Advancements in Detection Approaches of Severe Acute Respiratory Syndrome Coronavirus 2.

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.

Current trends in polymerase chain reaction based detection of three major human pathogenic vibrios.

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.

Authentication of Halal and Kosher meat and meat products: Analytical approaches, current progresses and future prospects.

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.

Short targeting multiplex PCR assay to detect and discriminate beef, buffalo, chicken, duck, goat, sheep and pork DNA in food products.

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.

Heptaplex Polymerase Chain Reaction Assay for the Simultaneous Detection of Beef, Buffalo, Chicken, Cat, Dog, Pork, and Fish in Raw and Heat-Treated Food Products.

Species authentication of meat and fish products is crucial to safeguard public health, economic investment, and religious sanctity. We developed a heptaplex polymerase chain reaction assay targeting short amplicon length (73-198 bp) for the simultaneous detection and differentiation of cow, buffalo, chicken, cat, dog, pig, and fish species in raw and processed food using species-specific primers targeting mitochondrial cytb, ND5, and 16s rRNA genes. Assay validation of adulterated and various heat-treated meatball matrices showed excellent stability and sensitivity under all processing conditions. The detection limit was 0.01-0.001 ng of DNA under pure states and 0.5% meat in meatball products. Buffalo was detected in 86.7% (13 out of 15) of tested commercial beef products, while chicken, pork, and fish products were found to be pure. The developed assay was efficient enough to detect target species simultaneously, even in highly degraded and processed food products at reduced time.

Universal mitochondrial 16s rRNA biomarker for mini-barcode to identify fish species in Malaysian fish products.

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.