Invasive alien fish species have become a silent treat towards the ecosystem especially the native fish population in Malaysia. There has been a need to develop rapid identification methods that can aid management teams in identifying fish species that are not native to our ecosystem. Current visual identification methods are highly tedious and require time, delaying action towards curbing the invasion. The LAMP assay successfully identified six popular invasive fish species in Malaysia. None of the LAMP assays showed false positives and the Limit of Detection of the LAMP primers were highly sensitive and could detect DNA samples up to 1 × 10-15 ng/μl. The LAMP primers designed were highly specific to the target species and did not amplify non target species. DNA sequencing was done to ensure the accuracy of LAMP assay results. This study demonstrates that LAMP is a suitable tool in species identification efforts of invasive fish species in Malaysia.
Gelatin is widely used in pharmaceuticals as a protective coating, such as soft and hard capsule shells. However, the animal source of gelatin is a sensitive issue because certain gelatins such as porcine and bovine gelatins are not welcome in Halal, Kosher and Hindus' consumer goods. Recently, we have documented DNA barcoding and multiplex PCR platforms for discriminating porcine, bovine and fish gelatins in various fish and confectionary products; but those assays were not self-authenticating and also not tested in highly refined pharmaceutical products. To address this knowledge gap, here we report a self-authenticating multiplex PCR-restriction fragment length polymorphism (RFLP) assay to identify animal sources of various gelatin in pharmaceutical capsules. Three different restriction enzymes, BsaAI, Hpy188I and BcoDI were used to yield distinctive RFLP patterns for gelatin-based bovine (26, 94 bp), fish (97, 198 bp) and porcine (17, 70 bp) DNA in control experiments. The specificity was cross-tested against 16 non-target species and the optimised assay was used to screen gelatin sources in 30 halal-branded pharmaceuticals capsule shells. Bovine and porcine DNA was found in 27 and 3 of the 30 different capsules products. The assay was suitable for detecting 0.1 to 0.01 ng total DNA extracted from pure and mixed gelatins. The study might be useful to authenticate and monitor halal, kosher, vegetarian and Hindu compliant pharmaceuticals, foods and cosmetics.
Food fraud is a global problem raising increased concerns during the past decades and food authenticity is now a burning issue. Beef, buffalo, chicken, duck, goat, sheep, and pork are heavily consumed meats bearing nutritional, economic and cultural/religious importance and are often found to be adulterated in raw and processed states. To authenticate these species, we developed and validated a highly specific multiplex (heptaplex) PCR assay targeting short length amplicons (73-263 bp) using seven pairs of species-specific primer sets targeting mitochondrial cytochrome b (cytb) and NADH dehydrogenase subunit 5 (ND5) genes. Specificity checking (in silico and in vitro) against 25 non-target species revealed no cross-species amplification. The developed multiplex assay was validated with various adulterated and heat-treated (boiled, microwaved and autoclaved) meatball products and were found to show high sensitivity and stability under all processing conditions. The assay was sensitive enough to detect 0.01-0.005 ng of DNA from raw meat and 0.5% (w/w) adulterated meat in mixed matrices. A market survey revealed mislabelling of 95% beef and 15% chicken products while pork products were found pure. Given some advantageous features including short sizes of amplicons, exceptional stability and superior sensitivity, the developed assay could be conveniently used for discriminatory detection of target species with a variety of raw meat as well as processed meat products undergoing extreme processing treatments.
Cattle, buffalo, and porcine materials are widely adulterated, and their quantification might safeguard health, religious, economic, and social sanctity. Recently, conventional polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) assays have been documented but they are just suitable for identification, cannot quantify adulterations. We described here a quantitative tetraplex real-time PCR assay with TaqMan Probes to quantify contributions from cattle, buffalo, and porcine materials simultaneously. Amplicon-sizes were very short (106-, 90-, and 146-bp for cattle, buffalo, and porcine) because longer targets could be broken down, bringing serious ambiguity in molecular diagnostics. False negative detection was eliminated through an endogenous control (141-bp site of eukaryotic 18S rRNA). Analysis of 27 frankfurters and 27 meatballs reflected 84-115% target recovery at 0.1-10% adulterations. Finally, a test of 36 commercial products revealed 71% beef frankfurters, 100% meatballs, and 85% burgers contained buffalo adulteration, but no porcine was found in beef products.
Rabbit meat is receiving increasing attention because it contains a high level of proteins with relatively little fat. On the other hand, squirrel meat is served in upper-class meals in certain countries, so is sold at higher prices. The other side of the coin is rat meat, which has family ties with rabbit and squirrel but poses substantial threats to public health because it is a potential carrier of several zoonotic organisms. Recently, rat meat was mislabelled and sold as lamb after chemical modification. Thus, the chances of rabbit and squirrel meat substitution by rat meat cannot be ruled out. For the first time, a multiplex PCR assay was developed in Malaysia for the discriminatory identification of rat, rabbit and squirrel in the food chain. Rabbit (123 bp), rat (108 bp) and squirrel (243 bp) targets were amplified from ATP6 and cytb genes, along with a eukaryotic internal control (141bp). The products were sequenced and cross-tested against 22 species. A total of 81 reference samples and 72 meatball specimens were screened to validate the assay. Analyte stability was evaluated through boiling, autoclaving and micro-oven cooking. The tested lower limits of detection were 0.01 ng DNA for pure meat and 0.1% for meatballs.
Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus are the most significant aquatic pathogens of the genera Vibrio, account for most Vibrio-associated outbreaks worldwide. Rapid identification of these pathogens is of great importance for disease surveillance, outbreak investigations and food safety maintenance. Traditional culture dependent methods are time-consuming and labor-intensive whereas culture-independent polymerase chain reaction (PCR) based assays are reliable, consistent, rapid and reproducible. This review covers the recent development and applications of PCR based techniques, which have accelerated advances in the analysis of nucleic acids to identify three major pathogenic vibrios. Emphasis has been given to analytical approaches as well as advantages and limits of the available methods. Overall, this review article possesses the substantial merit to be used as a reference guide for the researchers to develop improved PCR based techniques for the differential detection and quantification of Vibrio species.
Meat and meat products are widely consumed worldwide as a source of high-quality proteins, essential amino acids, vitamins, and necessary minerals. The acceptability of Halal and Kosher meat products relies not only on the species origin but also on the manner of slaughtering of animals. Both Islam and Judaism have their own dietary laws in their holy books regarding acceptance and forbiddance of dietary items particularly meat and meat products. They also include many strictures to follow for ritual cleanliness of foods. Since the authenticity of Halal and Kosher food created increased concerns among consumers, the integrity of Halal and Kosher meat and meat products must be assured so that consumers can be accomplished with the originality of products. There is an increasing demand for reliable and sensitive techniques for the authentication of various Halal and Kosher meat products. This up-to-date review intends to provide an updated and extensive overview critically on the present situation, progress, and challenges of analytical techniques to authenticate animal species in meat items. It also addresses slaughtering procedure with brief discussion on Halal and Kosher laws with a view to creating consumer awareness against fraudulent practices. The available methods are schematically presented, and their salient features are comparatively elucidated in tables. Potential future technologies are predicted, and probable challenges are summarized. Overall, the present review article possesses substantial merits to be served as a reference guide in the field of academia and industry for the preparation/processing and identification of Halal and Kosher meat and meat products as well as may act as a platform to help improve existing authentication methods.
Species substitution, the use of a low value fish in place of a high value fish, is the biggest problem in international trade and the leading cause of fraud in the fisheries arena sector. Current DNA barcoding systems have partly solved this problem but also failed in many instances to amplify PCR targets from highly processed products because of the degradation of a longer barcode marker (~650bp). In the present study, a novel mini barcode marker (295bp) was developed to discriminate fish species in raw and processed states forms. The barcode primers were cross-tested against 33 fish species and 15 other animal species and found to be universal for all the tested fish varieties. When 20 commercial fish products of five different categories were screened, all commercial fish sample yielded positive bands for the novel fish barcode. PCR product was sequenced to retrieve the species IDs that reflected 55% (11/20) of Malaysian fish products were mislabeled.
The demand for crocodile meat is quickly growing because of its exotic and organoleptic appeal and also the low content of cholesterol and lipids. Moreover, crocodile oil and blood have been used in alternative medicines for treating asthma and several other ailments since ancient times. Furthermore, crocodile hides have great demand in leather industries. All of these have collectively contributed to the extensive hunting, illegal trading and consequent decline of crocodiles in most parts of the world. To keep space with the growing demands, some crocodile species such as Crocodylus porosus have been raised in farms and its commercial trades have been legalised. However, demand for wild crocodiles in foods and medicines has continued in high gear. Recently, several DNA-based methods have been proposed for crocodile detection, but those assays are based on single gene and longer-sized amplicon targets that break down during extensive processing. To address this gap, here we developed and validated a highly stable double gene targeted multiplex PCR assay for the identification of C. porosus materials in commercial products. The assay involved two short sites from C. porosus atp6 (77 bp) and cytb (127 bp) genes and a universal internal control (99 bp) for eukaryotes. The PCR primers were cross-tested against 18 species and validated under pure and mixed matrices under extensive boiling, autoclaving and microwave cooking conditions. Finally, it was used to identify five crocodile-based commercial products. The lower limits of detection for atp6 and cytb genes were 0.001 ng and 0.01 ng DNA, respectively, in pure meat and 1% under mixed matrices. Some inherent features, such as 77-127 bp amplicon sizes, exceptional stability and superior sensitivity, suggested the assay could be used for the identification of C. porosus in any forensic specimen.
The Southeast Asian box turtle, Cuora amboinensis, is an ecologically important endangered species which needs an onsite monitoring device to protect it from extinction. An electrochemical DNA biosensor was developed to detect the C. amboinensis mitochondrial cytochrome b gene based on an in silico designed probe using bioinformatics tools, and it was also validated in wet-lab experiments. As a detection platform, a screen-printed carbon electrode (SPCE) enhanced with a nanocomposite containing gold nanoparticles and graphene was used. The morphology of the nanoparticles was analysed by field-emission scanning electron microscopy and structural characteristics were analysed by using energy-dispersive X-ray, UV-vis, and Fourier-transform infrared spectroscopy. The electrochemical characteristics of the modified electrodes were studied by cyclic voltammetry, differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy. The thiol-modified synthetic DNA probe was immobilised on modified SPCEs to facilitate hybridisation with the reverse complementary DNA. The turtle DNA was distinguished based on hybridisation-induced electrochemical change in the presence of methylene blue compared to their mismatches, noncomplementary, and nontarget species DNA measured by DPV. The developed biosensor exhibited a selective response towards reverse complementary DNAs and was able to discriminate turtles from other species. The modified electrode displayed good linearity for reverse complementary DNAs in the range of 1 × 10-11-5 × 10-6 M with a limit of detection of 0.85 × 10-12 M. This indicates that the proposed biosensor has the potential to be applied for the detection of real turtle species.
Detection of animal materials in gelatin-based products is required to address religious and cultural concerns, because porcine and bovine gelatins are prohibited in Halal, Kosher and Hindus consumer goods. In this paper, multiplex quantitative polymerase chain reaction (qPCR) assay using TaqMan probe was developed to discriminate bovine, porcine and fish gelatin species in a single assay platform. The assay was specific to cattle, pigs and fish, having been tested against 14 non-target species. The limit of detection, under gelatin admixed conditions, was 0.005 ng/µL. Finally, a pilot survey was undertaken testing 35 Halal branded processed food and dietary items. Out of 35 samples, only two were found to be positive for porcine species. The authenticity of these two qPCR products was confirmed by DNA sequencing analysis, which showed 99-100% similarity with Sus scrofa (Wild boar) species.
Diagnostic testing to identify individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a key role in selecting appropriate treatments, saving people's lives and preventing the global pandemic of COVID-19. By testing on a massive scale, some countries could successfully contain the disease spread. Since early viral detection may provide the best approach to curb the disease outbreak, the rapid and reliable detection of coronavirus (CoV) is therefore becoming increasingly important. Nucleic acid detection methods, especially real-time reverse transcription polymerase chain reaction (RT-PCR)-based assays are considered the gold standard for COVID-19 diagnostics. Some non-PCR-based molecular methods without thermocycler operation, such as isothermal nucleic acid amplification have been proved promising. Serologic immunoassays are also available. A variety of novel and improved methods based on biosensors, Clustered-Regularly Interspaced Short Palindromic Repeats (CRISPR) technology, lateral flow assay (LFA), microarray, aptamer etc. have also been developed. Several integrated, random-access, point-of-care (POC) molecular devices are rapidly emerging for quick and accurate detection of SARS-CoV-2 that can be used in the local hospitals and clinics. This review intends to summarize the currently available detection approaches of SARS-CoV-2, highlight gaps in existing diagnostic capacity, and propose potential solutions and thus may assist clinicians and researchers develop better technologies for rapid and authentic diagnosis of CoV infection.
Mislabelling in fish products is a highly significant emerging issue in world fish trade in terms of health and economic concerns. DNA barcoding is an efficient sequencing-based tool for detecting fish species substitution but due to DNA degradation, it is in many cases difficult to amplify PCR products of the full-length barcode marker (~650 bp), especially in severely processed products. In the present study, a pair of universal primers targeting a 198 bp sequence of the mitochondrial 16s rRNA gene was designed for identification of fish species in the processed fish products commonly consumed in Malaysia. The specificity of the universal primers was tested by both in-silico studies using bioinformatics software and through cross-reaction assessment by practical PCR experiments against the DNA from 38 fish species and 22 other non-target species (animals and plants) and found to be specific for all the tested fish species. To eliminate the possibility of any false-negative detection, eukaryotic endogenous control was used during specificity evaluation. The developed primer set was validated with various heat-treated (boiled, autoclaved and microwaved) fish samples and was found to show high stability under all processing conditions. The newly developed marker successfully identified 92% of the tested commercial fish products with 96-100% sequence similarities. This study reveals a considerable degree of species mislabelling (20.8%); 5 out of 24 fish products were found to be mislabelled. The new marker developed in this work is a reliable tool to identify fish species even in highly processed products and might be useful in detecting fish species substitution thus protecting consumers' health and economic interests.
Clinical diagnostic tests should be quick, reliable, simple to perform, and affordable for diagnosis and treatment of diseases. In this regard, owing to their novel properties, biosensors have attracted the attention of scientists as well as end-users. They are efficient, stable, and relatively cheap. Biosensors have broad applications in medical diagnosis, including point-of-care (POC) monitoring, forensics, and biomedical research. The electrochemical nucleic acid (NA) biosensor, the latest invention in this field, combines the sensitivity of electroanalytical methods with the inherent bioselectivity of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The NA biosensor exploits the affinity of single-stranded DNA/RNA for its complementary strand and is used to detect complementary sequences of NA based on hybridization. After the NA component in the sensor detects the analyte, a catalytic reaction or binding event that generates an electrical signal in the transducer ensues. Since 2000, much progress has been made in this field, but there are still numerous challenges. This critical review describes the advances, challenges, and prospects of NA-based electrochemical biosensors for clinical diagnosis. It includes the basic principles, classification, sensing enhancement strategies, and applications of biosensors as well as their advantages, limitations, and future prospects, and thus it should be useful to academics as well as industry in the improvement and application of EC NA biosensors.
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.