Defining the multiplex probe panel for detecting mutating viruses with high clinical sensitivity

Abstract

Nucleic acid technology has emerged as an important diagnostic for infectious diseases, cancer, cardiovascular diseases, and other diseases. However, mismatches between the probes and targets can lead to misdiagnosis. Here we determine how many mismatches between the probe and target lead to poor clinical performance and respond by developing a rationale multiplex strategy to overcome this detection problem. We found that the probe-target mismatches of greater than 20% yielded clinical sensitivity of 22% or less, rendering the diagnostic test useless. We designed probe panels to improve the clinical sensitivity. We tested our multiplex probe strategy using hepatitis C virus as the model pathogen because this virus has high mutation rates. We showed that we can improve the clinical sensitivity for detecting hepatitis C virus from 31 to 89% when we designed and applied a four-probe panel to the diagnostic test instead of a single probe system. Interestingly, increasing beyond four probes did not significantly increase the clinical sensitivity. We present a strategy to overcome the poor clinical sensitivity of nucleic acid tests for mutating genetic targets. Incorporating this panel design strategy can lead to improved diagnostic test performance, fewer false negatives and more accurate treatment planning for patients.