Regular testing and systematic investigation play a vital role to ensure product safety. Until now, the existing food authentication techniques have been based on proteins, lipids, and nucleic acid-based assays. Among various deoxyribonucleic acid (DNA)-based methods, the recently developed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) based bio-sensing is an innovative and fast-expanding technology. The CRISPR/Cas-9 is known as Clustered Regularly Interspaced Short Palindromic Repeats due to the flexibility and simplicity of the CRISPR/Cas9 site-specific editing tool has been applied in many biological research areas such as Gene therapy, cell line development, discovering mechanisms of disease, and drug discovery. Nowadays, the CRISPR-Cas system has also been introduced into food authentication via detecting DNA barcodes of poultry and livestock both in processed and unprocessed food samples. This review documents various DNA based approaches, in an accessible format. Future CRISPR technologies are forecast while challenges are outlined.
The inclusion of ingredients derived from pigs in highly processed consumer products poses a significant challenge for DNA-targeted analytical enforcement, which could be overcome by using digital PCR. However, most species detection methods use digital PCR to target single-copy nuclear genes, which limits their sensitivity. In this work, we examined the performance of a nanoplate-based digital PCR method that targets multi-copy nuclear (MPRE42) and mitochondrial (Cytb) genes. Poor separation of positive and negative partitions, as well as a 'rain effect' were obtained in the porcine-specific MPRE42 assay. Among the optimization strategies examined, the inclusion of restriction enzymes slightly improved the separation of positive and negative partitions, but a more extensive 'rain effect' was observed. The high copy number of the MPRE42 amplicon is hypothesized to contribute to the saturation of the positive signal. In contrast, the porcine-specific Cytb assay achieved perfect separation of positive and negative partitions with no 'rain effect'. This assay can detect as little as 0.4 pg of pork DNA, with a sensitivity of 0.05% (w/w) in a pork-chicken mixture, proving its applicability for detecting pork in meat and meat-based products. For the MPRE42 assay, potential applications in highly degraded products such as gelatin and lard are anticipated.
The onus of proof in criminal cases is beyond any reasonable doubt, and the issue on the lack of complete internal validation data can be manipulated when it comes to justifying the validity and reliability of the X-chromosomal short tandem repeats analysis for court representation. Therefore, this research evaluated the efficiency of the optimized 60% reduced volumes for polymerase chain reaction (PCR) amplification using the Qiagen Investigator® Argus X-12 QS Kit, as well as the capillary electrophoresis (CE) sample preparation for blood samples on Flinder's Technology Associates (FTA) cards. Good-quality DNA profile (3000-12,000 RFU) from the purified blood sample on FTA card (1.2 mm) were obtained using the optimized PCR (10.0 μL of PCR reaction volume and 21 cycles) and CE (9.0 μL Hi-Di™ Formamide and 0.3 μL DNA Size Standard 550 [BTO] and 27 s injection time) conditions. The analytical and stochastic thresholds were 100 and 200 RFU, respectively. Hence, the internal validation data supported the use of the optimized 60% reduced PCR amplification reaction volume of the Qiagen Investigator® Argus X-12 QS Kit as well as the CE sample preparation for producing reliable DNA profiles that comply with the quality assurance standards for forensic DNA testing laboratories, while optimizing the analytical cost.
This study investigates the effect of DNA hairpins on the stabilization of gold nanoparticles (AuNPs) against salt-induced aggregation (SIA) in label-free colorimetric biosensors. AuNPs were incubated with DNA hairpins of varying stem lengths and toehold sequences, followed by the addition of NaCl, before being subjected to ultraviolet-visible (UV-vis) measurement. Results showed that hairpins with longer stems generally provide better stabilization of AuNPs (18-bp >14-bp >10-bp). No improvement was observed for 14- and 18-bp hairpins with a toehold beyond 8A, which may be attributed to saturated adsorption of hairpins on the gold surface. For 14-bp hairpins with an 8-mer homopolymeric toehold, we observed a stabilization trend of A > C > G > T, similar to the reported trend of ssDNA. For variants containing ≥50% adenine as terminal bases, introducing cytosine or guanine as preceding bases could also result in strong stabilization. As the proportion of adenine decreases, variants with guanine or thymine provide less protection against SIA, especially for guanine-rich hairpins (≥6G) that could form G-quadruplexes. Such findings could serve as guidelines for researchers to design suitable DNA hairpins for label-free AuNP-based biosensors.
Tissue specimen quality assurance is a major issue of precision medicine for rare cancers. However, the laboratory standards and quality of pathological specimens prepared in Asian hospitals remain unknown. To understand the methods in Southeast Asian oncology hospitals and to clarify how pre-analytics affect the quality of formalin-fixed paraffin-embedded (FFPE) specimens, a questionnaire surveying pre-analytical procedures (Part I) was administered, quality assessment of immunohistochemistry (IHC) staining and DNA/RNA extracted from the representative FFPE specimens from each hospital (Part II) was conducted, and the quality of DNA/RNA extracted from FFPE of rare-cancer patients for genomic sequencing (Part III) was examined. Quality measurements for DNA/RNA included ΔΔCt, DV200, and cDNA yield. Six major cancer hospitals from Malaysia, Philippines, and Vietnam participated. One hospital showed unacceptable quality for the DNA/RNA assessment, but improved by revising laboratory procedures. Only 57% (n = 73) of the 128 rare-cancer patients' specimens met both DNA and RNA quality criteria for next-generation sequencing. Median DV200 was 80.7% and 64.3% for qualified and failed RNA, respectively. Median ΔΔCt was 1.25 for qualified and 4.89 for failed DNA. Longer storage period was significantly associated with poor DNA (fail to qualify ratio = 1579:321 days, p
A fluorescence biosensor has been developed based on hybridisation chain reaction (HCR) amplification coupled with silver nanoclusters (AgNCs) for nucleic acid detection. The fluorescence was activated via end-to-end transfer of dark AgNCs caged within a DNA template to another DNA sequence that could enhance their red fluorescence emission at 611 nm. Such cluster-transfer approach allows us to introduce fluorogenic AgNCs as external signal transducers, thereby enabling HCR to perform in a predictable manner. The resulted HCR-AgNC biosensor was able to detect target DNA with a detection limit of 3.35 fM, and distinguish the DNA target from single-base mismatch sequences. Moreover, the bright red fluorescence emission was detectable with the naked eye, with concentration of target DNA down to 1 pM. The biosensor also performed well in human serum samples with good recovery. Overall, our cluster-transfer approach provides a good alternative to construct HCR-AgNC assay with less risk of circuit leakage and produce AgNCs in a controllable manner.
Point-of-care diagnostic methods for animal species determination are critical for rapid, simple, and accurate enforcement of food labelling. PCR is the most common method for species identification. However, the requirement of using a thermal cycler created drawbacks for the PCR application, particularly in low-resource settings. Hence, in this study, a method for porcine DNA detection using recombinase polymerase amplification (RPA), coupled with nucleic acid lateral flow immunoassay (NALFIA), was developed. Porcine-specific primers targeting pig (Sus scrofa) cytochrome b gene fragments specifically amplify a 197 bp fragment of the mitochondrial gene as being visualized by 2% agarose gel and PCRD NALFIA. The reaction temperature and time were 39 °C and 20 min, respectively. Herein, the specificity of the primers to porcine was confirmed after being assayed against six animal species, namely cow, goat, chicken, duck, dog, and rabbit. The porcine-specific RPA assay shows a high limit of detection of 0.01 ng/µL pork DNA. Based on the preliminary performance data obtained from this study, the potential of this method as a rapid and sensitive tool for porcine DNA detection in meat-based products is foreseen.
The realization that ancient biomolecules are preserved in "fossil" samples has revolutionized archaeological science. Protein sequences survive longer than DNA, but their phylogenetic resolution is inferior; therefore, careful assessment of the research questions is required. Here, we show the potential of ancient proteins preserved in Pleistocene eggshell in addressing a longstanding controversy in human and animal evolution: the identity of the extinct bird that laid large eggs which were exploited by Australia's indigenous people. The eggs had been originally attributed to the iconic extinct flightless bird Genyornis newtoni (†Dromornithidae, Galloanseres) and were subsequently dated to before 50 ± 5 ka by Miller et al. [Nat. Commun. 7, 10496 (2016)]. This was taken to represent the likely extinction date for this endemic megafaunal species and thus implied a role of humans in its demise. A contrasting hypothesis, according to which the eggs were laid by a large mound-builder megapode (Megapodiidae, Galliformes), would therefore acquit humans of their responsibility in the extinction of Genyornis. Ancient protein sequences were reconstructed and used to assess the evolutionary proximity of the undetermined eggshell to extant birds, rejecting the megapode hypothesis. Authentic ancient DNA could not be confirmed from these highly degraded samples, but morphometric data also support the attribution of the eggshell to Genyornis. When used in triangulation to address well-defined hypotheses, paleoproteomics is a powerful tool for reconstructing the evolutionary history in ancient samples. In addition to the clarification of phylogenetic placement, these data provide a more nuanced understanding of the modes of interactions between humans and their environment.
miRNA (miR)-155 is a potential biomarker for breast cancers. We aimed at developing a nanosensor for miR-155 detection by integrating hybridization chain reaction (HCR) and silver nanoclusters (AgNCs). HCR serves as an enzyme-free and isothermal amplification method, whereas AgNCs provide a built-in fluorogenic detection probe that could simplify the downstream analysis. The two components were integrated by adding a nucleation sequence of AgNCs to the hairpin of HCR. The working principle was based on the influence of microenvironment towards the hosted AgNCs, whereby unfolding of hairpin upon HCR has manipulated the distance between the hosted AgNCs and cytosine-rich toehold region of hairpin. As such, the dominant emission of AgNCs changed from red to yellow in the absence and presence of miR-155, enabling a ratiometric measurement of miR with high sensitivity. The limit of detection (LOD) of our HCR-AgNCs nanosensor is 1.13 fM in buffered solution. We have also tested the assay in diluted serum samples, with comparable LOD of 1.58 fM obtained. This shows the great promise of our HCR-AgNCs nanosensor for clinical application.
We report a highly sensitive and selective multiplex assay by empowering an electrochemical DNA sensor with isothermal rolling circle amplification. The assay could simultaneously detect and discriminate three common entero-pathogens in a single reaction, with femtomolar sensitivity. It is useful for field- or resource-limited settings.
The effect of DNA topology on transfection efficiency of mammalian cells has been widely tested on plasmids smaller than 10 kb, but little is known for larger DNA vectors carrying intact genomic DNA containing introns, exons, and regulatory regions. Here, we demonstrate that circular BACs transfect more efficiently than covalently closed linear BACs. We found up to 3.1- and 8.9- fold higher eGFP expression from circular 11 kb and 100 kb BACs, respectively, compared to linear BACs. These findings provide insights for improved vector development for gene delivery and expression studies of large intact transgenes in mammalian cells.
Authentication, detection and quantification of ingredients, and adulterants in food, meat, and meat products are of high importance these days. The conventional techniques for the detection of meat species based on lipid, protein and DNA biomarkers are facing challenges due to the poor selectivity, sensitivity and unsuitability for processed food products or complex food matrices. On the other hand, DNA based molecular techniques and nanoparticle based DNA biosensing strategies are gathering huge attention from the scientific communities, researchers and are considered as one of the best alternatives to the conventional strategies. Though nucleic acid based molecular techniques such as PCR and DNA sequencing are getting greater successes in species detection, they are still facing problems from its point-of-care applications. In this context, nanoparticle based DNA biosensors have gathered successes in some extent but not to a satisfactory stage to mark with. In recent years, many articles have been published in the area of progressive nucleic acid-based technologies, however there are very few review articles on DNA nanobiosensors in food science and technology. In this review, we present the fundamentals of DNA based molecular techniques such as PCR, DNA sequencing and their applications in food science. Moreover, the in-depth discussions of different DNA biosensing strategies or more specifically electrochemical and optical DNA nanobiosensors are presented. In addition, the significance of DNA nanobiosensors over other advanced detection technologies is discussed, focusing on the deficiencies, advantages as well as current challenges to ameliorate with the direction for future development.
The Merbok Estuary comprises one of the largest remaining mangrove forests in Peninsular Malaysia. Its value is significant as it provides important services to local and global communities. It also offers a unique opportunity to study the structure and functioning of mangrove ecosystems. However, its biodiversity is still partially inventoried, limiting its research value. A recent checklist based on morphological examination, reported 138 fish species residing, frequenting or subject to entering the Merbok Estuary. In this work, we reassessed the fish diversity of the Merbok Estuary by DNA barcoding 350 specimens assignable to 134 species initially identified based on morphology. Our results consistently revealed the presence of 139 Molecular Operational Taxonomic Units (MOTUs). 123 of them are congruent with morphology-based species delimitation (one species = one MOTU). In two cases, two morphological species share the same MOTU (two species = one MOTU), while we unveiled cryptic diversity (i.e. COI-based genetic variability > 2%) within seven other species (one species = two MOTUs), calling for further taxonomic investigations. This study provides a comprehensive core-list of fish taxa in Merbok Estuary, demonstrating the advantages of combining morphological and molecular evidence to describe diverse but still poorly studied tropical fish communities. It also delivers a large DNA reference collection for brackish fishes occurring in this region which will facilitate further biodiversity-oriented research studies and management activities.
Atherosclerosis represents one of the major causes of death globally. The high mortality rates and limitations of current therapeutic modalities have urged researchers to explore potential alternative therapies. The clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) system is commonly deployed for investigating the genetic aspects of Atherosclerosis. Besides, advances in CRISPR/Cas system has led to extensive options for researchers to study the pathogenesis of this disease. The recent discovery of Cas9 variants, such as dCas9, Cas9n, and xCas9 have been established for various applications, including single base editing, regulation of gene expression, live-cell imaging, epigenetic modification, and genome landscaping. Meanwhile, other Cas proteins, such as Cas12 and Cas13, are gaining popularity for their applications in nucleic acid detection and single-base DNA/RNA modifications. To date, many studies have utilized the CRISPR/Cas9 system to generate disease models of atherosclerosis and identify potential molecular targets that are associated with atherosclerosis. These studies provided proof-of-concept evidence which have established the feasibility of implementing the CRISPR/Cas system in correcting disease-causing alleles. The CRISPR/Cas system holds great potential to be developed as a targeted treatment for patients who are suffering from atherosclerosis. This review highlights the advances in CRISPR/Cas systems and their applications in establishing pathogenetic and therapeutic role of specific genes in atherosclerosis.
The application of metabarcoding to environmental and invertebrate-derived DNA (eDNA and iDNA) is a new and increasingly applied method for monitoring biodiversity across a diverse range of habitats. This approach is particularly promising for sampling in the biodiverse humid tropics, where rapid land-use change for agriculture means there is a growing need to understand the conservation value of the remaining mosaic and degraded landscapes. Here we use iDNA from blood-feeding leeches (Haemadipsa picta) to assess differences in mammalian diversity across a gradient of forest degradation in Sabah, Malaysian Borneo. We screened 557 individual leeches for mammal DNA by targeting fragments of the 16S rRNA gene and detected 14 mammalian genera. We recorded lower mammal diversity in the most heavily degraded forest compared to higher quality twice logged forest. Although the accumulation curves of diversity estimates were comparable across these habitat types, diversity was higher in twice logged forest, with more taxa of conservation concern. In addition, our analysis revealed differences between the community recorded in the heavily logged forest and that of the twice logged forest. By revealing differences in mammal diversity across a human-modified tropical landscape, our study demonstrates the value of iDNA as a noninvasive biomonitoring approach in conservation assessments.
One of the current issues with thyroid tumor is early diagnosis as it makes the higher possibility of curing. This research was focused to detect and quantify the level of specific target sequence complementation of miR-222 with capture DNA sequence on interdigitated electrode (IDE) sensor. The aluminum electrode with the gap and finger sizes of 10 µm was fabricated on silicon wafer, further the surface was amine-functionalized for accommodating carboxylated-DNA probe. With DNA-target RNA complementation, the detection limit was attained to be 1 fM as estimated by a linear regression analysis [y = 1.5325x - 2.1171 R² = 0.9065] and the sensitivity was at the similar level. Current responses were higher by increasing the target RNA sequence concentrations. Control experiments with mismatched/noncomplementary sequences were failed to complement the capture DNA sequence immobilized on IDE, indicating the specific target validation. This research helps diagnosing and identifying the progression with thyroid tumor and miRNA being a potential "marker" in atypia diagnosis.
With the development of new metagenomic techniques, the microbial community structure of common bed bugs, Cimex lectularius, is well-studied, while information regarding the constituents of the bacterial communities associated with tropical bed bugs, Cimex hemipterus, is lacking. In this study, the bacteria communities in the blood-fed and starved tropical bed bugs were analysed and characterized by amplifying the v3-v4 hypervariable region of the 16S rRNA gene region, followed by MiSeq Illumina sequencing. Across all samples, Proteobacteria made up more than 99% of the microbial community. An alpha-proteobacterium Wolbachia and gamma-proteobacterium, including Dickeya chrysanthemi and Pseudomonas, were the dominant OTUs at the genus level. Although the dominant OTUs of bacterial communities of blood-fed and starved bed bugs were the same, bacterial genera present in lower numbers were varied. The bacteria load in starved bed bugs was also higher than blood-fed bed bugs.
Plasmodium knowlesi is the main cause of malaria in Sarawak, where studies on vectors of P. knowlesi have been conducted in only two districts. Anopheles balabacensis and An. donaldi were incriminated as vectors in Lawas and An. latens in Kapit. We studied a third location in Sarawak, Betong, where of 2169 mosquitoes collected over 36 days using human-landing catches, 169 (7.8%) were Anopheles spp. PCR and phylogenetic analyses identified P. knowlesi and/or P. cynomolgi, P. fieldi, P. inui, P. coatneyi and possibly novel Plasmodium spp. in salivary glands of An. latens and An. introlatus from the Leucosphyrus Group and in An. collessi and An. roperi from the Umbrosus Group. Phylogenetic analyses of cytochrome oxidase subunit I sequences indicated three P. knowlesi-positive An. introlatus had been misidentified morphologically as An. latens, while An. collessi and An. roperi could not be delineated using the region sequenced. Almost all vectors from the Leucosphyrus Group were biting after 1800 h but those belonging to the Umbrosus Group were also biting between 0700 and 1100 h. Our study incriminated new vectors of knowlesi malaria in Sarawak and underscores the importance of including entomological studies during the daytime to obtain a comprehensive understanding of the transmission dynamics of malaria.
Laboratory pedagogy is moving away from step-by-step instructions and toward inquiry-based learning, but only now developing methods for integrating inquiry-based writing (IBW) practices into the laboratory course. Based on an earlier proposal (Science 2011;332:919), we designed and implemented an IBW sequence in a university bioinformatics course. We automatically generated unique, double-blinded, biologically plausible DNA sequences for each student. After guided instruction, students investigated sequences independently and responded through IBW writing assignments. IBW assignments were structured as condensed versions of a scientific research article, and because the sequences were double blinded, they were also assessed as authentic science and evaluated on clarity and persuasiveness. We piloted the approach in a seven-day workshop (35 students) at Perdana University in Malaysia. We observed dramatically improved student engagement and indirect evidence of improved learning outcomes over a similar workshop without IBW. Based on student feedback, initial discomfort with the writing component abated in favor of an overall positive response and increasing comfort with the high demands of student writing. Similarly, encouraging results were found in a semester length undergraduate module at the National University of Singapore (155 students).
Infectious Bronchitis (IB) is an economically important avian disease that considerably threatens the global poultry industry. This is partly, as a result of its negative consequences on egg production, weight gain as well as mortality rate.The disease is caused by a constantly evolving avian infectious bronchitis virus whose isolates are classified into several serotypes and genotypes that demonstrate little or no cross protection. In order to curb the menace of the disease therefore, broad based vaccines are urgently needed. The aim of this study was to develop a recombinant DNA vaccine candidate for improved protection of avian infectious bronchitis in poultry. Using bioinformatics and molecular cloning procedures, sets of monovalent and bivalent DNA vaccine constructs were developed based on the S1 glycoprotein from classical and variants IBV strains namely, M41 and CR88 respectively. The candidate vaccine was then encapsulated with a chitosan and saponin formulated nanoparticle for enhanced immunogenicity and protective capacity. RT-PCR assay and IFAT were used to confirm the transcriptional and translational expression of the encoded proteins respectively, while ELISA and Flow-cytometry were used to evaluate the immunogenicity of the candidate vaccine following immunization of various SPF chicken groups (A-F). Furthermore, histopathological changes and virus shedding were determined by quantitative realtime PCR assay and lesion scoring procedure respectively following challenge of various subgroups with respective wild-type IBV viruses. Results obtained from this study showed that, groups vaccinated with a bivalent DNA vaccine construct (pBudCR88-S1/M41-S1) had a significant increase in anti-IBV antibodies, CD3+ and CD8+ T-cells responses as compared to non-vaccinated groups. Likewise, the bivalent vaccine candidate significantly decreased the oropharyngeal and cloacal virus shedding (p < 0.05) compared to non-vaccinated control. Chickens immunized with the bivalent vaccine also exhibited milder clinical signs as well as low tracheal and kidney lesion scores following virus challenge when compared to control groups. Collectively, the present study demonstrated that bivalent DNA vaccine co-expressing dual S1 glycoprotein induced strong immune responses capable of protecting chickens against infection with both M41 and CR88 IBV strains. Moreso, it was evident that encapsulation of the vaccine with chitosan-saponin nanoparticle further enhanced immune responses and abrogates the need for multiple booster administration of vaccine. Therefore, the bivalent DNA vaccine could serve as efficient and effective alternative strategy for the control of IB in poultry.