Advanced Techniques in Diagnostic Microbiology

16. Direct Nucleotide Sequencing 273
eight untreated HIV-1–infected individuals. The method had a limit of detection
of 20% to 25% for minor sequence variants. Pattern recognition (i.e., comparing
actual sequence data with expected wild-type and mutant sequence patterns) simplified
the identification of minor sequence variants. This real-time pyrosequencing
method was applied in a longitudinal study monitoring the development of PI resistance
in plasma samples obtained from four patients over a 2 1 -year period.
2
Pyrosequencing identified eight primary protease inhibitor resistance mutations as
well as several secondary mutations.
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