Detection of anthrax and other pathogens using a unique liquid array technology.

A bead-based liquid hybridization assay, Luminex(®) 100™, was used to identify four pathogenic bacteria, Bacillus anthracis, Clostridium botulinum, Francisella tularensis subsp. tularensis, and Yersinia pestis, and several close relatives. Hybridization between PCR-amplified target sequences and probe sequences (located within the 23S ribosomal RNA gene rrl and the genes related to the toxicity of each bacterium) was detected in single-probe or multiple-probe assays, depending on the organism. The lower limits of detection (LLDs) for the probes ranged from 0.1 to 10 ng. Sensitivity was improved using lambda exonuclease to digest the noncomplementary target strand. All contributors in 33 binary, ternary, and quaternary mixtures in which all components were present in a 1:1 ratio were identified with an 80% success rate. Twenty-eight binary mixtures in which the two components were combined in various ratios were further studied. All target sequences were detected, even when the minor component was overshadowed by a tenfold excess of the major component.

Pathogen detection using a liquid array technology.

Low concentrations of microbial pathogens in pure and mixed samples were detected using a bead-based, liquid array technology. A 20-bp sequence in the 23S rRNA gene, rrl, was amplified in four microorganisms: Bacillus cereus, Escherichia coli, Salmonella enterica and Staphylococcus aureus. PCR products were positively identified with the Luminex(®) 100™ system. The system could detect very low amounts of DNA and the instrument response was proportional to the input concentration. The lower limit of detection (LLD) was determined to be 0.5 ng for B. cereus and E. coli and 2 ng for S. enterica. The LLD for S. aureus was not determined as the instrument response was still above the threshold when quantities of DNA as low as 0.25 ng were used. The platform positively identified organisms present in mixed samples even when the minor component was overshadowed by a 10-fold excess of the major component.

Forensic utilization of familial searches in DNA databases.

DNA evidence is widely recognized as an invaluable tool in the process of investigation and identification, as well as one of the most sought after types of evidence for presentation to a jury. In the United States, the development of state and federal DNA databases has greatly impacted the forensic community by creating an efficient, searchable system that can be used to eliminate or include suspects in an investigation based on matching DNA profiles - the profile already in the database to the profile of the unknown sample in evidence. Recent changes in legislation have begun to allow for the possibility to expand the parameters of DNA database searches, taking into account the possibility of familial searches. This article discusses prospective positive outcomes of utilizing familial DNA searches and acknowledges potential negative outcomes, thereby presenting both sides of this very complicated, rapidly evolving situation.

Effect of 1,2-indanedione on PCR-STR typing of fingerprints deposited on thermal and carbonless paper.

1,2-Indanedione treated fingerprints deposited on two substrates (thermal and carbonless paper) were swabbed and tested to determine the effect of the chemical on PCR-STR DNA typing. Samples from 10 post-treatment intervals were analyzed. Two extraction methods, Chelex and Qiamp, were used to determine the effect of extraction procedure on the quality of the DNA profiles. The LCN DNA samples were concentrated using Microcon 100 and amplified using the Profiler Plus amplification kit. 1,2-Indanedione did not adversely affect the DNA profiles obtained from the treated fingerprints. Partial DNA profiles were obtained at all post-development time frames. Both extraction methods produced comparable profiles although more "drop-ins" were observed with the Qiamp method. More "drop-outs" occurred in the DNA profiles from samples deposited on the thermal substrate compared with carbonless substrate.

Identification of Acer rubrum using amplified fragment length polymorphism.

Amplified fragment length polymorphism (AFLP) analysis of botanical forensic evidence provides a means of obtaining a reproducible DNA profile in a relatively short period of time in species for which no sequence information is available. AFLP profiles were obtained for 40 Acer rubrum trees. Leaf material from five additional species was also typed. Genomic DNA was isolated using the DNeasy Plant Miniprep Kit (Qiagen, Valencia, CA), double-digested by two restriction endonucleases (EcoRI and MseI) and ligated to oligonucleotide adapters. Two consecutive PCR reactions (pre-amplification and selective amplification) were performed using a modification of the AFLP protocol described by Gibco (Invitrogen, Rockville, MD). The DNA fragments were separated by capillary electrophoresis using the CEQ 8000 DNA Fragment Analyzer. A number of Acer rubrum species-specific peaks were identified. In addition, within this closed set of samples, 15 of 16 (93.8%) blind samples were correctly identified. AFLP data can be used to determine the species of botanical evidence or to associate a sample to a source. This information can be used in forensic investigations to link a piece of evidence with a particular location or suspect.

A comparison study of hair examination methodologies.

A study was conducted to investigate the accuracy between two methods of hair analysis: PCR-STR DNA analysis and microscopic comparison analysis. Standard sets of pubic hairs were collected from volunteers, and unknown sets were generated from these samples. Three out of five (60%) of the hairs analyzed produced full DNA profiles that were correctly matched to the standard sets. DNA analysis was inconclusive (partial or no DNA profile) for two out of five (40%) of the samples. In contrast, the microscopic comparison analysis correctly matched four out of five (80%) of the samples to the standard sets but mis-identified one out of five (20%) of the samples. These results reinforce the practice of preliminary microscopic hair examination in narrowing down a set of hairs for DNA analysis. Microscopic comparison analysis is sufficiently reliable to remain a rapid and inexpensive method for forensic hair analysis.

Evaluation of an automated liquid hybridization method for DNA quantitation.

The AluQuant (Promega Corporation) liquid hybridization DNA quantitation method was evaluated on an automated robotic platform (Biomek 2000, Beckman Coulter, Fullerton, CA) for use in forensic PCR-STR systems. DNA from bloodstains and buccal swabs was extracted by three different methods: Chelex, Qiagen and DNA IQ (Promega). Samples were quantitated using both the Quantiblot and the AluQuant systems. Concordance between methods was determined by comparing the average AluQuant DNA concentrations for samples having matching (binned) Quantiblot values. Studies testing the "accuracy" (STR analysis), precision, sensitivity, and specifies specificity of the AluQuant method were also conducted. The effect of inhibitors (carpet, denim, and suede) was evaluated. The results indicate that the AluQuant quantitation system equals the Quantiblot system in "accuracy", sensitivity, precision, and primate-specificity. While extracts from denim and suede affected (inhibited) both systems minimally, the carpet extracts produced a sharp increase in DNA quantitation values in the AluQuant but not the Quantiblot system. The speed and user-friendliness of the AluQuant system on a robotic platform offer specific advantages to the forensic community.

Characterization of a novel dimorphism in the 5' flanking region of the short tandem repeat (STR) locus, c-fes/fps (FES).

The FES short tandem repeat (STR) locus contains seven to 14 repeats of the tetranucleotide sequence ATTT. A novel 10 base pair dimorphism in the 5' flanking region of the FES locus was characterized in four broad populations: African-American, Hispanic, Caucasian, and Asian. The absence of the 10 base pair sequence, or (-) allele, was closely linked to FES STR alleles with 10 or fewer repeats. The presence of the 10 base pair sequence, or (+) allele, was closely linked to FES STR alleles with 12 or more repeats. The (-) and (+) alleles occurred equally often in FES STR allele 11. The nucleotide sequence (5'-GGCTGTTTTG-3') of the (+) allele, located 179 base pairs upstream of the FES STR, was determined to be consistent within and among the four populations. Statistical and sequence analysis confirmed the linkage between the two polymorphic sites. The results indicate that the exclusion rate of the FES locus is increased, above that for the STR alone, when both polymorphic characteristics are considered.

Characterization of pMa025, a plasmid from the cyanobacterium Microcystis aeruginosa UV025.

The characterization of pMa025, a plasmid isolated from the unicellular, toxin-producing cyanobacterium Microcystis aeruginosa UV025, is described. A recombinant plasmid, pMaL [pMa025-pBluescript II SK(-)] was constructed for mapping, sequencing, and development of shuttle vectors capable of transforming both Escherichia coli and M. aeruginosa. pMa025 is 8,018 bp in length and has a G+C content of 62.3 mol%. Nineteen presumptive ORFs, ORF A - ORF S were identified using ATG or GTG as initiation codons. Fifteen different ORFs, ORF a - ORF o were identified using TGA as a degenerate codon for tryptophan. GTG was the start codon in two-thirds of the putative ORFs when TGA was the termination codon. GTG was the start codon in one-third of the putative ORFs when TGA was used as a codon for tryptophan. The deduced amino acid sequence from ORF j (3,114 bp) was significantly similar to that of a putative plasmid replication protein, RepA, from plasmid pUH24 of Synecho coccus sp. strain PCC7942. M. aeruginosa UV027 and E. coli were transformed to carbenicillin resistance with pMaL-D7, a 6.4-kb hybrid plasmid (3.46 kb pMa025, 2.95 kb pBluescript II) generated from the nested deletion strategy. pMaL-D7 will be used as a shuttle vector.