Targeting double genes in multiplex PCR for discriminating bovine, buffalo and porcine materials in food chain

Beef, buffalo and pork are the major meat of economic, religious and health concern. Current methods to authenticate these materials in food chain are based on mainly single gene targets which are susceptible to break down by food processing treatments. We, for the first time, described here a double gene targeting short-amplicon length multiplex polymerase chain reaction assay for discriminating bovine, buffalo and porcine materials in a single assay platform. The advantage of the assay is evidenced in terms of fidelity, cost and time since it is highly unlikely that two different targets would be missing even in a decomposed specimen. Detection of multiple targets in a single assay definitely saves analytical cost and time. Mitochondrial cytochrome b (cytb) and ND5 genes were targeted and six different targets (length: 90–146 bp), two for each of cow (120 and 106bp), buffalo (90 and 138bp) and pig (73 and 146bp), were amplified from raw, boiled, autoclaved and microwaved cooked meat under pure and mixed matrices. The detection limit was 0.02 ng DNA under pure states and 0.1% meat in binary mixtures and meatball products. Screening of Malaysian meatball products revealed all beef products were buffalo positive in which 35% were totally replaced. In contrast, all pork products were found uncontaminated from beef and buffalo

Development and evaluation of double genes targeted multiplex PCR assays for the determination of bovine, buffalo and porcine materials in food products / M. A. Motalib Hossain

Bovine, buffalo and porcine materials in food products are sensitive to religions and a big threat to health and fair economic practices. Current methods to authenticate these animal materials in food chain are based on mainly single gene target which are generally longer in length and thus breakdown during food processing treatments. For the first time, here I targeted double gene sites in short-amplicon length multiplex polymerase chain reaction (mPCR) assays for the detection and differentiation of bovine, buffalo and porcine materials in food chain. Multiple targets detection in single assay saves analytical cost and time. Both the conventional and real-time PCR platforms were developed and authentic target detection was confirmed through sequencing and Restriction Fragment Length Polymorphism analysis. Mitochondrial cytochrome b (cytb) and NADH dehydrogenase subunit 5 (ND5) genes were targeted and six different targets (length: 73-146 bp), two for each of cow (121 and 106 bp), buffalo (90 and 138 bp) and pig (73 and 146 bp), were amplified from raw, boiled, autoclaved and microwaved cooked meat under pure and mixed matrices. The specificity of the PCR assays were checked against three targets and 25 non-target species. Specific PCR products were found only from beef, buffalo, and pork that were targeted in this assay. To eliminate the possibility of any false-negative detection, eukaryotic endogenous control was used for specificity testing. The detection limit was 0.01 ng DNA for tetraplex and 0.02 ng DNA for hexaplex under pure states and 0.1% target meat in mixed and commercial matrices. Complete sequence matching was found for five the PCR products but 98.5% for buffalo ND5 gene. The PCR products were digested by four restriction enzymes, namely AluI, EciI, FatI and CviKI-1 and clear restriction fingerprints were obtained. The developed methods were used for the screening of bovine, buffalo and porcine materials in various commercial meat curries and processed foods, namely, meatballs and frankfurters. Survey results revealed about 80% of beef meatballs were adulterated with buffalo and about 20% of beef products were totally replaced with buffalo. Moreover, the analysis of 20 beef frankfurters revealed the presence of both beef and buffalo in all specimens. This demonstrated that all beef frankfurter products are adulterated with buffalo in Malaysia. However, the analysis of 7 beef curries reflected only 2 them were beef and 5 were buffalo. In contrast, porcine meatball and frankfurter were found 100% authentic and also no pork was detected in halal branded beef curries, meatballs and frankfurters and chicken frankfurters. Finally, the developed TaqMan probe multiplex real-time PCR (mqPCR) assay successfully detected 0.003 ng DNA in a pure state and 0.1% target meat in mixed and commercial matrices. Analysis of commercial products under mqPCR assay revealed 71% and 100% of beef frankfurters, meatballs and 85% burgers contained buffalo adulteration but no pork in Malaysian markets. The advantage of the method was evidenced in terms of fidelity, cost and time since all the three species were detected and the option of alternative targets could complement missing targets even in decomposed specimens

Application of near- and mid-infrared spectroscopy combined with chemometrics for discrimination and authentication of herbal products: A review

Herbal medicines along with its preparations have been commonly used as preventive and promotive agents around the world, especially in developing countries. Motivated by economic profits, the high-priced value of herbal medicines may be substituted or adulterated with less expensive ones; therefore, the authentication methods must be developed to overcome the adulteration practices. Due to their properties as fingerprint analytical techniques, near-infrared (NIR) and mid-infrared (MIR) spectroscopies offered fast and reliable techniques for authentication of herbal medicine. The data generated during authentication of herbal medicines were complex and difficult to be interpreted; therefore, the statistical approach called chemometrics has been used to treat data. The objective of the present review was to highlight the updates on the application of NIR and MIR spectroscopies and chemometrics techniques (discrimination, classification, and quantification) for discrimination and authentication of herbal medicine

Impact of Foliar Application of Zinc and Zinc Oxide Nanoparticles on Growth, Yield, Nutrient Uptake and Quality of Tomato

Appropriate foliar application of zinc (Zn) and zinc oxide nanoparticles (ZnO-NPs) is important for the proper growth and yield of tomato. However, the effects of foliar application of Zn and ZnO-NPs were not well-studied on tomato production. A pot experiment was conducted at glasshouse (8D) conditions under the Faculty of Agriculture, Universiti Putra Malaysia (UPM) to evaluate the effectiveness of Zn and ZnO-NPs on growth, yield, nutrient uptake, and fruit quality of tomatoes and to compare between the Zn nutrient and ZnO-NPs. Treatment combinations were 14 viz. T1 = 0 (control), T2 = 1500 ppm (mg/L) Zn nutrient, T3 = 2000 ppm (mg/L) Zn nutrient, T4 = 2500 ppm (mg/L) Zn nutrient, T5 = 75 ppm ZnO nanoparticle, T6 = 100 ppm ZnO nanoparticle, and T7 = 125 ppm ZnO nanoparticle along with two tomato varieties. The experimental design was a split plot with four replications. Results indicated that foliar application of 100 ppm ZnO-NPs performed best in terms of growth parameters, physiological traits, yield attributes, yield, and quality traits of tomatoes. The same treatment (100 ppm ZnO-NPs) contributed to attain the highest nutrient uptake. Recovery use efficiency of Zn was highest with foliar application of 75 ppm ZnO-NPs. The highest yield increment (200%) over control was from foliar sprayed with 100 ppm ZnO-NPs. Comparing the two varieties, MARDI Tomato-3 (MT3) showed better than MARDI Tomato-1 (MT1). As is appears from the results, foliar application of zinc oxide nanoparticles was more efficient than conventional zinc fertilizer. Therefore, the foliar sprayed with 100 ppm ZnO-NPs can be suggested to improve quantity and quality of tomato in glasshouse soil conditions

DEVELOPMENT OF MULTIPLEX PCR ASSAY FOR MEAT PRODUCTS AUTHENTICATION: TARGETING DOUBLE GENE

Authentication of the species origins of animal-originated food products is a rapidly growing field because of its direct relevance to public health as well as people’s religious and cultural traditions. Current polymerase chain reaction assay (PCR) based methods to authenticate the animal materials in food chain are based on mainly single gene targets which are generally longer in length and thus breakdown during food processing treatments. Consequently, there is a chance of a false negative result. For the first time, here we targeted double gene sites in short-amplicon length multiplex PCR (mPCR) for confirmed detection and differentiation of bovine, buffalo and porcine materials in food chain. Multiple targets detection in single assay saves analytical cost and time. The design of primer sets for mPCR assay is more complex and complicated because all biomarkers are annealed to their respective targets under a single set of PCR conditions. Inaccurately designed primers might prompt less amplification or formation of primer-dimer and/or non-specific products. Here we approached the techniques to design biomarkers for the development of double gene targeted mPCR assay. Mitochondrial cytochrome b (cytb) and NADH dehydrogenase subunit 5 (ND5) genes were targeted to design six different biomarkers, two for each of cow (121 and 106 bp), buffalo (90 and 138 bp) and pig (73 and 146 bp). The in-silico specificity of the developed primers was checked against three targets and 28 non-target species. Complete sequence matching was found only with target species, and 3−18 nucleotides (12.5−80%) mismatches were found with other species. The pairwise distance was also computed using the neighbour-joining method; the lowest and highest distances were observed between 0.144 and 1.993. These indicated adequate genetic distances among the studied species, eliminating the probability of any cross-target detection and thus facilitated the target detection through mPCR assay

Circulating cell free DNA as a predictor of systemic lupus erythematosus severity and monitoring of therapy

Background: Systemic lupus erythematosus (SLE) is the most heterogeneous chronic autoimmune disease; it is characterized by the presence of auto reactive B and T cells, responsible for the aberrant production of a broad and heterogeneous group of autoantibodies. Recent studies using various detection methods have demonstrated the elevations of circulating DNA in SLE patients. Aim of the study: The current study aimed to measure cell-free DNA (cf-DNA) in SLE patients as a potential tool to predict disease activity and treatment follow up. Subjects and methods: 52 of SLE patients with age ranging from 10 to 48 years were randomly selected and 25 healthy subjects with age and gender matched with the patients were included as a control group. Thorough clinical examination stressing on the central nervous system, vascular, renal, rash, musculoskeletal, mucocutaneous manifestations, and fever was done for patients. The following investigations were done: Complete blood count (CBC), kidney function tests, C-reactive protein (CRP), routine autoantibodies for autoimmune diseases, complements (C3 & C4), anti-nucleosome antibodies and cf-DNA by real time PCR (RT-PCR). Results: The levels of anti-double stranded DNA (anti-dsDNA), anti-nucleosome Ab, and cf-DNA were significantly increased in SLE patients compared to controls. The cf-DNA level was correlated to markers of disease severity namely CRP and anti-nucleosome. A significant reduction in levels of cf-DNA, anti-nucleosome Ab and anti-dsDNA was noticed after therapy. Conclusion: Our findings support that the measurement of cf-DNA appears to be a useful marker in addition to laboratory tests used in SLE diagnosis. High correlation with markers of disease severity suggesting its role in disease pathogenesis and decreasing its level after therapy makes it to be a marker of treatment follow-up

Current trends in the green syntheses of tin oxide nanoparticles and their biomedical applications

Metal oxide nanoparticles had found a variety of applications in numerous fields of industrial, medical, and environmental technologies, attributable to recent advances nanotechnology field. Tin oxide nanoparticles (SnO _2 NPs) have gained importance as metal oxide nanoparticles due to their potential in various fields, particularly nanomedicine and other biomedicine fields. Tin oxide nanoparticles can be made using a variety of biological, chemical, and physical methods. Physicochemical methods are costly, emit high levels of toxic chemicals into the atmosphere, and consume a lot of energy. On the other hand, the biological approach is an environmentally safe, cost-effective, dependable, convenient, and easy way to synthesize tin oxide nanoparticles. In this review, the bio-mediated synthesis, as well as various biomedical applications of tin oxide nanoparticles, were discussed

Impact of Foliar Application of Zinc and Zinc Oxide Nanoparticles on Growth, Yield, Nutrient Uptake and Quality of Tomato

Appropriate foliar application of zinc (Zn) and zinc oxide nanoparticles (ZnO-NPs) is important for the proper growth and yield of tomato. However, the effects of foliar application of Zn and ZnO-NPs were not well-studied on tomato production. A pot experiment was conducted at glasshouse (8D) conditions under the Faculty of Agriculture, Universiti Putra Malaysia (UPM) to evaluate the effectiveness of Zn and ZnO-NPs on growth, yield, nutrient uptake, and fruit quality of tomatoes and to compare between the Zn nutrient and ZnO-NPs. Treatment combinations were 14 viz. T1 = 0 (control), T2 = 1500 ppm (mg/L) Zn nutrient, T3 = 2000 ppm (mg/L) Zn nutrient, T4 = 2500 ppm (mg/L) Zn nutrient, T5 = 75 ppm ZnO nanoparticle, T6 = 100 ppm ZnO nanoparticle, and T7 = 125 ppm ZnO nanoparticle along with two tomato varieties. The experimental design was a split plot with four replications. Results indicated that foliar application of 100 ppm ZnO-NPs performed best in terms of growth parameters, physiological traits, yield attributes, yield, and quality traits of tomatoes. The same treatment (100 ppm ZnO-NPs) contributed to attain the highest nutrient uptake. Recovery use efficiency of Zn was highest with foliar application of 75 ppm ZnO-NPs. The highest yield increment (200%) over control was from foliar sprayed with 100 ppm ZnO-NPs. Comparing the two varieties, MARDI Tomato-3 (MT3) showed better than MARDI Tomato-1 (MT1). As is appears from the results, foliar application of zinc oxide nanoparticles was more efficient than conventional zinc fertilizer. Therefore, the foliar sprayed with 100 ppm ZnO-NPs can be suggested to improve quantity and quality of tomato in glasshouse soil conditions

Multiplex PCR assay discriminates rabbit, rat and squirrel meat in food chain

<p>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 <i>ATP6</i> and <i>cytb</i> 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.</p