DEVELOPMENT OF NOVEL ISOTHERMAL NUCLEIC ACID BASED AMPLIFICATION ASSAY FOR RAPID AND VISUAL DETECTION OF PORK
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Date
2022
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KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR
Abstract
Meat adulteration or fraud is one of the major issues in global meat market as it has economic, social, religious and health impact on the consumers. Hence, meat authenticity is one of the prime necessities to gain consumer confidence and to meet the statutory requirements. A plethora of techniques are available, however a requirement for rapid assay with on-field application is the need of the hour. Hence, in this study an attempt was made to develop a rapid, nucleic acid, isothermal amplification method, with SYBR Green I visualization called Polymerase Spiral Reaction (PSR) to identify tissues of pig origin. Primers were designed specifically to amplify the mitochondrial cytochrome b gene. The reaction temperature of PSR and component of reaction mixture were optimized at 64°C amplification temperature, 12 U/μL concentration of Bst DNA polymerase, 6 mM concentration of MgSO4, 1.2 mM concentration of dNTP, 0.8 M concentration of betaine, and 18 μM each of forward primer and reverse primers. Successful amplifications were confirmed using SYBR Green I dye and agarose gel electrophoresis. The specificity of assay was tested with DNA from beef, cara beef, mutton, chevon and chicken, where amplification was observed only in pork. The assay was also capable of detecting tissues of pig origin in heat treated and processed pork meat products. Analytical sensitivity revealed that PSR was able to detect 760 femto grams (fg) as compared to pork specific PCR which could detect only up to 7.6 pico gram (pg). The PSR assay was found to be 10 times more sensitive compared to PCR in detection of genomic DNA from tissues of pig origin. Hence, the developed pig-specific PSR assay can be effectively used in detection of tissue of pig origin.