Evaluation of a sensitive reverse transcription PCR-enzymelinked immunosorbent assay for detection of hepatitis A virus in oysters (Saccostrea glomerata) on the east coast of the Gulf of Thailand.
Hepatitis A virus (HAV) contamination in food can lead to major health problems. We developed a combination reverse transcription (RT) PCR method plus enzyme-linked immunosorbent assay (ELISA) to detect HAV in fresh oysters harvested along the east coast of the Gulf of Thailand. Viral nucleic acid was extracted via the glycine-arginine-polyethylene glycol method followed by RT-PCR amplification with specifically designed primers against HAV and an ELISA to detect the digoxigenin-labeled RT-PCR products. The ELISA in concert with the RT-PCR protocol further increased the detection sensitivity by 100-fold for the HAV genome and 10-fold in artificially contaminated oysters. The overall sensitivity of the RT-PCR in combination with the ELISA was 31.88 pg and 16 PFU/g, respectively. The ELISA increases the specificity of the RT-PCR assay for detecting naturally occurring HAV in oysters. This combined RT-PCR-ELISA approach is a practical and sensitive method for HAV detection and can be utilized in routine screening for HAV in shellfish.
Faculty of Allied Health Sciences, Burapha University, 169 Longhard-Bansaen Road, Bangsaen, Chon Buri 20131, Thailand. uintamaso@yahoo.com uraiwani@buu.ac.th.
Uraiwan
U
Intamaso
Faculty of Allied Health Sciences, Burapha University, 169 Longhard-Bansaen Road, Bangsaen, Chon Buri 20131, Thailand.
Sitthisak
S
Ketkhunthod
D000818:Animals; D017931:DNA Primers / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005506:Food Contamination / Q000032:analysis*; D030041:Hepatitis A virus / Q000235:genetics / Q000302:isolation & purification*; D010125:Ostreidae / Q000821:virology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012758:Shellfish / Q000821:virology*; D013785:Thailand
D017931:DNA Primers