Development of rapid and sensitive loop-mediated isothermal method for on-site visual identification of tissue origin of pig by using mitochondrial COI gene sequences
Sarita Kumari
A * , R. R. Kumar A , S. K. Mendiratta A , Anurag Mahala B , Arun Kumar B and Jyoti Jawla A
+ Author Affiliations
- Author Affiliations
A Indian Veterinary Research Institute (IVRI), Bareilly, Uttar Pradesh, India.
B Rajasthan University of Veterinary and Animal Sciences (RAJUVAS), Bikaner, Rajasthan, India.
Animal Production Science 63(7) 704-711 https://doi.org/10.1071/AN22298
Submitted: 29 July 2022 Accepted: 6 December 2022 Published: 5 January 2023
© 2023 The Author(s) (or their employer(s)). Published by CSIRO Publishing
Abstract
Context: The loop-mediated isothermal amplification (LAMP) methods have great potential to identify the species origin of the tissue in meat and meat products at isothermal temperature and are also suitable for field conditions.
Aim: The present study aimed to develop a rapid, specific, and sensitive assay based on the LAMP technique for identification of tissue of pig origin.
Methods: The pig-specific primers were designed by targeting the mitochondrial COI gene. The amplification temperature and time for the LAMP reaction were optimised as 64°C and 45 min. The analysis of the amplified product was performed on the basis of the development of colour after the addition of intercalating SYBR Green I dye, and also by the ladder-like pattern on agarose-gel electrophoresis.
Key results: The assay was found to be highly specific for DNA templates of pig origin and showed no cross-reactivity with other food animals, viz. cattle, buffalo, sheep, and goats. The analytical sensitivities of the LAMP and PCR assays were recorded as up to 0.00001 ng and 0.1 ng respectively, of the absolute DNA content. The laboratory validation of the developed method was performed on blind samples and an admixture of meat from different food animals, viz. cattle, buffalo, sheep, goat and pig. The analysis could be performed in an hour by using supernatant from Phire Animal Tissue Direct PCR kit-treated tissue, excluding the complex process of nucleic acid extraction.
Conclusion: The LAMP assay was found to be cost-effective, easy to perform, and highly species-specific for pig tissue in meat and meat admixture. The result of the assay can be analysed with the naked eye without the need for sophisticated equipment. Compared with pre-standardised PCR assay, the developed LAMP method was quite sensitive and could be performed within 1 h, from sampling to analysis results.
Implications: The developed LAMP assay is low resource-based single-tube approach that could be exploited significantly in the fields of diagnostics, agriculture, and aquaculture.
Keywords: adulteration, gel electrophoresis, loop-mediated isothermal amplification, polymerase chain reaction, pork, sensitive, specific, SYBR Green I dye.
References
Abdulmawjood A, Grabowski N, Fohler S, Kittler S, Nagengast H, Klein G (2014) Development of loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive identification of ostrich meat. PLoS ONE 9, e100717| Development of loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive identification of ostrich meat.Crossref | GoogleScholarGoogle Scholar |
Ahmed MU, Hasan Q, Hossain MM, Saito M, Tamiya E (2010) Meat species identification based on the loop mediated isothermal amplification and electrochemical DNA sensor. Food Control 21, 599–605.
| Meat species identification based on the loop mediated isothermal amplification and electrochemical DNA sensor.Crossref | GoogleScholarGoogle Scholar |
Aida AA, Man YC, Wong CMVL, Raha AR, Son R (2005) Analysis of raw meats and fats of pigs using polymerase chain reaction for Halal authentication. Meat Science 69, 47–52.
| Analysis of raw meats and fats of pigs using polymerase chain reaction for Halal authentication.Crossref | GoogleScholarGoogle Scholar |
Dooley JJ, Paine KE, Garrett SD, Brown HM (2004) Detection of meat species using TaqMan real-time PCR assays. Meat Science 68, 431–438.
| Detection of meat species using TaqMan real-time PCR assays.Crossref | GoogleScholarGoogle Scholar |
Erwanto Y, Abidin MZ, Sugiyono EYPM, Rohman A (2014) Identification of pork contamination in meatballs of Indonesia local market using polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) analysis. Asian-Australasian Journal of Animal Sciences 27, 1487–1492.
| Identification of pork contamination in meatballs of Indonesia local market using polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) analysis.Crossref | GoogleScholarGoogle Scholar |
Font-i-Furnols M, Guerrero L (2014) Consumer preference, behavior and perception about meat and meat products: an overview. Meat Science 98, 361–371.
| Consumer preference, behavior and perception about meat and meat products: an overview.Crossref | GoogleScholarGoogle Scholar |
Jensen MA, Fukushima M, Davis RW (2010) DMSO and betaine greatly improve amplification of GC-rich constructs in de novo synthesis. PLoS ONE 5, e11024
| DMSO and betaine greatly improve amplification of GC-rich constructs in de novo synthesis.Crossref | GoogleScholarGoogle Scholar |
Karabasanavar NS, Singh SP, Kumar D, Shebannavar SN (2014) Detection of pork adulteration by highly-specific PCR assay of mitochondrial D-loop. Food Chemistry 145, 530–534.
| Detection of pork adulteration by highly-specific PCR assay of mitochondrial D-loop.Crossref | GoogleScholarGoogle Scholar |
Kesmen Z, Yetiman AE, Şahin F, Yetim H (2012) Detection of chicken and turkey meat in meat mixtures by using real-time PCR assays. Journal of Food Science 77, C167–C173.
| Detection of chicken and turkey meat in meat mixtures by using real-time PCR assays.Crossref | GoogleScholarGoogle Scholar |
Koh B-R-D, Kim J-Y, Na H-M, Park S-D, Kim Y-H (2011) Development of species-specific multiplex PCR assays of mitochondrial 12S rRNA and 16S rRNA for the identification of animal species. Korean Journal of Veterinary Service 34, 417–428.
| Development of species-specific multiplex PCR assays of mitochondrial 12S rRNA and 16S rRNA for the identification of animal species.Crossref | GoogleScholarGoogle Scholar |
Kumar A, Kumar RR, Sharma BD, Mendiratta SK, Sharma D, Gokulakrishnan P (2012) Species specific polymerase chain reaction (PCR) assay for identification of pig (Sus domesticus) meat. African Journal of Biotechnology 11, 15590–15595.
| Species specific polymerase chain reaction (PCR) assay for identification of pig (Sus domesticus) meat.Crossref | GoogleScholarGoogle Scholar |
Kumar D, Kumar RR, Kumar A, Mendiratta SK, Lalawampuii H, Kumbhar V, Choudhary A, Kumari S, Rana P (2017) A PCR assay for identification of buffalo origin of tissue by amplification of the mt. D-loop gene. Journal of Animal Research 7, 7–14.
| A PCR assay for identification of buffalo origin of tissue by amplification of the mt. D-loop gene.Crossref | GoogleScholarGoogle Scholar |
Kumari S, Kumar RR, Mendiratta SK, Kumar D, Rana P, Kumar D, Jawla J (2019) Species-specific loop-mediated isothermal amplification (LAMP) assay for identification of tissue of cattle origin by targeting mitochondrial gene sequences. 3 Biotech 9, 69
| Species-specific loop-mediated isothermal amplification (LAMP) assay for identification of tissue of cattle origin by targeting mitochondrial gene sequences.Crossref | GoogleScholarGoogle Scholar |
Kumari S, Kumar RR, Mendiratta SK, Kumar D, Kumar A, Kumar D, Rana P, Jawla J (2020) On-site detection of tissues of buffalo origin by loop-mediated isothermal amplification (LAMP) assay targeting mitochondrial gene sequences. Food Analytical Methods 13, 1060–1068.
| On-site detection of tissues of buffalo origin by loop-mediated isothermal amplification (LAMP) assay targeting mitochondrial gene sequences.Crossref | GoogleScholarGoogle Scholar |
Kumari S, Kumar RR, Mendiratta SK, Kumar D, Kumar A, Jawla J, Anurag , Rana P, Kumar D (2021) Development of loop-mediated isothermal method and comparison with conventional PCR assay for rapid on spot identification of tissue of cattle origin. Journal of Food Science and Technology 58, 4608–4615.
| Development of loop-mediated isothermal method and comparison with conventional PCR assay for rapid on spot identification of tissue of cattle origin.Crossref | GoogleScholarGoogle Scholar |
Kumbhar VH, Kumar RR, Mendiratta SK, Choudhary A, Kumar D, Kumari S, Rana P (2020) Duplex PCR assay for identification of tissue of cattle and buffalo origin targeting mitochondrial cytochrome b gene. Proceedings of the National Academy of Sciences, India Section B: Biological Sciences 90, 287–291.
| Duplex PCR assay for identification of tissue of cattle and buffalo origin targeting mitochondrial cytochrome b gene.Crossref | GoogleScholarGoogle Scholar |
Lee M-F, Chen Y-H, Peng C-F (2009) Evaluation of reverse transcription loop-mediated isothermal amplification in conjunction with ELISA–hybridization assay for molecular detection of Mycobacterium tuberculosis. Journal of Microbiological Methods 76, 174–180.
| Evaluation of reverse transcription loop-mediated isothermal amplification in conjunction with ELISA–hybridization assay for molecular detection of Mycobacterium tuberculosis.Crossref | GoogleScholarGoogle Scholar |
Lee S-Y, Kim M-J, Hong Y, Kim H-Y (2016) Development of a rapid on-site detection method for pork in processed meat products using real-time loop-mediated isothermal amplification. Food Control 66, 53–61.
| Development of a rapid on-site detection method for pork in processed meat products using real-time loop-mediated isothermal amplification.Crossref | GoogleScholarGoogle Scholar |
Ma B, Dai M, Fang J, Wu Y, Zhang M (2016) Visual loop-mediated isothermal amplification (LAMP) method for identification bovine and ovine gene in animal foodstuff. American Journal of Food Technology 11, 193–203.
| Visual loop-mediated isothermal amplification (LAMP) method for identification bovine and ovine gene in animal foodstuff.Crossref | GoogleScholarGoogle Scholar |
Nakyinsige K, Man YBC, Sazili AQ (2012) Halal authenticity issues in meat and meat products. Meat Science 91, 207–214.
| Halal authenticity issues in meat and meat products.Crossref | GoogleScholarGoogle Scholar |
Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Research 28, e63
| Loop-mediated isothermal amplification of DNA.Crossref | GoogleScholarGoogle Scholar |
Rohman A, Che Man YB (2012) Analysis of pig derivatives for halal authentication studies. Food Reviews International 28, 97–112.
| Analysis of pig derivatives for halal authentication studies.Crossref | GoogleScholarGoogle Scholar |
Sharma S, Pandey M, Onteru SK, Singh D (2020) Loop-mediated isothermal amplification (LAMP) for detection of Ureaplasma diversum from cervico-vaginal swab of buffaloes. Applied Biochemistry and Biotechnology 190, 1201–1211.
| Loop-mediated isothermal amplification (LAMP) for detection of Ureaplasma diversum from cervico-vaginal swab of buffaloes.Crossref | GoogleScholarGoogle Scholar |
Soares S, Amaral JS, Oliveira MBP, Mafra I (2013) A SYBR Green real-time PCR assay to detect and quantify pork meat in processed poultry meat products. Meat Science 94, 115–120.
| A SYBR Green real-time PCR assay to detect and quantify pork meat in processed poultry meat products.Crossref | GoogleScholarGoogle Scholar |
Thangsunan P, Temisak S, Morris P, Rios-Solis L, Suree N (2021) Combination of loop-mediated isothermal amplification and AuNP-oligoprobe colourimetric assay for pork authentication in processed meat products. Food Analytical Methods 14, 568–580.
| Combination of loop-mediated isothermal amplification and AuNP-oligoprobe colourimetric assay for pork authentication in processed meat products.Crossref | GoogleScholarGoogle Scholar |
Wissiack R, de la Calle B, Bordin G, Rodriguez AR (2003) Screening test to detect meat adulteration through the determination of hemoglobin by cation exchange chromatography with diode array detection. Meat Science 64, 427–432.
| Screening test to detect meat adulteration through the determination of hemoglobin by cation exchange chromatography with diode array detection.Crossref | GoogleScholarGoogle Scholar |
Yang L, Fu S, Peng X, Li L, Song T, Li L (2014) Identification of pork in meat products using real-time loop-mediated isothermal amplification. Biotechnology & Biotechnological Equipment 28, 882–888.
| Identification of pork in meat products using real-time loop-mediated isothermal amplification.Crossref | GoogleScholarGoogle Scholar |
Zhang H, Feng J, Xue R, Du X-J, Lu X, Wang S (2014) Loop-mediated isothermal amplification assays for detecting Yersinia pseudotuberculosis in milk powders. Journal of Food Science 79, M967–M971.
| Loop-mediated isothermal amplification assays for detecting Yersinia pseudotuberculosis in milk powders.Crossref | GoogleScholarGoogle Scholar |
