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Identification of Species with DNA-Based Technology: Current Progress and Challenges
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One of the grand challenges of modern biology is to develop accurate and reliable technologies for a rapid screening of DNA sequence variation. This topic of research is of prime importance for the detection and identification of species in numerous fields of investigation, such as taxonomy, epidemiology, forensics, archaeology or ecology. Molecular identification is also central for the diagnosis, treatment and control of infections caused by different pathogens. In recent years, a variety of DNA-based approaches have been developed for the identification of individuals in a myriad of taxonomic groups. Here, we provide an overview of most commonly used assays, with emphasis on those based on DNA hybridizations, restriction enzymes, random PCR amplifications, species-specific PCR primers and DNA sequencing. A critical evaluation of all methods is presented focusing on their discriminatory power, reproducibility and user-friendliness. Having in mind that the current trend is to develop small-scale devices with a high-throughput capacity, we briefly review recent technological achievements for DNA analysis that offer great potentials for the identification of species.
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... Although DNA-based studies have provided ample evidence to support morphological based taxonomic schemes, there are circumstances where morphologically identical species have been reported to be genetically distinct and considered as other species. This has obviously culminated in better understanding of species diversity and numbers (Pereira et al. 2008;Maharachchikumbura et al. 2021). Thanks to advances in taxonomy, we have greater capabilities to identify species boundaries because of the wealth of data and tools available (Lücking et al. 2020(Lücking et al. , 2021a(Lücking et al. , b, 2022. ...
Estimates of global fungal diversity have varied widely, suggesting a range from fewer than one million to over 10 million species, with each of the estimates drawing data from various criteria. In 2022, Fungal Diversity published a special issue on fungal numbers. It had been hoped that the editorial would provide a more accurate account of the numbers of fungi. Instead, it was concluded that this was not possible based on present evidence and instead, some of the data necessary for accurate assessments was put forward, and the present paper expands on this short article. The review first looks at estimates of fungal numbers and what these estimates are based on. It then presents future research needs that will help us to gain a more accurate estimate of fungal numbers. This includes work that needs to be done in tropical rainforests, where the greatest diversity is expected, where whole rainforests, canopy diversity, and palm fungi are addressed. Case studies for lichens and associated fungi, soil and litter fungi, evidence from particle filtration, freshwater fungi, marine fungi, mushrooms, and yeasts will also be given. Once we have such information, we can obtain a more accurate estimate of fungal numbers.
... As an alternative to this method, molecular techniques such as DNA sequencing and polymerase chain reaction (PCR) have been reported to be more efficient, as they use a single process for all the DNA markers [24,25]. These molecular technologies are now critical for the diagnosis, treatment and control of multiple PPNs [26]. ...
Accurate identification and estimation of the population densities of microscopic, soil-dwelling plant-parasitic nematodes (PPNs) are essential, as PPNs cause significant economic losses in agricultural production systems worldwide. This study presents a comprehensive review of emerging techniques used for the identification of PPNs, including morphological identification, molecular diagnostics such as polymerase chain reaction (PCR), high-throughput sequencing, meta barcoding, remote sensing, hyperspectral analysis, and image processing. Classical morphological methods require a microscope and nematode taxonomist to identify species, which is laborious and time-consuming. Alternatively, quantitative polymerase chain reaction (qPCR) has emerged as a reliable and efficient approach for PPN identification and quantification; however, the cost associated with the reagents, instrumentation, and careful optimisation of reaction conditions can be prohibitive. High-throughput sequencing and meta-barcoding are used to study the biodiversity of all tropical groups of nematodes, not just PPNs, and are useful for describing changes in soil ecology. Convolutional neural network (CNN) methods are necessary to automate the detection and counting of PPNs from microscopic images, including complex cases like tangled nematodes. Remote sensing and hyperspectral methods offer non-invasive approaches to estimate nematode infestations and facilitate early diagnosis of plant stress caused by nematodes and rapid management of PPNs. This review provides a valuable resource for researchers, practitioners, and policymakers involved in nematology and plant protection. It highlights the importance of fast, efficient, and robust identification protocols and decision-support tools in mitigating the impact of PPNs on global agriculture and food security.
... Finally, the identification of Rattus species (R. norvegicus or R. rattus) was done by restriction fragment length polymorphism (RFLP) analysis on their mitochondrial cytb gene. Based on the work of Galan et al. [12] who targeted a small region of the mitochondrial cytb gene (130 bp) sufficient for rodent species identification using next-generation sequencing, we designed primers to amplify a larger region (643 bp) in the cytb gene in order to perform RFLP analysis on this fragment to visually identify rodent species directly on gels, without any sequencing required [29,33]. To validate this approach, we performed in silico PCR using Primer Blast (NCBI) and we obtained 153 different species (birds, mammals, including rodents). ...
Angiostrongylus cantonensis, commonly known as the rat lungworm, causes Eosinophilic meningitis in humans. Our study aimed to investigate the prevalence and distribution of this parasite in rats in Haiti. Rats were trapped at 8 sites, 7 in Artibonite (rural region) and one in an urban area of Port-au-Prince. After euthanasia, hearts and lungs were sampled and preserved in 70% ethanol. Subsequently, the organs were dissected to detect adult worms. Parasite DNA was amplified using PCR targeting either the nematode ITS2 gene for rodent lung tissue or cox1 for isolated worms. Subsequent sequencing allowed parasite identification. A total of 70 rats were captured, i.e. 23 Rattus norvegicus and 47 Rattus rattus. Adult nematodes morphologically compatible with A. cantonensis were isolated from 5/70 rats (7%) and identification was confirmed by sequencing. Molecular analysis of lung tissue revealed a parasite prevalence of 31.4% (22/70), and its presence at 4 of the 8 sites investigated, including Port-au-Prince. The molecular approach on lung tissue targeting the ITS2 gene enabled us to detect a prevalence 4 times higher than the visual search for adult worms alone. Only one COX1 haplotype was identified, belonging to genotype II-G, widely distributed in Brazil, the French Antilles (Guadeloupe), French Polynesia, Hawaii, and Japan. These results confirm that A. cantonensis is an endemic parasite in Haiti not only in the capital Port-au-Prince, but also in several rural areas. Direct molecular screening for Angiostrongylus DNA in rat lung tissue showed higher sensitivity than visual detection of worms during dissection and could be useful for further prevalence studies.
... Ants can also be analyzed genotypically. DNA is useful for identifying species, as described by Pereira et al. (2008) in their study. Their research contends that DNA is a stable and long-lived biological molecule that can be recovered from biological material that has been subjected to stress conditions. ...
... In fact, the robustness of multiplex PCR assay for species specific identification of different food products like milk and milk products is primarily determined by lengths of the PCR products and optimum lengths of the target fragments should range between 100 and 400 bp (Sangthong, Suwannarat, Samipak, & Sangthong, 2021). The PCR products with >500 bp are adversely affected by DNA fragmentation that can be caused by food processing steps (Pereira, Carneiro, & Amorim, 2008). On the other hand PCR products with length <100 bp are not very suitable for analysis by agarose gel electrophoresis (Sangthong et al., 2021). ...
... Biological characteristics are important signs of the human body. Through the detection and matching of human biological attributes such as DNA [63,64] and body odor [65,66], accurate human identity recognition can be realized. In 2020, Budowle et al. [67] created an HDID for the identity recognition of human remains and missing persons. ...
In recent years, Wi-Fi sensing technology has become an emerging research direction of human-computer interaction due to its advantages of low cost, contactless, illumination insensitivity, and privacy preservation. At present, Wi-Fi sensing research has been expanded from target location to action recognition and identity recognition, among others. This paper summarizes and analyzes the research of Wi-Fi sensing technology in human identity recognition. Firstly, we overview the history of Wi-Fi sensing technology, compare it with traditional identity-recognition technologies and other wireless sensing technologies, and highlight its advantages for identity recognition. Secondly, we introduce the steps of the Wi-Fi sensing process in detail, including data acquisition, data pre-processing, feature extraction, and identity classification. After that, we review state-of-the-art approaches using Wi-Fi sensing for single-and multi-target identity recognition. In particular, three kinds of approaches (pattern-based, model-based, and deep learning-based) for single-target identity recognition and two kinds of approaches (direct recognition and separated recognition) for multi-target identity recognition are introduced and analyzed. Finally, future research directions are discussed, which include transfer learning, improved multi-target recognition, and unified dataset construction.
The objective of this review is to summarize numerous studies on the use of the random amplified polymorphic DNA (RAPD) technique on rice, corn, wheat, sorghum, barley, rye, and oats to examine its feasibility and alidity for assessment of genetic variation, population genetics, mapping, linkage and marker assisted selection, phylogenetic analysis, and the detection of somaclonal variation. Also we discuss the advantages and limitations of RAPD. Molecular markers have entered the scene of genetic improvement in different fields of agricultural research. The simplicity of the RAPD technique made it ideal for genetic mapping, plant and animal breeding programs, and DNA fingerprinting, with particular utility in the field of population genetics.
The recent explosion of interest in DNA-based tools for species identification has prompted widespread speculation on the
future availability of inexpensive, rapid, and accurate means of identifying specimens and assessing biodiversity. One applied
field that may benefit dramatically from the development of such technologies is the detection, identification, and monitoring
of invasive species. Recent studies have demonstrated the feasibility of DNA-based tools for such important tasks as confirmation
of specimen identity and targeted screening for known or anticipated invaders. However, significant technological hurdles
must be overcome before more ambitious applications, including estimation of propagule pressure and comprehensive surveys
of complex environmental samples, are to be realized. Here we review existing methods, examine the technical difficulties
associated with development of more sophisticated tools, and consider the potential utility of these DNA-based technologies
for various applications relevant to invasive species monitoring.
A convenient DNA-based identification system is described for testing the species origin of meat samples. Probes are generated
by PCR with primers binding to species-specific satellite DNA and hybridized to DNA purified from meat. This method is more
robust and versatile than methods based on oligonucleotide hybridization. With the exception of a slight cross-reaction of
mutton and beef, each probe only recognized the species from which it was derived. Purifying the DNA with a DNA-binding resin
improved the sensitivity. Admixtures of 0.1–0.5% can be detected in raw meat and 0.5–5% in autoclaved meat samples. The method
can be adapted to detect any eukaryotic species for which species-specific DNA sequences are available. This method has proven
its value in the routine inspection of meat samples by revealing more cases of deliberate or accidental species substitution
and admixture than conventional techniques.
Single-nucleotide polymorphisms (SNPs) are the most abundant type of human genetic variation. These variable sites are present at high density in the genome, making them powerful tools for mapping and diagnosing disease-related alleles. We have developed a sensitive and rapid flow cytometry-based assay for the multiplexed analysis of SNPs based on polymerase-mediated primer extension, or minisequencing, using microspheres as solid supports. The new method involves subnanomolar concentrations of sample in small volumes (∼10 μl) which can be analyzed at rates of one sample per minute or faster, without a wash step. Further, genomic analysis using multiplexing microsphere arrays (GAMMArrays), enables the simultaneous analysis of dozens, and potentially hundreds of SNPs per sample. We have tested the new method by genotyping the Glu69 variant from the HLA DPB1 locus, a SNP associated with chronic beryllium disease, as well as HLA DPA1 alleles using the multiplexed method. The results demonstrate the sensitivity and accuracy of flow cytometry-based minisequencing, a powerful new tool for genome- and global-scale SNP analysis.
Bicyclic nucleoside analogues with a fixed N-type conformation, 2′-O,4′-C-methyleneuridine and -cytidine, were incorporated into oligonucleotides, and the binding efficiency of the modified oligonucleotides to the complementary DNA and RNA as well as the CD spectra of the modified DNA-DNA and modified DNA-RNA duplexes were studied.
Recent trends and challenges in the electrochemical methods for the detection of DNA hybridization are reviewed. Electrochemistry has superior properties over the other existing measurement systems, because electrochemical biosensors can provide rapid, simple and low-cost on-field detection. Electrochemical measurement protocols are also suitable for mass fabrication of miniaturized devices. Electrochemical detection of hybridization is mainly based on the differences in the electrochemical behaviour of the labels towards the hybridization reaction on the electrode surface or in the solution. Basic criteria for electrochemical DNA biosensor technology, and already commercialized products, are also introduced. Future prospects towards PCR-free DNA chips are discussed.
Amplified fragment length polymorphism (AFLP) is a novel molecular fingerprinting technique that can be applied to DNAs of
any source or complexity. Total genomic DNA is digested using two restriction enzymes. Double-stranded nucleotide adapters
are ligated to the DNA fragments to serve as primer binding sites for PCR amplification. Primers complementary to the adapter
and restriction site sequence, with additional nucleotides at the 3′-end, are used as selective agents to amplify a subset
of ligated fragments. Polymorphisms are identified by the presence or absence of DNA fragments following analysis on polyacrylamide
gels. This technique has been extensively used with plant DNA for the development of high-resolution genetic maps and for
the positional cloning of genes of interest. However, its application is rapidly expanding in bacteria and higher eukaryotes
for determining genetic relationships and for epidemiological typing. This review describes the AFLP procedure, and recent,
novel applications in the molecular fingerprinting of DNA from both eukaryotic and prokaryotic organisms.