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Development and validation of a universal primer pair for the taxonomic and phylogenetic studies of vertebrates

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Abstract

Background

Recent studies in the field of molecular identification have described 16S rRNA gene as a highly informative fragment of mitochondrial DNA for species discrimination. This study presents a newly developed universal primer pair yielding an approximately 350 bp fragment of mitochondrial 16S rRNA, variable enough to encompass and identify all vertebrate classes.

Methods and results

The primers were designed by aligning and analyzing over 1500 16S rRNA sequences downloaded from the NCBI nucleotide database. A total of 93 vertebrate species, spanning 27 orders and 55 families, were PCR-amplified to validate the primers. All the target species were successfully amplified and identified when aligned with reference sequences from the NCBI nucleotide database. Using the Kimura 2-parameter model, low intra-species genetic divergence of the target region was observed – from 0 to 4.63%, whereas relatively higher inter-species genetic divergence was observed, ranging from 4.88% to 69.81%. Moreover, the newly developed primers were successfully applied to a direct PCR protocol, making the workflow very cost-effective, time-saving and less laborious in comparison to conventional PCR.

Conclusions

The short length, high variability and conserved priming sites of the target fragment across all vertebrate species make it a highly desirable marker for species identification and discrimination.

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Data availability

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

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Funding

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

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Authors and Affiliations

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

    Sana Jafar, Muhammad Yasir Zahoor, Wasim Shehzad & Muhammad Imran

  2. Department of Wildlife and Ecology, University of Veterinary and Animal Sciences, Ravi Campus, Pattoki, Kasur, Punjab, Pakistan

    Khalid Mahmood Anjum

  3. Department of Zoology, Institute of Molecular Biology and Biotechnology, The University of Lahore (Sargodha Campus), Sargodha, Punjab, Pakistan

    Asif Naseem

Authors
  1. Sana Jafar
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  2. Khalid Mahmood Anjum
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  3. Muhammad Yasir Zahoor
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Contributions

SJ carried out the experiments, collected the data, performed data analysis and wrote the manuscript. WS and KMA supervised and verified the experimental results and revised the manuscript. MYZ and AN provided technical support and revised the manuscript. MI designed the methodology, performed data analysis, supervised the experiments and revised the manuscript. All authors have read and approved the final manuscript.

Corresponding author

Correspondence to Muhammad Imran.

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Competing interests

The authors declare no competing interests.

Ethical approval

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

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Jafar, S., Anjum, K.M., Zahoor, M.Y. et al. Development and validation of a universal primer pair for the taxonomic and phylogenetic studies of vertebrates. Mol Biol Rep 51, 332 (2024). https://doi.org/10.1007/s11033-023-09175-w

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