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Updated: May 5, 2026
Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis
Published on: February 5, 2014
Rapid molecular typing of Prototheca zopfii by high resolution melting real-time PCR (PCR-HRM)
Hideto Sobukawa1, Masato Ibaraki, Rui Kano
1Department of Pathobiology, Nihon University School of Veterinary Medicine.
Abstract:
Prototheca zopfii is an achlorophyllic alga that is ubiquitous around cow sheds. The alga is associated with bovine mastitis, which causes a reduction in milk production and secretion of thin watery milk containing white flakes. Isolates of P. zopfii from bovine mastitis were almost all identified as P. zopfii genotype 2, suggesting that it is the main causative agent of bovine protothecal mastitis. The ability to differentiate between genotype 1 and genotype 2 is therefore very important for preventing bovine mastitis. In this study, high resolution melting real-time PCR (PCR-HRM) analysis of the protothecal 18S rDNA domain successfully differentiated between genotypes of P. zopfii in less than 3 hours, while conventional sequence analysis requires more than 48 hours to differentiate between genotypes. PCR-HRM analysis clustered P. zopfii genotype 1 isolates separately from P. zopfii genotype 2 isolates, indicating that this molecular typing method is an effective tool for rapidly diagnosing bovine protothecal mastitis.
Insights
Prototheca zopfii causes bovine mastitis, with genotype 2 being the main culprit. High-resolution melting real-time PCR rapidly differentiates P. zopfii genotypes, aiding in diagnosing this costly dairy cattle disease.
Area of Science:
- Veterinary Microbiology
- Molecular Diagnostics
Background:
- Prototheca zopfii, an achlorophyllic alga, is linked to bovine mastitis, impacting milk production.
- Bovine mastitis caused by P. zopfii leads to reduced milk yield and abnormal milk secretion.
- Most bovine mastitis isolates are P. zopfii genotype 2, highlighting its significance.
Purpose of the Study:
- To develop a rapid method for differentiating P. zopfii genotypes 1 and 2.
- To establish an effective diagnostic tool for bovine protothecal mastitis.
Main Methods:
- High-resolution melting real-time PCR (PCR-HRM) analysis targeting the protothecal 18S rDNA domain.
- Conventional sequence analysis for comparison.
Main Results:
- PCR-HRM successfully differentiated P. zopfii genotypes within 3 hours.
- Conventional sequencing required over 48 hours for genotype differentiation.
- PCR-HRM analysis effectively clustered P. zopfii genotype 1 and genotype 2 isolates separately.
Conclusions:
- PCR-HRM is a rapid and effective molecular typing method for P. zopfii.
- This technique enables quick diagnosis of bovine protothecal mastitis.
- Differentiating P. zopfii genotypes is crucial for managing and preventing bovine mastitis.
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