Visual detection and differentiation of Classic Swine Fever Virus strains using nucleic acid sequence-based amplification (NASBA) and G-quadruplex DNAzyme assay

Xiaolu Lu,Tiantian Wu,Yi Wang,Rui Qin,Xueyao Shi,Gege Wu

doi:10.1038/srep44211

Abstract

The split G-quadruplex DNAzyme has emerged as a valuable tool for visual DNA detection. Here, we successfully integrated colorimetric split G-quadruplex DNAzyme assay with nucleic acid sequence-based amplification to generate a novel detection approach, allowing visual and rapid detection for the RNA of Shimen and HCLV strains of Classic Swine Fever Virus (CSFV). CSFV is a RNA virus that causes a highly contagious disease in domestic pigs and wild boar. With this method, we were able to detect as little as 10 copies/ml of CSF viral RNA within 3 h in serum samples taken from the field. No interference was encountered in the amplification and detection of Classic Swine Fever Virus in the presence of non-target RNA or DNA. Moreover, Shimen and HCLV strains of Classic Swine Fever Virus could be easily differentiated using the NASBA-DNAzyme system. These findings indicate the NASBA-DNAzyme system is a rapid and practical technique for detecting and discriminating CSFV strains and may be applied to the detection of other RNA viruses.

Highlights

The G-quadruplex/hemin complex, acting as an activated horseradish peroxidase (HRP)-mimicking DNAzyme, can catalyze the oxidation of 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS2−) by H2O2 to produce a colorimetric signal that can be visualized by the naked eye[5,6,7,8,9]
We developed a novel method by integrating Nucleic acid sequence-based amplification (NASBA) and split G-quadruplex DNAzyme analysis to detect and differentiate Classic swine fever virus (CSFV) Shimen and hog cholera virus (HCLV) strains
Traditional methods for CSFV detection relied on virus isolation or serological diagnosis

Summary

Introduction

The G-quadruplex/hemin complex, acting as an activated horseradish peroxidase (HRP)-mimicking DNAzyme, can catalyze the oxidation of 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS2−) by H2O2 to produce a colorimetric signal that can be visualized by the naked eye[5,6,7,8,9]. We utilized the colorimetric split G-quadruplex DNAzyme system combined with NASBA technique to generate a novel CSFV detection approach This approach adopted a split probe targeting system, designed with G-rich sequences, which reassembles in the presence of target RNA amplified by NASBA, and producing G-quadruplexes with a catalytic activity. With this approach, we could detect Shimen strain (the most widespread and highly virulent wide type CSFV strain in China) in serum samples and distinguish them from the HCLV strain (the most used vaccine strain in China) within 3 hours through a simple colorimetric change

Methods

Results

Conclusion