Species-specific loop-mediated isothermal amplification (LAMP) assay for identification of tissue of cattle origin by targeting mitochondrial gene sequences

Abstract

The present study was carried out with the objective of development of species-specific loop-mediated isothermal amplification (LAMP) assay for identification of tissue of cattle origin. The cattle-specific LAMP primer set was designed by targeting mitochondrial D-loop gene. The conditions for LAMP reaction for amplification of template DNA from cattle using designed cattle-specific primer set were optimized for the components of mixture and temperature of reaction. Amplified products were analysed using SYBR Green I dye and by agarose gel electrophoresis. The developed species-specific LAMP assay was evaluated for its specificity, sensitivity and validated in laboratory on samples from known, coded, binary meat admixture with other than cattle at relative percentage of 20%, 10%, 5% and 1%, Phire tissue direct PCR master mix treated tissues of cattle and on species-specific polymerase chain reaction assay positive samples. The developed LAMP assay using self-designed primer set was highly specific, amplifying the DNA template exclusively from cattle tissue under the optimized LAMP reaction conditions. The sensitivity assay using serially diluted DNA templates revealed lowest level of detection as 0.01 ng of absolute DNA from target species. Laboratory validation substantiated the accuracy of assay in known/unknown (coded) samples and up to the 1% level of admixture in binary meat sample. DNA present in supernatant of Phire Animal tissue kit treated samples were also amplified successfully eliminating the extra step of extraction of genomic DNA. The developed assays exhibited comparable results with previously established species-specific PCR assay taken as gold standards. Thus, it was concluded that developed species-specific loop-mediated isothermal amplification assay was effective in identification of tissue of cattle origin.

Recombinase Polymerase Amplification Based Multiplex Lateral Flow Dipstick for Fast Identification of Duck Ingredient in Adulterated Beef

“…The results had shown that all samples could be detected by two T lines and found that the 5% duck in beef can be easily detected ( Figure 4 ). We were not pursuing a lower proportion because it was difficult to obtain any commercial profit from the 5% adulteration of low-quality meat in actual manufacturing and sales, which was in good agreement with the data reported in many previous reports [ 35 , 36 ]. In addition, the sensitivity test was also conducted for each species, a dilution series of beef and duck DNA with concentrations ranging from 100 to 0.01 ng were performed.…”

Loop-Mediated Isothermal Amplification (LAMP): The Better Sibling of PCR?

“…Such assessments usually involve the analysis of mitochondrial genes, i.e., cytochrome b (cytb) and cytochrome c oxidase subunits I and II (cox1 and cox2), and the control region (CR) [62]. Food frauds are especially prevalent in poor and developing countries with high populations, where the demand for meat and meat products is rising, which inflates their prices [63]. Subject literature contains an increasing number of reports on pork frauds and their detectability with LAMP [64][65][66][67][68].…”

“…The most common targets of fraud are pork and beef products [63]; however, not enough tests have been designed to date. Some of the available tests can detect pork at a concentration of 0.01% in the mixture, which corresponds to 0.1 g of pork in 1 kg of meat [64,69,70].…”

“…Another test can determine the composition of bovine tissues in mixtures with closely related species, i.e., buffaloes, goats, and sheep. The results of the test have demonstrated that LAMP is also highly specific and effective at identifying bovine tissue, being able to detect it in mixtures of meat from two species at a minimum concentration of 0.01 ng [63]. Other popular targets of fraud include ostrich meat.…”

A cost-effective plan for global testing - an infection rate stratified, algorithm guided, multiple-level, continuously pooled testing strategy

“…The test accuracy is the primary concern. At present, the accuracy of PCR testing technology has reached near 100% ( Dharavath et al, 2020 ; Garg et al, 2020 ), while its capability to detect a positive individual in a pooled sample is between 100 and 1000 individuals ( Dharavath et al, 2020 ; Kumari et al, 2019 ). Thus, within a mixture of 1000 samples, the applied PCR method is sensitive enough to identify the presence of the virus from a single sample.…”