3 Biotech

, 9:69 | Cite as

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

  • Sarita Kumari
  • Rajiv Ranjan KumarEmail author
  • Sanjod Kumar Mendiratta
  • Deepak Kumar
  • Preeti Rana
  • Dhananjay Kumar
  • Jyoti Jawla
Original Article


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.


Species-specific Loop-mediated isothermal amplification (LAMP) Cattle Sensitivity and laboratory validation 



The authors gratefully acknowledge Indian council of Agricultural research-Indian Veterinary Research Institute (IVRI) Izatnagar, India for providing necessary facilities and financial support to accomplish this research.

Author contribution

The authors’ contributions are as follows: Sarita Kumari: carried out the majority of the research work, DNA isolation and species-specific LAMP assay; RRK: responsible for the conception of the project, designed the study and planned the experiments, manuscript drafting. SKM and DK coordinated and carried out data interpretation, Preeti Rana: DNA isolation from diverse samples, field samples, Dhananjay Kumar and Jyoti Jawla: validation of findings by repeating the experiments. All authors read and approved this manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest in the publication.

Ethical statement

The authors declare their commitment for scientific integrity and research ethics of reported research work and assure the absence of scientific fraud and misrepresentations, inappropriate manipulation of graphics files, redundant publications, and plagiarism in the manuscript.


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Copyright information

© King Abdulaziz City for Science and Technology 2019

Authors and Affiliations

  1. 1.Department of Livestock Products TechnologyPGIVER, RAJUVASJaipurIndia
  2. 2.Division of Livestock Products TechnologyIndian Veterinary Research InstituteBareillyIndia
  3. 3.Division of Veterinary BiotechnologyIVRIIzatnagarIndia
  4. 4.Department of Livestock Products TechnologyCVASc, DUVASUMathuraIndia

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