Steps for Using a HEV Test
1. Sample Preparation
Specimen Types: Serum (preferred), plasma (EDTA), or stool* (*RNA testing only)
Processing Requirements:
Centrifuge blood samples at 3000×g for 10 min
For stool samples: prepare 10% suspension in PBS
Store at 2-8°C (≤24h) or -20°C (long-term)
2. Test Procedures
A. Rapid Antibody Test (Lateral Flow)
Equilibrate kit to room temperature (15-30°C)
Add 50μL serum/plasma to sample well
Immediately add 2 drops running buffer
Interpret at 15 min (IgM/IgG separate lines)
B. ELISA (IgM/IgG)
Load 100μL samples/controls into antigen-coated wells
Incubate:
IgM: 60 min at 37°C + RF absorbent
IgG: 30 min at 37°C
Wash 5× with 300μL wash buffer
Add HRP-conjugate, incubate 30 min at 37°C
Develop with TMB for 10 min, stop with H₂SO₄
Read at 450/630nm
C. RT-PCR (RNA Detection)
Extract RNA using viral RNA kit
Prepare reaction mix:
5μL RNA + 15μL master mix (ORF2/3 primers)
Run real-time PCR:
Reverse transcription: 50°C for 15 min
Amplification: 45 cycles of 95°C/10 sec → 60°C/45 sec
3. Interpretation
Rapid Test:
Positive: Control line + IgM/IgG line(s)
Acute infection: IgM+ only
Past infection: IgG+ only
ELISA:
Cutoff = NC mean + 0.3 (IgM) / 0.2 (IgG)
Gray zone: Repeat with fresh sample
PCR:
Positive: Ct ≤40 with S-shaped curve
Quantitation: Use standard curve (IU/mL)
4. Quality Control
Include HEV-positive serum and no-template controls
For PCR: Monitor internal control amplification
Record lot numbers and expiration dates
Critical Notes:
• IgM tests may cross-react with CMV/RFV
• RNA testing is essential for immunocompromised patients
• Heat-inactivate serum at 56°C for 30 min if RF interference suspected
