Thitika Kitpipit

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. Validation showed that the assay is robust, rapid, economical, reproducible, specific, and sensitive down to 12,500 mitochondrial copy (equating to seven fg). It could be used with a variety of raw meats and products, including highly degraded and processed food samples. This proposed method will be greatly beneficial to the consumers, food industry, and law enforcement.

The development and validation of a single SNaPshot multiplex for tiger species and subspecies identification—Implications for forensic purposes

The tiger (Panthera tigris) is currently listed on Appendix I of the Convention on the International Trade in Endangered Species of Wild Fauna and Flora; this affords it the highest level of international protection. To aid in the investigation of alleged illegal trade in tiger body parts and derivatives, molecular approaches have been developed to identify biological material as being of tiger in origin. Some countries also require knowledge of the exact tiger subspecies present in order to prosecute anyone alleged to be trading in tiger products. In this study we aimed to develop and validate a reliable single assay to identify tiger species and subspecies simultaneously; this test is based on identification of single nucleotide polymorphisms (SNPs) within the tiger mitochondrial genome. The mitochondrial DNA sequence from four of the five extant putative tiger subspecies that currently exist in the wild were obtained and combined with DNA sequence data from 492 tiger and 349 other mammalian species available on GenBank. From the sequence data a total of 11 SNP loci were identified as suitable for further analyses. Five SNPs were species-specific for tiger and six amplify one of the tiger subspecies-specific SNPs, three of which were specific to P. t. sumatrae and the other three were specific to P. t. tigris. The multiplex assay was able to reliably identify 15 voucher tiger samples. The sensitivity of the test was 15,000 mitochondrial DNA copies (approximately 0.26 pg), indicating that it will work on trace amounts of tissue, bone or hair samples. This simple test will add to the DNA-based methods currently being used to identify the presence of tiger within mixed samples.

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. Three smartphones (Samsung Galaxy S Plus, Apple iPhone 4, and Apple iPad 2) were compared. The environmental effects - temperature, humidity, light exposure, and anticoagulant - on the bloodstain age estimation process were explored. The color values from the digital images were extracted and correlated with time since deposition. Magenta had the highest correlation (R(2)=0.966) and was used in subsequent experiments. The Samsung Galaxy S Plus was the most suitable smartphone as its magenta decreased exponentially with increasing time and had highest repeatability (low variation within and between pictures). The quantifiable color change observed is consistent with well-established hemoglobin denaturation process. Using a statistical classification technique called Random Forests™, we could predict bloodstain age accurately up to 42 days with an error rate of 12%. Additionally, the age of forty blind stains were all correctly predicted, and 83% of mock casework samples were correctly classified. No within- and between-person variations were observed (p>0.05), while smartphone camera, temperature, humidity, and substrate color influenced the age determination process in different ways. Our technique provides a cheap, rapid, easy-to-use, and truly portable alternative to more complicated analysis using specialized equipment, e.g. spectroscopy and HPLC. No training is necessary with our method, and we envision a smartphone application that could take user inputs of environmental factors and provide an accurate estimate of bloodstain age.

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. Six swab types and six moistening agents were used to collect dried buffy coat stains on four common IED substrates. The most efficient swab/moistening agent combinations were then compared with tape-lifting using three brands of adhesive tape and also with direct DNA extraction from evidence. The most efficient collection methods for different IED substrates (post-study protocol) were then implemented for IED casework and compared with the pre-study protocol using 195 pieces of IED evidence. There was no single best swab type or moistening agent. Swab type had the largest effect on DNA recovery percentages, but moistening agents, substrates, and the interactions between factors all affected DNA recovery. The most efficient swab/moistening agent combinations performed equally well when compared with the best adhesive tape and direct extraction. The post-study protocol significantly improved STR profiles obtained from IED evidence. This paper outlines a comprehensive study of DNA collection methods for trace DNA and the validation of the most efficient collection methods with IED evidence. The findings from both parts of this study emphasize the need to continuously re-evaluate standard operating protocols with empirical studies.

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. In this study, we aimed to reveal the genetic structure and genetic affinity between MUS and BUD by analyzing 15 autosomal short tandem repeats. Both distance and model-based clustering methods showed significant genetic homogeneity between these two populations, suggesting a common biological ancestry. After Islamization in this region during the fourteenth century AD, gradual albeit nonstatistically significant genetic changes occurred within these two populations. Cultural barriers possibly influenced these genetic changes. MUS have closer admixture to Malaysian-Malay Muslims than BUD countrywide. Admixture proportions also support certain degree of genetic dissimilarity between the two studied populations, as shown by the unequal genetic contribution from Malaysian-Malay Muslims. Cultural transformation and recent minor genetic admixture are the likely processes that shaped the genetic structure of both MUS and BUD.

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. In this study, a real-time PCR assay using melt curve analysis was developed and fully validated for forensic use. The presence or absence of three Elephantidae-specific and elephant species-specific melting peaks was used to identify the elephant species. Using 141 blood and ivory samples from the three extant elephant species, the assay demonstrated very high reproducibility and accuracy. The limit of detection was as low as 0.031ng of input DNA for conventional amplification and 0.002ng for nested amplification. Both DNA concentrations are typically encountered in forensic casework, especially for degraded samples. No cross-reactivity was observed for non-target species. Evaluation of direct amplification and nested amplification demonstrated the assays flexibility and capability of analyzing low-template DNA samples and aged samples. Additionally, blind trial testing showed the assays suitability application in real casework. In conclusion, wildlife forensic laboratories could use this novel, quick, and low-cost assay to help combat the continuing poaching crises leading to the collapse of elephant numbers in the wild.

Mini-SNaPshot multiplex assays authenticate elephant ivory and simultaneously identify the species origin

Illegal trading of ivory is mainly responsible for the dramatic decline in elephant populations. Thailand is one of the largest laundering hotspots for African ivory, as the domestic Asian elephant ivory can be legally traded. So, to help combat ivory poaching and smuggling, an efficient method is needed to identify the elephant species from its ivory and ivory products. In this study, a mini-SNaPshot® multiplex assay was developed and fully validated for the identification of confiscated ivory and low DNA template ivory products. Elephantid- and elephant species-specific mitochondrial single nucleotide polymorphisms (SNPs) were identified from 207 mammalian and 1705 elephant/mammoth cytochrome b sequence alignments. Seven informative SNPs were used for assay development. The assay unambiguously and accurately identified authentic elephant ivory and its species of origin on the basis of peak size and color observed in the haplotype profile. The assay was highly efficient for analysis of confiscated ivory and low-template ivory products with a 99.29% success rate (N=140). It was highly reproducible, exhibited no cross-reaction with eight other mammalian DNA; and had 100% identification accuracy. In addition, nested and direct PCR amplification were also compatible with the developed assay. This efficient assay should benefit wildlife forensic laboratories and aid in the prosecution of elephant-related crimes.

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. Validation of the assay showed that it is robust, reproducible, specific, and sensitive down to 0.32ng of DNA template. It takes less than one hour from sample to result and costs less than one USD per sample. We also successfully amplified 92.5% of market samples, demonstrating the assays robustness. The developed assay has the potential to be implemented in food testing laboratories worldwide.

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. Additionally, due to the inefficient DNA extraction process, the amount of DNA may not be enough for STR typing, especially if parts of the evidence had been subjected to presumptive testing. To overcome these problems, we developed a direct PCR method for STR profiling of stains (blood, saliva, and semen) that had been subjected to presumptive tests and also those that had not undergone presumptive tests. Using the optimized protocols, 86 of 90 untreated samples (95.6%) resulted in a full DNA profile. For presumptively-tested samples, both the type of presumptive test used and the surfaces where the stains are deposited affected the quality of the STR profiles. With blood, we obtained full STR profiles from 88% of samples tested with luminol and 78% with Hemastix. The acid phosphatase test for semen and Phadebas test for saliva resulted in full STR profiles from 85% and 73% of samples, respectively. Different substrates also affected the resulting STR profiles, but there was no clear trend based on absorbency or texture. The interactions of types of body fluids, presumptive tests, and substrates must be considered together. Our direct PCR protocol can be used to detect DNA even with 6 months-old biological samples. The benefits of the developed protocol include increasing amount of DNA obtained from evidence, decreasing chances of DNA contamination from complex or lengthy extraction steps, using minimal sample amount for analysis, and most importantly, improving STR profiles. Also, the process could save analysis time and cost due to the omission of DNA extraction and quantification. Our developed method could be beneficial to cases with limited stains available, as forensic analysts can perform indirect presumptive testing on the suspected stains and direct PCR could be carried out from the filter paper used, thus leaving the original stain for subsequent DNA extraction or re-analysis.

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. QIAGENs IDplex Plus kit targets the same loci, but the PCR cycling time is shorter by about 90min. A direct comparison that assesses forensic parameters and applicability to casework between the two kits has never been carried out. In this study, we performed a direct comparison between the two kits using serial dilutions of two control DNA samples and 60 randomly selected casework samples, including samples taken from improvised explosive devices and terrorist raids. We statistically compared the performance of the two kits in terms of peak height, number of allele detected (allelic drop-out), intra-locus balance, inter-locus balance, inhibitor tolerance, stutter ratio, concordance, and allelic drop-in. The results demonstrate that both kits are statistically similar in performance. IDplex Plus gave higher peak heights in sensitivity test and tolerated inhibitors better, but had slightly worse inter-locus balances and stutter ratios. However, these differences were not practically significant, as seen by the resulting profiles of the casework samples (p=0.601). The performance on low-template samples also was not different. In conclusion, laboratories looking to replace the aging Identifiler® Plus might consider the IDplex Plus as a faster, more robust alternative that fits right into their existing structure without further investment in instrument and DNA database. Having more kits available worldwide by different companies could help bring the technology to different forensic laboratories and the justice system as a whole.