Zika Virus Detection in Semen Using rRT-PCR Assay

The patent extracts and amplifies only the specific viral genetic sequences of interest from the complex virus isolation and serology processes. By using PCR to amplify targeted viral DNA regions, the method isolates the critical diagnostic information from the lengthy traditional diagnostic workflow, achieving rapid detection without sacrificing accuracy.

The patent replaces the mechanical and biochemical processes of virus isolation and serology with a molecular biology-based PCR detection system. This substitution uses temperature-cycled DNA amplification and hybridization detection to replace traditional cell culture and antibody-based methods, dramatically reducing testing time while maintaining diagnostic precision.

The patent performs preliminary amplification of viral genetic material from semen samples before detection. By using PCR to amplify trace amounts of viral DNA present in semen to detectable levels, the method prepares the sample in advance to overcome the low viral titer challenge, enabling subsequent sensitive detection that would otherwise be impossible with conventional methods.

The patent changes the detection parameter from direct viral particle detection to amplified genetic sequence detection. By transforming the detectable parameter through PCR amplification, the method increases the effective concentration of target material from low viral titers in semen to detectable levels, thereby improving measurement precision without requiring changes to sample collection or handling procedures.

The patent implements continuous viral DNA amplification through multiple PCR cycles, maintaining the useful action of target sequence replication throughout the detection process. This continuous amplification ensures that even trace amounts of persistent low-titer viral DNA in semen samples are progressively enriched to detectable levels, achieving both rapid detection and high sensitivity simultaneously.

The patent uses nested PCR primers that bind to overlapping regions of the viral genome, creating a nested amplification system. This nested structure allows for highly specific and sensitive detection by requiring two separate binding events, reducing false positives while maintaining rapid detection capability. The nested design enables detection of persistent low-titer infections through cumulative amplification of specific viral sequences.