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Analysis of the Frequency of Mutations at Diagnostic Oligonucleotide Sites and Their Impact on the Efficiency of PCR for HIV-1.

Elena Vladimirovna BogoslovskayaGalina M TsyganovaAnastasiia O NosovaGerman A Shipulin
Published in: Microorganisms (2023)
The development of effective diagnostic kits for HIV-1 remains a pressing concern. We designed diagnostic oligonucleotides for HIV-1 real-time PCR to target the most conserved region of the HIV-1 genome and assessed the mutation frequency at annealing sites. Two databases of nucleotide sequences, Los Alamos and NCBI, were analyzed, revealing that more than 99% of the sequences either lack mutations or contain 1-2 mutations at the binding site of the forward and reverse primers. Additionally, 98.5% of the sequences either lack mutations or contain 1-2 mutations at the binding site of the TaqMan probe. To evaluate the efficiency of primers and the probe in real-time PCR in the case of mutations at their binding sites, we constructed several plasmids containing the most common mutations and, in a model experiment, showed how different mutations affect the efficiency of PCR. Our analysis demonstrated that about 98.5% of HIV-1 strains can be efficiently detected using a single pair of selected primers. For the remaining 1.5% of strains, a more careful selection of the second target is needed.
Keyphrases
  • real time pcr
  • antiretroviral therapy
  • hiv positive
  • hiv infected
  • hiv testing
  • human immunodeficiency virus
  • hepatitis c virus
  • hiv aids
  • men who have sex with men
  • escherichia coli
  • living cells
  • fluorescent probe