Establishment of a sensitive and specific hyper-branched rolling circle amplification assay and test strip for TSV

Abstract

A specific padlock probe (PLP) and detection probe were designed to target the capsid protein gene of Taura syndrome virus (TSV), and a hyper-branched rolling circle amplification (HRCA) assay and a corresponding strip-based test were produced. The reaction time and temperature were optimized for both. The PLPs were linked with the target sequence via T4 DNA ligase under conditions of 37°C for 30 min, followed by reaction with Bst DNA polymerase Large Fragment at 61°C for 30 min, and then the test strip was made using the detection probe. Both the assay and the strip had similarly high accuracy and specificity, and their sensitivity was close to 10 copies, which is 100 times higher than that of conventional RT-PCR. We tested 89 batches of shrimps for TSV to assess the HRCA assay and test strip; the results indicated that the TSV HRCA assay and the test strip are rapid diagnostic tools for detection in the field, and have potential for early diagnosis of TSV.

Keywords: HRCA; HRCA test strip; PLP; RT-PCR; Shrimp; TSV.