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Successful application of modified crude DNA extraction from muscle tissues for various types of PCR amplifications

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Abstract

Background

One of the most challenging aspects of nucleic acid amplification tests is the extraction of genomic DNA. However, achieving satisfactory quality and quantity of genomic DNA is not always easy, while the demand for rapid, low-cost and less laborious DNA isolation methods is ever-increasing.

Methods and results

We have developed a rapid (⁓2 min) crude DNA extraction method leading to direct-PCR that requires minimum reagents and laboratory equipment. It was developed by eliminating the time-consuming purification steps of DNA extraction, by processing the sample in optimized amounts of Taq KCl PCR buffer and DNARelease Additive/Proteinase K in only two minutes and carrying out amplification using conventional Taq DNA polymerase. The DNA preparation method was validated on muscle tissue samples from 12 different species as well as 48 cooked meat samples. Its compatibility was also successfully tested with different types of PCR amplification platforms extensively used for genetic analysis, such as simplex PCR, PCR-RFLP (Restriction Fragment Length Polymorphism), multiplex PCR, isothermal amplification, real-time PCR and DNA sequencing.

Conclusions

The developed protocol provides sufficient amount of crude DNA from muscle tissues of different species for PCR amplifications to identify species-of-origin via different techniques coupled with PCR. The simplicity and robustness of this protocol make nucleic acid amplification assays more accessible and affordable to researchers and authorities for both laboratory and point-of-care tests.

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Data availability

All data generated or analyzed during this study are included in this published article.

References

  1. Petralia S, Conoci S (2017) PCR technologies for Point of Care Testing: Progress and perspectives. ACS Sens 2:876–891

    Article  CAS  PubMed  Google Scholar 

  2. Kumar Y, Narsaiah K (2021) Rapid point-of-care testing methods/devices for meat species identification: a review. Compr Rev Food Sci Food Saf 20:900–923

    Article  CAS  PubMed  Google Scholar 

  3. Bauer M, Patzelt D (2003) A method for simultaneous RNA and DNA isolation from dried blood and semen stains. Forensic Sci Int 136:76–78

    Article  CAS  PubMed  Google Scholar 

  4. Muhammad H, Iqbal Z, Iqbal MU, Younas T, Bashir Q (2016) An efficient method for DNA isolation from Fish Fin. Pakistan J Agricultural Sci 53:843–850

    Article  Google Scholar 

  5. Lopera-Barrero NM, Povh JA, Ribeiro RP, Gomes PC, Jacometo CB, Silva Lopes TD (2008) Comparison of DNA extraction protocols of fish fin and larvae samples: modified salt (NaCl) extraction. Ciencia E investigación Agrar 35:77–86

    Google Scholar 

  6. Sajali N, Wong SC, Hanapi UK, Abu Bakar Jamaluddin S, Tasrip NA, Mohd Desa MN (2018) The challenges of DNA extraction in different assorted food matrices: a review. J Food Sci 83:2409–2414

    Article  CAS  PubMed  Google Scholar 

  7. Besbes N, Sáiz-Abajo M-J, Sadok S (2022) Comparative study of DNA extraction to initiate harmonized protocol for a simple method of species identification: fresh and canned tuna case study. CyTA - J Food 20:39–49

    Article  CAS  Google Scholar 

  8. Mezzomo P, Mielniczki-Pereira AA, Sausen TL, Marinho JR, Cansian RL (2021) Evaluation of eight protocols for genomic DNA extraction of Hypostomus commersoni Valenciennes, 1836 (Loricariidae: Siluriformes). Braz J Biol 81:674–683

    Article  CAS  PubMed  Google Scholar 

  9. Chapela MJ, Sotelo CG, Pérez-Martín RI, Pardo MÁ, Pérez-Villareal B, Gilardi P, Riese J (2007) Comparison of DNA extraction methods from muscle of canned tuna for species identification. Food Control 18:1211–1215

    Article  CAS  Google Scholar 

  10. Chen H, Rangasamy M, Tan SY, Wang H, Siegfried BD (2010) Evaluation of five methods for total DNA extraction from western corn rootworm beetles. PLoS ONE 5:e11963

    Article  PubMed  PubMed Central  Google Scholar 

  11. Swaran YC, Welch L (2012) A comparison between direct PCR and extraction to generate DNA profiles from samples retrieved from various substrates. Forensic Sci Int Genet 6:407–412

    Article  CAS  PubMed  Google Scholar 

  12. Colussi A, Viegas M, Beltramo J, Lojo M (2009) Efficiency of DNA IQ System® in recovering semen DNA from cotton swabs. Forensic Sci International: Genet Supplement Ser 2:87–88

    Google Scholar 

  13. Kishore R, Reef Hardy W, Anderson VJ, Sanchez NA, Buoncristiani MR (2006) Optimization of DNA extraction from low-yield and degraded samples using the BioRobot EZ1 and BioRobot M48. J Forensic Sci 51:1055–1061

    Article  CAS  PubMed  Google Scholar 

  14. Guan F, Jin Y, Zhao J, Ai J, Luo Y (2018) A Novel Direct PCR lysis buffer can improve PCR from meat matrices. Food Anal Methods 12:100–107

    Article  Google Scholar 

  15. Thanakiatkrai P, Dechnakarin J, Ngasaman R, Kitpipit T (2019) Direct pentaplex PCR assay: an adjunct panel for meat species identification in Asian food products. Food Chem 271:767–772

    Article  CAS  PubMed  Google Scholar 

  16. Sepp R, Szabo I, Uda H, Sakamoto H (1994) Rapid techniques for DNA extraction from routinely processed archival tissue for use in PCR. J Clin Pathol 47:318–323

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Kitpipit T, Sittichan K, Thanakiatkrai P (2014) Direct-multiplex PCR assay for meat species identification in food products. Food Chem 163:77–82

    Article  CAS  PubMed  Google Scholar 

  18. Chomczynski P, Rymaszewski M (2006) Alkaline polyethylene glycol-based method for direct PCR from bacteria, eukaryotic tissue samples, and whole blood. Biotechniques 40(454):456

    PubMed  Google Scholar 

  19. Truett GE, Heeger P, Mynatt RL, Truett AA, Walker JA, Warman ML (2000) Preparation of PCR-quality mouse genomic DNA with hot sodium hydroxide and tris (HotSHOT). Biotechniques 29:52

    Article  CAS  PubMed  Google Scholar 

  20. Xue C, Wang P, Zhao J, Xu A, Guan F (2017) Development and validation of a universal primer pair for the simultaneous detection of eight animal species. Food Chem 221:790–796

    Article  CAS  PubMed  Google Scholar 

  21. Jafar S, Kabir F, Anjum KM, Zahoor MY, Shehzad W, Imran M (2023) Comparison of different DNA preparatory methods for development of a universal direct PCR-RFLP workflow for identification of meat origin in food products. Food Sci Technol 43

  22. Iqbal M, Saleem MS, Imran M, Khan WA, Ashraf K, Yasir Zahoor M, Rashid I, Rehman HU, Nadeem A, Ali S et al (2020) Single tube multiplex PCR assay for the identification of banned meat species. Food Addit Contam Part B Surveill 13:284–291

    Article  CAS  PubMed  Google Scholar 

  23. Wang X, Yan C, Wei M, Shi C, Niu S, Ma C (2019) On-site Method for Beef Detection based on strand exchange amplification. Anal Sci 35:337–341

    Article  CAS  PubMed  Google Scholar 

  24. Kellner MJ, Koob JG, Gootenberg JS, Abudayyeh OO, Zhang F (2019) SHERLOCK: nucleic acid detection with CRISPR nucleases. Nat Protoc 14:2986–3012

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. da Silva LE, Silva DD, Crispim BD, Vaini JO, Grisolia AB, Seno LD (2013) Variation in concentration of proteinase K for bovine DNA extraction protocols. Arch Vet Sci 18(2):15–19

    Article  Google Scholar 

  26. Ñique AM, Coronado-Marquina F, Mendez Rico JA, García Mendoza MP, Rojas-Serrano N, Simas PV, Cabezas Sanchez C, Drexler JF (2021) A faster and less costly alternative for RNA extraction of SARS-CoV-2 using proteinase K treatment followed by thermal shock. PLoS ONE 16(3):e0248885

    Article  PubMed  PubMed Central  Google Scholar 

  27. Meizarini A, Puteri A, Yasan YD, Hussaini HM (2023) Optimization of proteinase K incubation protocol duration during DNA extraction from oral squamous cell carcinoma FFPE samples. Maj Kedokt Gig 56(4):233–237

    Google Scholar 

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Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or non-profit sectors.

Author information

Author notes

  1. Sana Jafar and Salman Jamil have equally contributed to the study and should be considered as first authors

Authors and Affiliations

  1. Molecular Diagnostics Laboratory, Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore, 54000, Punjab, Pakistan

    Sana Jafar, Salman Jamil, Muhammad Sheraz Yasin, Muhammad Yasir Zahoor, Wasim Shehzad & Muhammad Imran

  2. Institute of Molecular Biology and Biotechnology, The University of Lahore (Sargodha campus), 10-km Lahore road, Sargodha, Punjab, Pakistan

    Asif Naseem

Authors
  1. Sana Jafar
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  2. Salman Jamil
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  3. Muhammad Sheraz Yasin
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  4. Asif Naseem
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  5. Muhammad Yasir Zahoor
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  6. Wasim Shehzad
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  7. Muhammad Imran
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Contributions

M.I. and W.S. conceived, designed and supervised the experiments; M.Y.Z., M.S.Y. and A.N. provided technical support and revised the manuscript; S.J. and S.J. performed experiments, contributed to data analysis and prepared the first draft of the manuscript. All authors critically reviewed the manuscript.

Corresponding author

Correspondence to Muhammad Imran.

Ethics declarations

Ethics approval

This study does not contain experiments that involve human or animals, and the authors have collected all the meat tissue samples from the postmortem department of University of Veterinary and Animal Sciences Lahore.

Declarations of interest

The authors declare that they have no personal or financial competing interest.

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Jafar, S., Jamil, S., Yasin, M.S. et al. Successful application of modified crude DNA extraction from muscle tissues for various types of PCR amplifications. Mol Biol Rep 51, 490 (2024). https://doi.org/10.1007/s11033-024-09356-1

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  • DOI: https://doi.org/10.1007/s11033-024-09356-1

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