Quantification of hepatitis A virus in shellfish by competitive reverse transcription-PCR with coextraction of standard RNA

Abstract

To quantify hepatitis A virus (HAV) in experimentally contaminated mussels, we developed an internal standard RNA with a 7-nucleotide deletion for competitive reverse transcription (RT)-PCR. Deposited directly into the sample, this standard was used both as extraction control and as quantification tool. After coextraction and competitive RT-PCR, standard and wild-type products were detected by differential hybridization with specific probes and a DNA enzyme immunoassay. The quantifiable range with this reproducible method was 10(4) to 10(7) copies of HAV/gram or 400 to 10(6) 50% tissue culture infective doses/ml.

FIG. 1

Construction of IS-RNA for use in quantitative HAV RT-PCR. After cDNA synthesis using antisense primer (HAL2), a 7-bp shortened DNA fragment with a 10-nucleotide extended antisense primer in which 7 internal nucleotides had been deleted (HALS1) was generated by PCR. Deleted product was reamplified using a sense primer (HALS2) carrying a 34-nucleotide SP6 RNA polymerase recognition sequence (SP6) and a biotin label at its 5′ end (B). Avidin-coated magnetic beads (Bead) were used to purify the product of the second round of amplification. The supernatant was replaced, and the in vitro transcription mixture was added directly to the solid phase. After reaction, beads retaining DNA template were trapped in the magnetic field (Magnet), and the supernatant containing RNA molecules was recovered. The RNA standard obtained harbors a seven-nucleotide deletion and could be amplified with the same primers (HAL1 and HAL2) as wild-type HAV.

FIG. 2

Estimation of linearity as a function of concentration of virus in the sample for the two quantification protocols. Results obtained with 2 × 10³ copies of IS-RNA in the RT-PCR tube (A) or with 2 × 10⁵ copies of IS-RNA added directly to the shellfish homogenate (B) are shown.