TY - JOUR
T1 - Single nucleotide polymorphism-mismatch primer development for rapid molecular identification of selected microalgal species
AU - Park, Sang Hyuck
AU - Steichen, Seth Alan
AU - Brown, Judith K.
N1 - Funding Information:
This project was funded by the US Department of Energy Office (DOE) of Biomass Program and was affiliated with the National Alliance for Advanced Biofuels and Bioproducts or NAABB consortium grant DE-EE0003046, and by DOE RAFT Project, funded by grant DE-EE0006269.
Funding Information:
The authors thank Dr. Juergen Polle, City University of New York, Brooklyn, for providing the Chlorella sorokiniana DOE1412 culture used in this study. We also thank the University of Arizona-ARID reactor cultivation team members for assistance in algal cultivation and harvesting
Publisher Copyright:
© 2021, The Author(s), under exclusive licence to Springer Nature B.V. part of Springer Nature.
PY - 2021/6
Y1 - 2021/6
N2 - Microalgae have been a great source for food, cosmetic, pharmacological, and biofuel production. The adoption of effective diagnostic assays for monitoring all stages of algal cultivation has become essential. In addition to microscopy identification, molecular assays can aid greatly in the identification and monitoring of algal species of interest. In this study the 18S ribosomal RNA (rRNA) sequences of 12 microalgal species and/or strains were used to design algal identification primers. Sequence alignment revealed five highly variable regions and multiple unique single nucleotide polymorphisms (SNPs). To design target algae specific primers, a SNP identified as unique to each microalgal species was incorporated into the 3’-terminus of forward and reverse primer pairs, respectively. To further enhance primer specificity, transverse mutation was introduced into each primer at the third base upstream of the respective SNP. The SNP-mismatch primer pairs yield size-specific amplicons, enabling the rapid molecular detection of 12 microalgae by circumventing cloning and sequencing. To verify the primer specificity, two SNP-mismatch primer pairs designed for Chlorella sorokiniana DOE1412 and wildtype species of Scenedesmus were tested in the outdoor reactor run inoculated with C. sorokiniana DOE1412. The primer pairs were able to identify C. sorokiniana DOE1412 as well as the environmental invader Scenedesmus sp. Furthermore, the “relative concentration” of two microalgae was accessed throughout the entire cultivation run. The use of SNPs primers designed in this study offers a cost-effective, easy to use alternative for routine monitoring of microalgal cultures in laboratories, in scale-ups, and in cultivation reactors, independent of the production platform.
AB - Microalgae have been a great source for food, cosmetic, pharmacological, and biofuel production. The adoption of effective diagnostic assays for monitoring all stages of algal cultivation has become essential. In addition to microscopy identification, molecular assays can aid greatly in the identification and monitoring of algal species of interest. In this study the 18S ribosomal RNA (rRNA) sequences of 12 microalgal species and/or strains were used to design algal identification primers. Sequence alignment revealed five highly variable regions and multiple unique single nucleotide polymorphisms (SNPs). To design target algae specific primers, a SNP identified as unique to each microalgal species was incorporated into the 3’-terminus of forward and reverse primer pairs, respectively. To further enhance primer specificity, transverse mutation was introduced into each primer at the third base upstream of the respective SNP. The SNP-mismatch primer pairs yield size-specific amplicons, enabling the rapid molecular detection of 12 microalgae by circumventing cloning and sequencing. To verify the primer specificity, two SNP-mismatch primer pairs designed for Chlorella sorokiniana DOE1412 and wildtype species of Scenedesmus were tested in the outdoor reactor run inoculated with C. sorokiniana DOE1412. The primer pairs were able to identify C. sorokiniana DOE1412 as well as the environmental invader Scenedesmus sp. Furthermore, the “relative concentration” of two microalgae was accessed throughout the entire cultivation run. The use of SNPs primers designed in this study offers a cost-effective, easy to use alternative for routine monitoring of microalgal cultures in laboratories, in scale-ups, and in cultivation reactors, independent of the production platform.
KW - Microalgae
KW - Molecular diagnostics
KW - Quality control
KW - Single nucleotide polymorphism
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U2 - 10.1007/s10811-021-02409-z
DO - 10.1007/s10811-021-02409-z
M3 - Article
AN - SCOPUS:85101552455
SN - 0921-8971
VL - 33
SP - 1685
EP - 1694
JO - Journal of Applied Phycology
JF - Journal of Applied Phycology
IS - 3
ER -