African and Asian elephant ivory discrimination using a portable strip test.

Currently, the global elephant population has significantly declined due to the poaching of elephants for their ivory, and this is the reason why elephants are listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). However, Thailand allows the legal trade of ivory from registered, domesticated Asian elephants, leading to the smuggling of African elephant ivory, and passing them off as Asian elephant ivory. Therefore, this research aims to develop and validate a portable strip test to discriminate between Asian and African elephants DNA, using Recombinase Polymerase Amplification (RPA) and Lateral Flow Dipstick assay (LFD) according to international standards.

Direct STR profiling from laundered bloodstains: an investigation of different factors of laundering.

Bloodstains on fabrics may be washed or cleaned to eliminate incriminating evidence. These actions reduce the chances of obtaining an interpretable DNA profile. Previous studies have shown that conventional short-tandem repeat (STR) typing is affected by various factors associated with washing or laundering of stains.

Direct STR typing from human bones.

Identification by STR analysis of bones is time-consuming, mainly due to the lengthy decalcification required and complex DNA extraction process. To streamline this process, we developed a direct STR typing protocol from bone samples. We optimized bone sample amounts using femur and tibia and two commercial PCR kits (Identifiler™ Plus and IDplex Plus kits).

Extraction and electrochemical detection for quantification of trace-level DNA.

A novel strategy was developed to extract, detect, and quantify trace-level DNA. For the extraction step, a composite of methylene blue (MB), poly(acrylic acid) (PAA), and modified iron oxide magnetic nanoparticles (IOMNPs) (PAA/IOMNPs) was used to adsorb DNA from the sample. MB-PAA/IOMNPs with adsorbed DNA were then separated from the solution with an external magnet and MB-DNA was eluted from PAA/IOMNPs with acetic acid.

Discrimination of highly degraded, aged Asian and African elephant ivory using denaturing gradient gel electrophoresis (DGGE).

Background: Elephant populations have greatly reduced mainly due to illegal poaching for their ivory. The trade in elephant products is protected by national laws and CITES agreements to prevent them from further decline. For instance, in Thailand, it is illegal to trade ivory from African elephants; however, the law allows possession of ivory from Asian elephants if permission has been obtained from the authorities.

A novel, 4-h DNA extraction method for STR typing of casework bone samples.

Bones are often found in mass grave crime scene. To increase DNA identification success rates, a highly efficient DNA extraction method should be selected. Several DNA extraction methods for human bones have been published yet never been systematically compared, and some are time-consuming or complex.

Direct STR typing from fired and unfired bullet casings.

Cartridges, bullets, and casings (CBCs) are commonly recovered after shooting incidents and could provide valuable DNA information from touch DNA that has been left behind during handling of bullets and loading of guns. Direct PCR, in which the DNA extraction and quantification steps are bypassed, has been shown to provide comparable and sometimes better results from touch DNA and trace DNA samples. Here, direct PCR was applied to touch DNA retrieved from bullet casings from three ammunition types and guns.

Improved STR profiles from improvised explosive device (IED): fluorescence latent DNA detection and direct PCR.

Bombing accounts for the largest share of terrorist incidents worldwide. Most involve an improvised explosive device (IED): a bomb made from household items. Touch DNA may be left on parts of an IED during assembly.

A new cost-effective and fast direct PCR protocol for insects based on PBS buffer.

Insect DNA barcoding is a species identification technique used in biodiversity assessment and ecological studies. However, DNA extraction can result in the loss of up to 70% of DNA. Recent research has reported that direct PCR can overcome this issue.

Direct pentaplex PCR assay: An adjunct panel for meat species identification in Asian food products.

A direct pentaplex PCR assay was developed for the identification of meat from sources other than those declared on the packaging. Species-specific primers were designed, based on the mitochondrial cytochrome oxidase I (COI) gene. The assay amplified specific DNA fragments from dog (230 bp), duck (283 bp), buffalo (363 bp), goat (396 bp), and sheep (477 bp).

Direct-STR typing from presumptively-tested and untreated body fluids.

Body fluids provide key pieces of information for a forensic investigation. However, sometimes only a small amount of body fluids is found and/or DNA are also degraded by environmental factors at the crime scene. In extreme cases, a forensic analyst may have to decide whether to perform a presumptive test on the stains or proceed straightaway to DNA profiling, which could be wasteful for non-biological stains.

Meat species identification by two direct-triplex real-time PCR assays using low resolution melting.

We developed and validated the first direct multiplex real-time PCR assay with melt curve analysis for the identification of different types of meat. The assay detects six commonly eaten meat species and provides the following benefits: ease of use, shortened analysis time, and decreased analysis cost. Species-specific primers were designed from cytochrome b, cytochrome oxidase I, and 16S rRNA genes to generate PCR products with specific melting peaks that differentiate pork, beef, horsemeat, duck meat, ostrich meat, and chicken.

Comparative performance of AmpFLSTR® Identifiler® Plus PCR amplification kit and QIAGEN® Investigator® IDplex Plus kit.

Many forensic STR kits are currently available in the market. The AmpFLSTR® Identifiler® Plus kit, which targets 15 STRs, is commonly used worldwide. The Thai forensic DNA community is built around it in terms of instrument, databases, and interpretation.

Ivory species identification using electrophoresis-based techniques.

Despite continuous conservation efforts by national and international organizations, the populations of the three extant elephant species are still dramatically declining due to the illegal trade in ivory leading to the killing of elephants. A requirement to aid investigations and prosecutions is the accurate identification of the elephant species from which the ivory was removed. We report on the development of the first fully validated multiplex PCR-electrophoresis assay for ivory DNA analysis that can be used as a screening or confirmatory test.

A novel real time PCR assay using melt curve analysis for ivory identification.

Demand for ivory and expansion of human settlements have resulted in a rapid decline in the number of elephants. Enforcement of local and international laws and regulations requires identification of the species from which any ivory, or ivory products, originated. Further geographical assignment of the dead elephant from which the ivory was taken can assist in forensic investigations.

Systematic study for DNA recovery and profiling from common IED substrates: From laboratory to casework.

Improvised explosive devices (IEDs) made from household items are encountered in terrorist attacks worldwide. Assembling an IED leaves trace DNA on its components, but deflagration degrades DNA. To maximize the amount of DNA recovered, a systematic evaluation of DNA collection methods was carried out and the most efficient methods were implemented with IED casework evidence as a validation exercise.

Forensic STR loci reveal common genetic ancestry of the Thai-Malay Muslims and Thai Buddhists in the deep Southern region of Thailand.

Among the people living in the five deep Southern Thai provinces, Thai-Malay Muslims (MUS) constitute the majority, while the remaining are Thai Buddhists (BUD). Cultural, linguistic and religious differences between these two populations have been previously reported. However, their biological relationship has never been investigated.

Direct-multiplex PCR assay for meat species identification in food products.

This is the first time that direct PCR - DNA amplification without prior DNA extraction - was successfully developed and fully validated for rapid and economical simultaneous identification of six commonly consumed meat species. To achieve this, six species-specific primers were selected from previous reports and newly designed from the mitochondrial cytochrome b (cyt b), cytochrome oxidase I (COI), and 12s rRNA gene. The assay generated PCR products of 100, 119, 133, 155, 253, and 311 bp for pork, lamb/mutton, chicken, ostrich meat, horsemeat and beef, respectively.

Forensic animal DNA analysis using economical two-step direct PCR.

Wildlife forensic DNA analysis by amplification of a mitochondrial locus followed by DNA sequencing is routine, yet suffers from being costly and time-consuming. To address these disadvantages we report on a low-cost two-step direct PCR assay to efficiently analyze 12 forensically relevant mammalian sample types without DNA extraction. A cytochrome oxidase I degenerate-universal primer pair was designed and validated for the developed assay.

Age estimation of bloodstains using smartphones and digital image analysis.

Recent studies on bloodstains have focused on determining the time since deposition of bloodstains, which can provide useful temporal information to forensic investigations. This study is the first to use smartphone cameras in combination with a truly low-cost illumination system as a tool to estimate the age of bloodstains. Bloodstains were deposited on various substrates and photographed with a smartphone camera.

Using the Taguchi method for rapid quantitative PCR optimization with SYBR Green I.

Here, we applied the Taguchi method, an engineering optimization process, to successfully determine the optimal conditions for three SYBR Green I-based quantitative PCR assays. This method balanced the effects of all factors and their associated levels by using an orthogonal array rather than a factorial array. Instead of running 27 experiments with the conventional factorial method, the Taguchi method achieved the same optimal conditions using only nine experiments, saving valuable resources.

Evaluation of nucleosome forming potentials (NFPs) of forensically important STRs.

Degraded forensic samples have proved difficult to analyze and interpret. New analysis techniques are constantly being discovered and improved but researchers have overlooked the structural properties that could prevent or slow the process of degradation. In theory, DNA that are bound to histones as nucleosomes are less prone to degradation, because nucleosomes prevent DNA from being exposed to degradative enzymes.