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A simple and rapid approach for human herpesvirus type 8 subtype characterization using single base extension
M L Hulaniuk1, D Corach2,3, J Trinks1,2
1Instituto de Medicina Traslacional e Ingeniería Biomédica (IMTIB), CONICET, Instituto Universitario del Hospital Italiano (IUHI), Hospital Italiano (HIBA), Buenos Aires, Argentina.
Letters in Applied Microbiology
|May 28, 2021
Summary
A new single base extension (SBE) method accurately genotypes human herpesvirus type 8 (HHV-8) ORFK1 subtypes. This rapid, low-cost technique offers an efficient alternative for viral genotyping, even with low-quality DNA.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Human herpesvirus type 8 (HHV-8) ORFK1 sequence analysis identifies six subtypes (A-F) linked to Kaposi's sarcoma progression.
- Phylogenetic analysis of ORFK1 is the standard for HHV-8 genotyping but presents challenges.
Purpose of the Study:
- To design and validate a single base extension (SBE) protocol for efficient HHV-8 ORFK1 subtype characterization.
- To provide a rapid, cost-effective, and reliable method for HHV-8 genotyping.
Main Methods:
- A nested polymerase chain reaction (PCR) amplified a 294-bp fragment of HHV-8 ORFK1 containing four single nucleotide polymorphisms.
- A multiplex single base extension (SBE) technique was developed and validated against 20 samples previously genotyped by phylogenetic analysis.
Main Results:
- The SBE protocol successfully discriminated between HHV-8 ORFK1 subtypes.
- Typing results from SBE completely matched the 'gold standard' phylogenetic method in all samples.
- The method reliably identified prevalent subtypes A, B, and C, as well as subtypes D, E, and F.
Conclusions:
- Single base extension (SBE) is an efficient, rapid, and low-cost screening method for HHV-8 genotyping.
- SBE is particularly useful for samples with low-quality DNA and offers easy data interpretation.
- This validated SBE protocol provides a reliable alternative for HHV-8 ORFK1 subtyping.
