Real-Time PCR-Based Detection of Hepatitis E Virus in Groundwater: Primer Performance and Method Validation.

Area of Science:

• Environmental Science
• Molecular Biology
• Public Health

Background:

• Hepatitis E virus (HEV) is a major cause of viral hepatitis.
• Transmission primarily occurs through contaminated water and food.
• Groundwater is a potential, yet understudied, HEV transmission vector.

Purpose of the Study:

• To optimize real-time polymerase chain reaction (rtPCR) for sensitive HEV detection.
• To compare TaqMan probe-based and SYBR Green-based rtPCR methods for HEV identification.
• To evaluate primer set performance for HEV detection in environmental samples.

Main Methods:

• Evaluated 12 primer sets for TaqMan probe-based rtPCR.
• Screened 41 primer sets for SYBR Green-based rtPCR.
• Assessed sensitivity and specificity using artificially spiked samples.

Main Results:

• Primer set #4 (TaqMan) and #21 (SYBR Green) showed optimal sensitivity and specificity.
• Both optimized methods detected HEV at 1 fg/μL in spiked samples.
• TaqMan offered rapid detection with low non-specific amplification; SYBR Green allowed broader primer screening via melting curve analysis.

Conclusions:

• Optimized rtPCR methods provide sensitive and specific HEV detection.
• These methods are valuable for environmental monitoring of HEV.
• The study contributes to developing enhanced diagnostic tools for public health surveillance of HEV.