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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
  • 18 Downloads

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.

Keywords

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

Notes

Acknowledgements

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.

References

  1. Abdullahi UF, Igwenagu E, Aliyu S, Mu’azu A, Naim R, Wan-Taib WR (2017) A rapid and sensitive Loop-mediated isothermal amplification assay for detection of pork DNA based on porcine tRNAlys and ATPase 8 genes. Int Food Res J 24(4):1357–1361Google Scholar
  2. 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(6):1007–1017CrossRefGoogle Scholar
  3. Cho AR, Dong HJ, Cho S (2014) Meat species identification using loop-mediated isothermal amplification assay targeting species-specific mitochondrial DNA. Korean J Food Sci Anim Resour 34(6):799–807CrossRefGoogle Scholar
  4. Cowan JA (2002) Structural and catalytic chemistry of magnesium-dependent enzymes. Biometals 15(3):225–235CrossRefGoogle Scholar
  5. Doosti A, Dehkordi PG, Rahimi E (2014) Molecular assay to fraud identification of meat products. J Food Sci Technol 51(1):148–152CrossRefGoogle Scholar
  6. En FX, Wei X, Jian L, Qin C (2008) Loop-mediated isothermal amplification establishment for detection of pseudorabies virus. J Virol Methods 151(1):35–39CrossRefGoogle Scholar
  7. Fajardo V, González I, López-Calleja I, Martín I, Hernández PE, García T (2006) PCR-RFLP authentication of meats from red deer (Cervuselaphus), fallow deer (Damadama), roe deer (Capreoluscapreolus), cattle (Bostaurus), sheep (Ovisaries), and goat (Capra hircus). J Agric Food Chem 54(4):1144–1150CrossRefGoogle Scholar
  8. Fajardo V, Gonzalez I, Martyn I, Rojas M, Hernandez PE, Garcya T (2008a) Real-time PCR for detection and quantification of red deer (Cervus elaphus), fallow deer (Dama dama), and roe deer (Capreolus capreolus) in meat mixtures. Meat Sci 79(2):289–298CrossRefGoogle Scholar
  9. Fernández Soto P, Mvoulouga PO, Akue JP, Abán JL, Santiago BV, Sánchez MC (2014) Development of a highly sensitive loop-mediated isothermal amplification (LAMP) method for the detection of Loa loa. PloS One 9(4):946–964CrossRefGoogle Scholar
  10. Fischbach J, Xander NC, Frohme M, Glökler JF (2015) Method summary. Biotechniques 58(4):189–194CrossRefGoogle Scholar
  11. George G, Mony P, Kenneth J (2011) Comparison of the efficacies of loop-mediated isothermal amplification, fluorescence smear microscopy and culture for the diagnosis of tuberculosis. PloS One 6(6):210–217CrossRefGoogle Scholar
  12. Ghatak S, Muthukumaran RB, Nachimuthu SK (2013) A simple method of genomic DNA extraction from human samples for PCR-RFLP analysis. J Biomol Tech JBT 24(4):224–231PubMedGoogle Scholar
  13. Ghovvati S, Nassiri MR, Mirhoseini SZ, Moussavi AH, Javadmanesh A (2009) Fraud identification in industrial meat products by multiplex PCR assay. Food Control 20(8):696–699CrossRefGoogle Scholar
  14. Girish PS, Anjaneyulu ASR, Viswas KN, Anand M, Rajkumar N, Shivakumar BM (2004) Sequence analysis of mitochondrial 12S rRNA gene can identify meat species. Meat Sci 66(3):551–556CrossRefGoogle Scholar
  15. Goto M, Honda E, Ogura A, Nomoto A, Hanaki KI (2009) Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxynaphthol blue. Biotechniques 46(3):167–172CrossRefGoogle Scholar
  16. Hou B, Meng X, Zhang L, Guo J, Li S, Jin H (2015) Development of a sensitive and specific multiplex PCR method for the simultaneous detection of chicken, duck and goose DNA in meat products. Meat Sci 101:90–94CrossRefGoogle Scholar
  17. Jeong J, Cho SY, Lee WH, Lee K, Ju HJ (2015) Development of a rapid detection method for Potato virus X by reverse transcription loop-mediated isothermal amplification. Plant Pathol J 31(3):219–225CrossRefGoogle Scholar
  18. Kanchanaphum P, Maneenin S, Chaiyana W (2014) Analysis of pork meat using loop mediated isothermal amplification (LAMP) to confirm halal status. Int J Biosci 4:62–68Google Scholar
  19. Kil EJ, Kim S, Lee YJ, Kang EH, Lee M, Cho SH (2015) Advanced loop-mediated isothermal amplification method for sensitive and specific detection of Tomato chlorosis virus using a uracil DNA glycosylase to control carry-over contamination. J Virol Methods 213:68–74CrossRefGoogle Scholar
  20. Koh BRD, Kim JY, Na HM, Park SD, Kim YH (2011) Development of species-specific multiplex PCR assays of mitochondrial 12S rRNA and 16S rRNA for the identification of animal species. Korean J Vet Serv 34(4):417–428CrossRefGoogle Scholar
  21. 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. Afr J Biotech 11(89):15590–15595CrossRefGoogle Scholar
  22. Lee SY, Kim MJ, Hong Y, Kim HY (2016) Development of a rapid on-site detection method for pork in processed meat products using real-time loop-mediated isothermal amplification. Food Control 66:53–61CrossRefGoogle Scholar
  23. Li YJ, Fan JY (2017) Rapid visual identification of bovine meat by loop mediated isothermal amplification combined with immunochromatographic strip. BioChip J 11(1):8–13CrossRefGoogle Scholar
  24. Li B, Bai S, Xu Y, Zhang W, Ma J (2006) Identification of sika deer and red deer using partial cytochrome b and 12 s ribosomal RNA genes. J For Res 17(2):160–162CrossRefGoogle Scholar
  25. Liu J, Huang C, Wu Z, Zhang X, Wang Y (2010) Detection of tomato aspermy virus infecting chrysanthemums by LAMP. Sci Agric Sin 43(6):1288–1294Google Scholar
  26. 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. Am J Food Technol 11:193–203CrossRefGoogle Scholar
  27. Mane BG, Mendiratta SK, Tiwari AK, Sharma BD, Bhilegaokar KN, Anjaneyulu ASR (2011) Detection of pork in admixed meat and meat products by species-specific PCR technique. Indian J Anim Sci 8:11–16Google Scholar
  28. Mohindra V, Khare P, Lal KK, Punia P, Singh RK, Barman AS (2007) Molecular discrimination of five Mahseer species from Indian peninsula using RAPD analysis. Acta Zool Sinica 53(4):725–732Google Scholar
  29. Nagdev KJ, Kashyap RS, Parida MM, Kapgate RC, Purohit HJ, Taori GM (2011) Loop-mediated isothermal amplification for rapid and reliable diagnosis of tuberculous meningitis. J Clin Microbiol 49(5):1861–1865CrossRefGoogle Scholar
  30. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28(12):63–69CrossRefGoogle Scholar
  31. Ortea I, Pascoal A, Cañas B, Gallardo JM, Barros-Velázquez J, Calo-Mata P (2012) Food authentication of commercially-relevant shrimp and prawn species: From classical methods to Foodomics. Electrophoresis 33(15):2201–2211CrossRefGoogle Scholar
  32. Parida M, Sannarangaiah S, Dash PK, Rao PVL, Morita K (2008) Loop mediated isothermal amplification (LAMP): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases. Rev Med Virol 18(6):407–421CrossRefGoogle Scholar
  33. Ran G, Ren L, Han X, Liu X, Li Z, Pang D (2016) Development of a rapid method for the visible detection of pork DNA in halal products by loop-mediated isothermal amplification. Food Anal Methods 9(3):565–570CrossRefGoogle Scholar
  34. Ranjbar R, Afshar D (2015) Development of a loop-mediated isothermal amplification assay for rapid detection of Yersinia enterocolitica via targeting a conserved locus. Iran J Microbiol 7(4):185–190PubMedPubMedCentralGoogle Scholar
  35. Rastogi G, Dharne MS, Walujkar S, Kumar A, Patole MS, Shouche YS (2007) Species identification and authentication of tissues of animal origin using mitochondrial and nuclear markers. Meat Sci 76(4):666–674CrossRefGoogle Scholar
  36. Rojas M, González I, Pavon MA, Pegels N, Hernández PE, García T (2011) Mitochondrial and nuclear markers for the authentication of partridge meat and the specific identification of red-legged partridge meat products by polymerase chain reaction. Poult Sci 90(1):211–222CrossRefGoogle Scholar
  37. Saini M, Das DK, Dhara A, Swarup D, Yadav MP, Gupta PK (2007) Characterisation of peacock (Pavocristatus) mitochondrial 12S rRNA sequence and its use in differentiation from closely related poultry species. Br Poult Sci 48(2):162–166CrossRefGoogle Scholar
  38. Spink J, Moyer DC (2011) Backgrounder: defining the public health threat of food fraud. Minneap Min Natl Cent Food Prot Def 76(9):157–162Google Scholar
  39. Tomlinson J, Boonham N (2008) Potential of LAMP for detection of plant pathogens. CAB Rev Perspect Agric Vet Sci Nutr Nat Resour 3(66):1–7Google Scholar
  40. Wang X, Seo DJ, Lee MH, Choi C (2014) Comparison of conventional PCR, multiplex PCR, and loop-mediated isothermal amplification assays for rapid detection of Arcobacter species. J Clin Microbiol 52(2):557–563CrossRefGoogle Scholar
  41. Yang L, Fu S, Peng X, Li LE, Song T, Li L (2014) Identification of pork in meat products using real-time loop-mediated isothermal amplification. Biotechnol Biotechnol Equip 28(5):882–888CrossRefGoogle Scholar
  42. Zahradnik C, Martzy R, Mach RL, Krska R, Farnleitner AH, Brunner K (2015) Loop-mediated isothermal amplification (LAMP) for the detection of horse meat in meat and processed meat products. Food Anal Methods 8(6):1576–1581CrossRefGoogle Scholar
  43. Zhang CL, Fowler MR, Scott NW, Lawson G, Slater A (2007) A TaqMan real-time PCR system for the identification and quantification of bovine DNA in meats, milks and cheeses. Food Control 18(9):1149–1158CrossRefGoogle Scholar
  44. Zhou D, Wang C, Li Z, Chen Y, Gao S, Guo J (2016) Detection of bar transgenic sugarcane with a rapid and visual loop-mediated isothermal amplification assay. Front Plant Sci 7:279–284PubMedPubMedCentralGoogle Scholar

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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