Forensically Informative Nucleotide Sequencing Research Articles

Value of FINS Depends on Choice of DNA Locus as Shown in Baihuasheshecao

Forensically informative nucleotide sequencing (FINS) is a molecular approach to identify biological materials based on DNA sequences. It focuses on the clustering of samples with reference species through an analysis of the phylogenetic relationship among the species. The choice of loci is critical to the successful use of FINS for identification. In this study, we investigated the influence of six loci (ITS, matK, rbcL, rps16, trnH-psbA and trnL-trnF) on the application of FINS for the identification of traditional Chinese medicine as demonstrated in Baihuasheshecao, a traditional anti-tumor herb derived from Hedyotis diffusa willd. A total of 36 plant samples of Hedyotis species and eight samples of Baihuasheshecao commodities were collected from Hong Kong, Macao, Taiwan, mainland China and the USA. Internal transcribed spacers (ITS) sequences displayed the highest rate of formation of single species cluster (89%), followed by rps16 (78%), matK (74%), trnL-trnF (72%), trnH-psbA (67%) and rbcL (61%). All the loci could discriminate H. diffusa from the common substitute species H. corymbosa, H. pinifolia and H. tenelliflora. FINS identification based on ITS, trnH-psbA and trnL-trnF revealed that five Baihuasheshecao commodities were genuine herb derived from H. diffusa, while the other three commodities were substitutes derived from H. corymbosa. MatK, rbcL and rps16 could not be amplified from all commodity samples and were thus inapplicable for FINS identification of dried the samples. In conclusion, the success of FINS identification depends on the DNA loci as they show different abilities to differentiate genuine species from substitutes, to discriminate closely related species and to generate quality sequences. In this study, the eligibility of DNA locus for FINS identification of Baihuasheshecao commodities, in descending order, is ITS ＞ trnL-trnF ＞ trnH-psbA ＞ rps16 ＞ matK ＞ rbcL.

Identification of selected wild felids using hair morphology and forensically informative nucleotide sequencing (FINS): Wildlife forensics prospective.

We describe the identification of the leopard cat (Prionailurus bengalensis), jungle cat (Felis chaus) fishing cat (Prionailurus viverrinusis), wild cat (Felis silvestris) and caracal (Caracal caracal) using combined approach of morphological and molecular based analysis. Three mitochondrial genes [12S rRNA, 16S rRNA and cytochrome b (Cyt b)] and hair characteristics (cuticle and medulla patterns) were analysed and variation was observed among few selected wild felids. We did not find conclusive morphological difference among these examined felids. We observed maximum variation in Cyt b in comparison to12S rRNA and 16S rRNA genes. Therefore, despite poor hair morphological difference, forensically informative nucleotide sequencing (FINS) exhibited unambiguous variation among the examined felids. We suggest the use of FINS for differentiating biological samples of closely related wild felids to avoid any false identification of species in illicit trade. Furthermore, the data generated from the present work would help in strengthening the DNA database of Indian small cats.

Authentication of nine snapper species by single-strand conformation polymorphism (SSCP) and forensically informative nucleotide sequencing (FINS) methods

PCR-SSCP and PCR-FINS methods were developed to authenticate nine species of snapper. A highly variable mitochondrial D-loop region was chosen as a target region and three new primers viz., one forward (Fish-DL-F) and two reverse primers (Fish-DL-R and DLFR) were designed. PCR-SSCP was first targeted to amplify 515 bp D-loop fragment using Fish-DL-F/Fish-DL-R primers, but it could not differentiate all the species. Another reverse primer (DLFR) could amplify a 360 bp region, which had differentiated all the species. A FINS method was also developed for snapper authentication using the same D-loop region of 515 bp. The developed FINS was validated with 20 market snapper samples that showed 100% specificity. This study helped to generate nucleotide sequences of D-loop region for 6 species of snapper for the first time for deposition in GenBank. Hence, to authenticate the snapper species, the developed PCR-SSCP and FINS were more specific and reliable.

Forensically informative nucleotide sequencing (FINS) for species and subspecies of genus Prionailurus (Mammalia: Carnivora: Felidae) through mitochondrial genes (12SrRNA and cytochrome b) by using old taxidermy samples

Genus Prionailurus comprises four species, i.e. Leopard cat, Rusty spotted cat, Fishing cat, Flat-headed cat, listed under IUCN as threatened species except P. bengalensis. In U.S.A., P. bengalensis is listed as Endangered. Subspecies of P. bengalensis, i.e. iriomotensis is listed as Critically Endangered under IUCN since 2008. The present study describes the use of two markers 12SrRNA and cytochrome b genes to differentiate the three species and three subspecies of Prionailurus. Old taxidermy samples (three skin samples) of P. viverrinus and P.b. horsfieldi were used from India for the study. The study done by using DNAsp v5, MEGA 6.0, and Network 5.0.0.1, proved that both gene markers are useful for differentiating the species and subspecies of Prionailurus. This study is also the first study to present forensically informative nucleotide sequence (FINS) for three species and three subspecies of Prionailurus.

Molecular identification of species and production origins of edible bird's nest using FINS and SYBR green I based real-time PCR

The increasing demand and consumption of edible bird's nest (EBN) by people worldwide has contributed to the food fraud issue. To ensure the authenticity of EBN in regard to their origin, rapid and accurate analytical methods are very much needed. In this study, forensically informative nucleotide sequencing (FINS) technique based on mitochondrial and nuclear DNA sequences, and phylogenetic analysis was performed to identify the species and production origins of raw and commercial EBNs. The cytochrome b (Cyt b), NADH dehydrogenase subunit 2 (ND2), 12S ribosomal RNA and beta-fibrinogen intron 7 gene markers used were able to identify and classify EBN produced by Aerodramus fuciphagus and Aerodramus maximus. It was newly discovered that EBN from man-made houses and natural caves were genetically differentiable using the mitochondrial Cyt b and ND2 genes. The phylogenetic results revealed that all EBN samples were well-separated into two groups following their species origin and production origin. A rapid and cost-effective identification alternative of SYBR green I based real-time PCR assay targeting a 177 bp of the mitochondrial Cyt b gene was developed and it efficiently differentiated genuine EBN from counterfeits. This FINS and SYBR green I based real-time PCR are highly sensitive, specific and reliable methods for identification of EBN origins and could be useful for preventing fraud substitution and mislabelling of EBN to ensure food safety.

Molecular identification of honey entomological origin based on bee mitochondrial 16S rRNA and COI gene sequences

The increasing recognition and production of stingless bee honey has raised the needs to develop rapid, sensitive and accurate methodologies for honey identification through bees' species from the DNA of bees present in honey. Genetic identification of raw honeys produced by different bee species from Malaysia was performed based on mitochondrial DNA sequences and phylogenetic analysis by means of forensically informative nucleotide sequencing (FINS) technique. The 300 bp of mitochondrial large subunit ribosomal RNA (16S rRNA) gene region and mitochondrial cytochrome c oxidase subunit I (COI) gene region were the gene markers used to classify and identify honey produced by Apis honey bees and Trigona stingless bees. The genetic identities of honey origin from Apis dorsata, Apis mellifera, Apis cerana, and Heterotrigona itama were accurately identified. Kelulut honey sourced from the stingless bee was distinguishable from other Apis type of honeys in phylogenetic analyses. This FINS technique is a specific and reliable method for identification of honey origin via its bee source that can be used to overcome the issue of false declaration of honey origin and mislabelling.

Forensically informative nucleotide sequencing (FINS) for the first time authentication of Indian Varanus species: implication in wildlife forensics and conservation

Monitor lizards are Varanus species widely distributed, endangered reptile in the IUCN red data list. In India, based on the morphological and ecological characteristic, it is divided into four species viz. Bengal monitor lizard, Yellow monitor lizard, Desert monitor lizard and Water monitor lizard. These four species listed as Schedule I species in Indian Wildlife (Protection) Act 1972. This paper first attempt to present Forensically Informative Nucleotide Sequencing (FINS) for the Indian Varanus based on three mitochondrial genes. The molecular framework will be useful for the identification of Indian Varanus species and trade products derived from monitors and as such, have important applications for wildlife management and conservation. Here, we used known 14 individual skin pieces of four species of monitor lizards; the partial fragment of three mitochondrial genes (Cyt b, 12S rRNA, and 16S rRNA) were amplified for genetic study. In Cyt b, 12S rRNA and 16s rRNA, we observed, 5, 5 and 4 Haplotypes; 71, 69, and 43 Variables sites; 90, 89, and 50 Parsimony Informative sites within four species of Indian monitor lizards, respectively. Despite it, the nucleotide composition was T 26.4, C 32.8, A 29.2 and G11.6; T 18.8, C 29.7, A 34.0 and G 17.5; T 21.7, C 27.3, A 32.5 and G 18.5 in Cyt b, 12S rRNA and 16S rRNA, respectively. The neighbor joining phylogenetic tree and maximum parsimony tree of three mitochondrial genes, showed similar results and reveal that, there are two major clades are present in Indian monitor lizards.

SNP-based PCR-RFLP, T-RFLP and FINS methodologies for the identification of commercial fish species in Egypt

Genetic fingerprinting is important for both certifying authenticity and traceability of fish species. In the present study, three methodologies were developed for the identification of aquaculture and fisheries Egyptian fish: forensically informative nucleotide sequencing (FINS), single nucleotide polymorphism-based polymerase chain reaction-terminal restriction fragment length polymorphism (SNP-based PCR-T-RFLP) capillary electrophoresis, and SNP-based PCR-RFLP. FINS based on mitochondrial 12S rDNA allowed the genetic identification of seventeen Egyptian species analyzed. Four SNPs, located in restriction sites, also enable identifying all species. The resolution power of agarose gel electrophoresis was not enough to detect small-sized fragments, whereas T-RFLP capillary electrophoresis produced complete species-specific patterns. The three methods are ready now and can be employed as inexpensive, rapid and effective tools for food authentication in Egypt and other countries where seafood contributes much to local animal production economics.

Assessment of the labelling accuracy of spanish semipreserved anchovies products by FINS (forensically informative nucleotide sequencing).

Anchovies have been traditionally captured and processed for human consumption for millennia. In the case of Spain, ripened and salted anchovies are a delicacy, which, in some cases, can reach high commercial values. Although there have been a number of studies presenting DNA methodologies for the identification of anchovies, this is one of the first studies investigating the level of mislabelling in this kind of products in Europe. Sixty-three commercial semipreserved anchovy products were collected in different types of food markets in four Spanish cities to check labelling accuracy. Species determination in these commercial products was performed by sequencing two different cyt-b mitochondrial DNA fragments. Results revealed mislabelling levels higher than 15%, what authors consider relatively high considering the importance of the product. The most frequent substitute species was the Argentine anchovy, Engraulis anchoita, which can be interpreted as an economic fraud.

Fish species identification in canned pet food by BLAST and Forensically Informative Nucleotide Sequencing (FINS) analysis of short fragments of the mitochondrial 16s ribosomal RNA gene (16S rRNA)

Nowadays, pet food claiming high-valued fish among ingredients is largely available on the market. Unfortunately, the modifications induced by processing make species identification by visual inspection difficult and hinder the enforcement of the legislation on traceability. In this work, after aligning 819 sequences of Clupeidae, Engraulidae, Salangidae and Scombridae families, we developed new universal primers for the amplification and sequencing of 2 short fragments (±118 and ±213) of the mitochondrial 16s ribosomal RNA (16S rRNA) gene. Once tested on 130 DNA reference samples, these primers were used in the analysis of highly degraded DNA extracted from 43 canned cat food containing whole minnows (whitebait) (M) and tuna, or bonito or mackerel fillets (F). Three M and 2 F samples were analyzed for each can. A BLAST and a FINS analysis, the latter performed only on the 118 bp fragment, were performed separately on the sequences obtained from M and F samples. All the M samples were identified at the species or genus level by both BLAST and FINS analysis. This allowed to highlight an impressive rate of mislabeling (100%). F samples, for which FINS was less performing in species identification, resulted mislabeled in 40% of the products.

Authentication of consumer fraud in Taiwanese fish products by molecular trace evidence and forensically informative nucleotide sequencing

An inexpensive, efficient, and reliable molecular method of authenticating commercial puffer fish-based products was developed to determine the origin of adulterated fish products and for food regulatory control. This system is based on molecular trace evidence obtained using a specific primer set with a short gene marker located on the mitochondrial cytochrome b sequence region. The method was successfully tested and validated in 12 specimens of puffer fish, simulated products, and 50 commercial samples. Fourteen percent of the collected commercial products were found to be puffer fish-based. However, of these 14%, 28% were identified as toxic varieties by further analysis with forensically informative nucleotide sequencing (FINS) and BLAST methods. These results reveal that the developed method is a rapid and efficient tool to unequivocally identify puffer fish, which may aid in the prevention of consumer fraud or mislabeling of fish products.

What is inside the jar? Forensically informative nucleotide sequencing (FINS) of a short mitochondrial COI gene fragment reveals a high percentage of mislabeling in jellyfish food products

The jellyfish belonging to the Rhizostomeae order are a typical Asian seafood and are nowadays also marketed in the Western countries. The jellyfish species used for food cannot be identified due to the loss of the morphological characteristics during processing. In this work, a pre-extraction treatment consisting of desalting under running water for 48h, followed by incubation in 400mM EDTA solution for 1h, was developed for products containing high concentration of salts. Moreover, considering the DNA degradation observed in both classical (CPs) and ready to eat (RE) commercial samples, five sets of primers for the amplification of fragments with different lengths belonging to the mitochondrial COI gene were designed and used to obtain the sequences from 54 reference specimens and 70 market samples. A phylogenetic analysis was then performed using the neighbor-joining method. It was found that a fragment of 142bp was enough informative to allow identification at the species level. The results showed that most of the products were made of Nemopilema nomurai (94% of RE and 45.5% of CPs). The analysis permitted to uncover an alarming level of mislabeling which reaches 79% for CPs and 100% for ready-to-eat products.

Application of Genetic Resources for the Molecular Identification of Traditional Chinese Medicinal Materials

With the rapid development of molecular biotechnology, DNA sequences of a large number of medicinal plants and animals have been published in recent years. The development of DNA barcodes of medicinal species has been one of the major focuses in this field. These public genetic resources have provided a valuable foundation for the development of novel molecular techniques for the identification of traditional Chinese medicines. In 2010, we established the first Medicinal Materials DNA Barcode Database (MMDBD) in the world which contains over 1,600 medicinal and related species recorded in the Pharmacopoeia of the People's Republic of China and the Pharmacopoeia of the United States. We have also established standard DNA barcodes for the identification of medicinal species used in the Chinese 'cooling' beverage (Liangcha) and differentiation of easily confused herbs retailed in the market. With the use of forensically informative nucleotide sequencing (FINS), the identity of unknown herbal samples can be properly identified. In addition, specific primers based on the polymorphic sites can be used in the development of novel isothermal amplification techniques for rapid molecular identification. These primers may be patented and developed into commercial identification kits.

Identification of Galliformes through Forensically Informative Nucleotide Sequencing (FINS) and its Implication in Wildlife Forensics

Galliformes are hunted for the demand of their attractive feathers and to supply a cheap animal food for the rural communities. In such cases, species identification through visual inspection of the meat or based on feather morphometrics is a challenging task for the law enforcement agencies to enforce the Wildlife Protection Act (WPA). Here, we extracted DNA from the individual feathers of unknown species encountered during field surveys and two mitochondrial genes (12S rRNA and Cytochrome b) were amplified using universal primers for species identification. Most homologous sequences were retrieved using NCBI-BLAST for each generated sequence of both the genes. Neighbor-Joining trees based on Kimura 2 parameter distance matrices in FINS analysis identified the species from the individual feather with strong bootstrap support. Nine species specific polymorphic sites were found in the partial sequence of Cytochrome b gene that differentiated Pavo cristatus to Pavo muticus imperator. Our study highlighted the importance of feathers in identifying the species and their applicability in wildlife offence cases using FINS approach.

Direct PCR-FINS: Wildlife species identification without DNA extraction

Forensically informative nucleotide sequencing (FINS) is commonly used in forensic laboratories worldwide for wildlife species identification. Recently direct PCR – DNA amplification without prior DNA extraction – has been gathering attention in the human forensic community due to its high efficiency, but it has never been applied to non-human forensic investigation. Therefore, we aimed to evaluate the possibility and efficiency of using direct PCR-FINS for species identification from various types of forensically relevant animal samples. Eight biological evidence types (hair, muscle tissue, bloodstain, bone, ear, skin, urine, and feces) from three common crime-related animals (tiger, white rhino, and Asian elephant) were analyzed with the three-step direct PCR-FINS method: direct PCR amplification of target DNA with 16S rRNA and cytochrome b universal primers, sequencing, and database searching (BLAST). All sample types were successfully amplified using direct PCR-FINS method and provided the expected PCR products. Each amplicon sequence and its relevant animal species were completely matched using BLAST, meaning that the species of the samples were correctly identified. In conclusion, the ‘direct PCR-FINS method’ is rapid, reliable, and meets the requirement of international forensic organizations.

Species identification of Cyprinidae fish in Taiwan by FINS and PCR–RFLP analysis

Cyprinidae fish are widely distributed in Taiwan, and processed products made from them are favored by consumers. To identify the species used for these processed products, complete mitochondrial cytochrome b genes (cytb, 1141 bp) of 8 Cyprinidae were established. Then, a rapid PCR–RFLP method was used to amplify a 426 bp fragment of cytb using primer set Lnew1/Hnew1, and the fragment was co-digested with NlaIII and BstXI. The results show that using this PCR–RFLP technique for the species identification of the 8 Cyprinidae was successful. The method was further applied to investigate 16 commercial samples of processed Cyprinidae products from traditional markets. The results indicate that 9 of the samples were Carassius auratus auratus, Cyprinus carpio carpio, or Ctenopharyngodon idella. The other 6 samples did not belong to the Cyprinidae family according to PCR–RFLP. These samples were further analyzed with the forensically informative nucleotide sequencing (FINS) and BLAST methods. The results show that these 6 processed products were Oreochromis spp., a monophyletic fish group. The last commercial product could not be amplified to obtain a 426 bp fragment, so its species remains unknown. This study shows that PCR–RFLP and FINS can be used to identify processed products fraudulently labeled as Cyprinidae.

Forensically informative nucleotide sequencing (FINS) for the authentication of Chinese medicinal materials.

Chinese medicinal materials may be authenticated by molecular identification. As a definitive approach to molecular identification of medicinal materials, forensically informative nucleotide sequencing (FINS) comprises four steps, namely (1) DNA extraction from biological samples, (2) selection and amplification of a specific DNA fragment, (3) determination of the sequence of the amplified DNA fragment and (4) cladistic analysis of the sample DNA sequence against a DNA database. Success of the FINS identification depends on the selection of DNA region and reference species. This article describes the techniques and applications of FINS for authenticating Chinese medicinal materials.

Application of FINS and multiplex PCR for detecting genuine abalone products

Abalones rank first on the list of the four treasures from the sea in Chinese cuisine. It is regarded as a tonic for yin-enrichment and nourishing the lungs and liver. Hong Kong is a major entrepôt of abalones, importing 48% of the global production volume. Rapid detection of substitutes in abalone products is essential for safeguarding consumer interests and ensuring proper delivery of healthcare. Two molecular tools are applied to authenticate abalone products. Firstly, forensically informative nucleotide sequencing (FINS) analysis based on mitochondrial 16S rDNA region of abalone products retailed in Hong Kong revealed that all sliced and one canned products matched the sequences of other gastropods but not Haliotis species, suggesting the presence of substitutions. Secondly, multiplex PCR with a Haliotis-specific primer was used to facilitate rapid and reliable exclusion of substitutes from abalone products.

Genetic Differentiation between Fake Abalone and Genuine Haliotis Species Using the Forensically Informative Nucleotide Sequencing (FINS) Method

Abalones ( Haliotis species) are a popular delicacy and commonly preserved in dried form either whole or in slices or small pieces for consumption in Asian countries. Driven by the huge profit from trading abalones, dishonest traders may substitute other molluscan species for processed abalone, of which the morphological characteristics are frequently lost in the processed form. For protection of consumer rights and law enforcement against fraud, there is a need for an effective methodology to differentiate between fake and genuine abalone. This paper describes a method (validated according to the international forensic guidelines provided by SWGDAM) for the identification of fake abalone species using forensically informative nucleotide sequence (FINS) analysis. A study of the local market revealed that many claimed "abalone slice" samples on sale are not genuine. The fake abalone samples were found to be either volutids of the genus Cymbium (93%) or the muricid Concholepas concholepas (7%). This is the first report of Cymbium species being used for the preparation and sale as "abalone" in dried sliced form in Hong Kong.

Authentication of species in meat products by genetic techniques

In the present work, a method for the authentication of meat products was developed, using Polymerase Chain Reaction (PCR) followed by Forensically Informative Nucleotide Sequencing (FINS). This study describes the use of sequencing of the cytochrome b gene of the mtDNA in a wide variety of species to diagnose adulteration of meat through the substitution from one species to another that have less commercial value. The main importance of this work is to deal with a wide variety of species that have not previously been analyzed. This methodology strategy allows the authentication of meat species in all kind of products, fresh, or precooked products, the more usual format for marketing in that species. This method shows a specificity of 100%. The developed methodology was validated and finally applied to 20 commercial samples including some that had been subjected to intensive thermal treatment. In 15% of the products analyzed, the name of the species displayed in the label was not in agreement with the identified species. The main novelty of this work lies in the fact that it allows the identification of a large number of meat species not analyzed so far in previous works. In this work are included meat species, which can be easily found in our markets, and a wide variety of others whose consumption is common in other parts of the world. Therefore, this technique can be used as a routine method to avoid the mislabeling in the marketing of these products and to assess their correct traceability.