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. 2025 Jul 30;26(15):7377.
doi: 10.3390/ijms26157377.

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

Jin-Ho Kim  1   2 , Siwon Lee  3 , Eung-Roh Park  4
Affiliations

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

Jin-Ho Kim et al. Int J Mol Sci. .

Abstract

Hepatitis E virus (HEV) is a leading cause of acute viral hepatitis and is primarily transmitted via contaminated water and food. Groundwater may also serve as a potential vector for HEV transmission. This study aimed to optimize real-time polymerase chain reaction (rtPCR) for the detection of HEV, employing both TaqMan probe- and SYBR Green-based methods. A total of 12 primer sets for the TaqMan probe-based method and 41 primer sets for the SYBR Green-based method were evaluated in order to identify the optimal combinations. Primer sets #4 (TaqMan probe-based) and #21 (SYBR Green-based) demonstrated the highest sensitivity and specificity, successfully detecting HEV in artificially spiked samples at 1 fg/μL. The TaqMan probe-based method facilitated rapid detection with minimized non-specific amplification, whereas the SYBR Green-based method allowed for broader primer exploration by leveraging melting curve analysis. Despite the absence of HEV detection in 123 groundwater samples, this study underscores the value of real-time PCR for environmental monitoring and paves the way for enhanced diagnostic tools for public health applications.

Keywords: groundwater monitoring; hepatitis E virus (HEV); molecular diagnostics; real-time (rt) PCR; viral detection.

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Conflict of interest statement

Siwon Lee was employed by the company LSLK Co., Ltd. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.

Figures

Figure 1
Figure 1
Sensitivity results of three HEV SYBR Green-based method primer sets using artificially spiked samples (ASSs).
Figure 2
Figure 2
Validation results of the HEV TaqMan probe-based real-time (rt) PCR assay by three analysts.
See this image and copyright information in PMC

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