items, undesired DNA mixtures were detected for all recovery techniques and all substrates. The overall research findings show first that the novel dry-vacuuming method is suited for DNA recovery from textiles. Secondly, they indicate that more attention should be paid to the substrate-collection dependency to ensure best practices in recovering genetic material in a precise, confident and targeted manner from the variety of forensic casework material.     

PCR inhibitor removal using the NucleoSpin® DNA Clean-Up XS kit

Forensic evidence samples are collected from an unlimited variety of substrates, which may contain compounds known to inhibit the polymerase chain reaction (PCR). These PCR inhibitors are co-extracted with the DNA sample and can negatively affect the DNA typing results, which can range from partial to complete inhibition of the short tandem repeat (STR) PCR. One potential solution is to remove the PCR inhibitors from the extracts prior to the STR PCR with the NucleoSpin^® DNA Clean-Up XS kit. The kit contains a NucleoSpin^® XS silica column that has a special funnel design of thrust rings along with a very small silica membrane, which allows for sample elution in a small volume that is appropriate for use with current STR typing kits. The NucleoSpin^® DNA Clean-Up XS kit was optimized for the best possible DNA recovery and then evaluated for its ability to remove eight commonly encountered PCR inhibitors including: bile salt, collagen, hematin, humic acid, indigo, melanin, tannic acid and urea. Each of these PCR inhibitors was effectively removed by the NucleoSpin^® DNA Clean-Up XS kit as demonstrated by generating more complete STR profiles from the cleaned up inhibitor samples than from the raw inhibitor samples.     

Validation and assessment of the Investigator® 24plex QS kit for forensic casework application: Comparison with the PowerPlex® fusion system and GlobalFiler™ PCR amplification kits

Casework evidence samples are likely to be placed under diverse and harsh environments as compared to quantified DNA samples including serial-diluted standard DNA samples. Internal validation of a novel STR kit using casework evidence sample, which is conducted according to various conditions such as DNA contamination and degradation, is crucial before being used as a forensic application. Therefore, this study aimed to elucidate the reliability of the Investigator® 24plex QS kit through DNA derived from casework evidence and to assess whether it is applicable to STR analysis together with PowerPlex® Fusion System and GlobalFiler™ PCR Amplification Kit. DNA was extracted from 189 casework evidence samples in a total of 77 cases. The mismatch of the allelic size of this kit through allelic sizing precision test, was suitable according to ENFSI guidelines. All heterozygous balance of the three kits were above 0.6 recommended value of ENFSI guideline. The number of allele drop-in was most frequent in the GlobalFiler™ PCR Amplification Kit. In addition, the number of allele drop-out was most frequent in the Investigator® 24plex QS kit. The cutoff concentration of DNA detected in three kits of one complete STR was approximately 45 pg/μL on average. Despite of several limitations, the Investigator® 24plex QS kit is considered to have the capability to be used for STR analysis of casework evidence samples.     

A comparison of methods for forensic DNA extraction: Chelex-100® and the QIAGEN DNA Investigator Kit (manual and automated)

Efficient isolation of DNA from a sample is the basis for successful forensic DNA profiling. There are many DNA extraction methods available and they vary in their ability to efficiently extract the DNA; as well as in processing time, operator intervention, contamination risk and ease of use. In recent years, automated robots have been made available which speed up processing time and decrease the amount of operator input. This project was set up to investigate the efficiency of three DNA extraction methods, two manual (Chelex^®-100 and the QIAGEN DNA Investigator Kit) and one automated (QIAcube), using both buccal cells and blood stains as the DNA source. Extracted DNA was quantified using real-time PCR in order to assess the amount of DNA present in each sample. Selected samples were then amplified using AmpFlSTR SGM Plus amplification kit. The results suggested that there was no statistical difference between results gained for the different methods investigated, but the automated QIAcube robot made sample processing much simpler and quicker without introducing DNA contamination.     

Evaluation of InnoTyper® 21 in a sample of Rio de Janeiro population as an alternative forensic panel

International Journal of Legal Medicine - The use of bi-allelic markers such as retrotransposable element insertion polymorphisms or Innuls (for insertion/null) can overcome some limitations of...     

Effects of humic acid on DNA quantification with Quantifiler? Human DNA Quantification kit and short tandem repeat amplification efficiency

Correct DNA quantification is an essential part to obtain reliable STR typing results. Forensic DNA analysts often use commercial kits for DNA quantification; among them, real-time-based DNA quantification kits are most frequently used. Incorrect DNA quantification due to the presence of PCR inhibitors may affect experiment results. In this study, we examined the alteration degree of DNA quantification results estimated in DNA samples containing a PCR inhibitor by using a Quantifiler? Human DNA Quantification kit. For experiments, we prepared approximately 0.25?ng/μl DNA samples containing various concentrations of humic acid (HA). The quantification results were 0.194-0.303?ng/μl at 0-1.6?ng/μl HA (final concentration in the Quantifiler reaction) and 0.003-0.168?ng/μl at 2.4-4.0?ng/μl HA. Most DNA quantity was undetermined when HA concentration was higher than 4.8?ng/μl HA. The C (T) values of an internal PCR control (IPC) were 28.0-31.0, 36.5-37.1, and undetermined at 0-1.6, 2.4, and 3.2?ng/μl HA. These results indicate that underestimated DNA quantification results may be obtained in the DNA sample with high C (T) values of IPC. Thus, researchers should carefully interpret the DNA quantification results. We additionally examined the effects of HA on the STR amplification by using an Identifiler? kit and a MiniFiler? kit. Based on the results of this study, it is thought that a better understanding of various effects of HA would help researchers recognize and manipulate samples containing HA.     

Are antimicrobial peptides an alternative for conventional antibiotics?

Antimicrobial peptides are widespread in living organisms and constitute an important component of innate immunity to microbial infections. By the early 1980s, more than 800 different antimicrobial peptides had been isolated from mammals, amphibians, fish, insects, plants and bacterial species. In humans, they are produced by granulocytes, macrophages and most epithelial and endothelial cells. Newly discovered antibiotics have antibacterial, antifungal, antiviral and even antiprotozoal activity. Occasionally, a single antibiotic may have a very wide spectrum of activity and may show activity towards various kinds of microorganisms. Although antimicrobial activity is the most typical function of peptides, they are also characterized by numerous other properties. They stimulate the immune system, have anti-neoplastic properties and participate in cell signalling and proliferation regulation. As antimicrobial peptides from higher eukaryotes differ structurally from conventional antibiotics produced by bacteria and fungi, they offer novel templates for pharmaceutical compounds, which could be used effectively against the increasing number of resistant microbes.     

Population data and genetic characteristics of 12 X-STR loci using the Investigator® Argus X-12 Quality Sensor kit for the Kedayan population of Borneo in Malaysia

International Journal of Legal Medicine - DNA profiling of X-chromosomal short tandem repeats (X-STR) has exceptional value in criminal investigations, especially for complex kinship and incest...     

The β-ray gauge as an evaporimeter

Diurnal variations of the net count rate through a turgid tobacco leaf measured by β-ray gauge system corresponded with the stomatal movement. Three distinct stages of evaporation rates were observed through a porous medium. Use of the instrument to study stomatal dynamics and transpiration non-destructively is suggested.     

The low-energy β(-) and electron emitter (161)Tb as an alternative to (177)Lu for targeted radionuclide therapy

Introduction

The low-energy β⁻ emitter ¹⁶¹Tb is very similar to ¹⁷⁷Lu with respect to half-life, beta energy and chemical properties. However, ¹⁶¹Tb also emits a significant amount of conversion and Auger electrons. Greater therapeutic effect can therefore be expected in comparison to ¹⁷⁷Lu. It also emits low-energy photons that are useful for gamma camera imaging.

Methods

The ¹⁶⁰Gd(n,γ)¹⁶¹Gd→¹⁶¹Tb production route was used to produce ¹⁶¹Tb by neutron irradiation of massive ¹⁶⁰Gd targets (up to 40 mg) in nuclear reactors. A semiautomated procedure based on cation exchange chromatography was developed and applied to isolate no carrier added (n.c.a.) ¹⁶¹Tb from the bulk of the ¹⁶⁰Gd target and from its stable decay product ¹⁶¹Dy. ¹⁶¹Tb was used for radiolabeling DOTA-Tyr3-octreotate; the radiolabeling profile was compared to the commercially available n.c.a. ¹⁷⁷Lu. A ¹⁶¹Tb Derenzo phantom was imaged using a small-animal single-photon emission computed tomography camera.

Results

Up to 15 GBq of ¹⁶¹Tb was produced by long-term irradiation of Gd targets. Using a cation exchange resin, we obtained 80%–90% of the available ¹⁶¹Tb with high specific activity, radionuclide and chemical purity and in quantities sufficient for therapeutic applications. The ¹⁶¹Tb obtained was of the quality required to prepare ¹⁶¹Tb–DOTA-Tyr3-octreotate.

Conclusions

We were able to produce ¹⁶¹Tb in n.c.a. form by irradiating highly enriched ¹⁶⁰Gd targets; it can be obtained in the quantity and quality required for the preparation of ¹⁶¹Tb-labeled therapeutic agents.     

Development of an LC–MS/MS method for quantification of 3-chloro-L-tyrosine as a candidate marker of chlorine poisoning

A simple and sensitive liquid chromatography coupled with electrospray ionization-tandem mass spectrometry (LC/ESI–MS/MS) method for the determination of 3-chloro-L-tyrosine (Cl-Tyr) was developed and validated. For sample preparation, 50 μL of the body fluids or tissue extracts were processed by protein precipitation followed by the derivatization with dansyl chloride. The calibration curve was linear over the concentration range of 2.0–200 ng/mL blood or 4.0–400 ng/g tissue. Our method allowed the reproducible and accurate quantification. That is, the intra- and inter-assay coefficients of variation were below 7.73 and 6.94%, respectively in both the blood and lung. We applied the developed method to the analysis of Cl-Tyr in the human autopsy samples, which were suspected of chlorine poisoning, and detected 55.2 ng/mL and 206.6 ng/g Cl-Tyr in left heart blood and lung, respectively. Furthermore, in more than 20 autopsy samples, which were obtained from other causes of death including burn, drowning, hanging, internal disease, trauma and drug poisoning, Cl-Tyr was almost not detected in their both body fluids and organ tissues. In conclusion, the data here reported demonstrate that the LC/ESI–MS/MS method allows the Cl-Tyr in the autopsy samples and that chlorine exposure strongly affects its level, providing a basis for novel identification tool of chlorine poisoning.     

Evaluation of the RapidHIT™ 200 and RapidHIT GlobalFiler® Express kit for fully automated STR genotyping

The RapidHIT™ 200 Human Identification System and RapidHIT GlobalFiler^® Express kit were evaluated and validated for use with single-source reference samples. It was of primary interest to evaluate the system for its efficacy as an expert system and to estimate a first pass success rate, as well as to identify the technical variables impacting that result. While results indicated that this instrument/kit combination can be used to accurately type single-source buccal samples, substantial variability in sensitivity and intra-color balance were observed, as were multiple artifacts, requiring extensive manual editing of the profiles. Artifacts included dye “blobs” and spectral overlap (pull-up) peaks that often originated from relatively low intensity allele peaks. Reduced intra-color balance, in combination with low sensitivity, occasionally resulted in instances of allelic dropout. Overall, 50% of the buccal samples analyzed in this study would have been successfully typed to give full GlobalFiler^® profiles without the need for manual review and editing.