Development of a Quantitative Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of Novel Goose Parvovirus.

Jing Yang,Xiaoyu Niu,Youxiang Diao,Zhenzhong Wang,Yi Tang,Xianglong Yu,Hao Chen

doi:10.3389/fmicb.2017.02472

Abstract

An infectious disease characterized with short bills and protruding tongues has attacked to meat ducks in China since March 2015, which has caused ducks poor growth and enormous economic losses to duck industry of China. It was eventually proved to be caused by parvovirus after pathogen isolation and identification. As the genomic sequence analysis showed, this pathogen shared 90.8–94.6% of nucleotide identity with goose parvovirus (GPV), and it was called duck-origin novel goose parvovirus (N-GPV). In this study, a quantitative loop-mediated isothermal amplification (qLAMP) assay was developed for the rapid diagnosis of N-GPV. A set of four specific primers, two inner and two outer, were designed targeting at VP3 gene, which could be completed within 60 min at 65°C in water bath or on a real-time PCR instrument for quantitative analysis. Specificity test of LAMP assay showed that there was no cross-reactivity between N-GPV and other duck pathogens, and the detection limit of qLAMP assay was 1.0 × 102 copies/μL. The repeatability of this method was confirmed by inter-assay and intra-assay tests with variability ranging from 0.74 to 2.25%. The results have indicated that the qLAMP assay was a simple, rapid, accurate, sensitive, and specific method for detecting N-GPV, especially on field detection.

Highlights

Waterfowl parvovirus causes high morbidity and mortality in geese and Muscovy ducks, and mortality rate ranges from 10 to 80%
The results showed that our assay could only detect N-goose parvovirus (GPV) and GPV, while no amplification signals were detected in other viruses (Figure 4A)
Phylogenetic trees based on VP3 genes showed that the SDLC strains were in the same branch with European GPV and vaccine isolates, and shared 93.4–98.9% identity with GPV isolates, while only shared 80.4–88.7% with Muscovy duck parvovirus (MDPV), which indicated that evolutionary relationship of SDLC strain is closer to GPV stains, and N-GPV is a novel variant of GPV

Summary

Introduction

Waterfowl parvovirus causes high morbidity and mortality in geese and Muscovy ducks, and mortality rate ranges from 10 to 80% These diseases can lead to enteric symptoms, watery diarrhea, prostration, and growth retardation, resulting in serious economic losses to waterfowl industry (Glávits et al, 2005). In 2015, a disease, which emerged in France and Poland in the early 1970s in mule and Muscovy ducks was first reported in Chinese mainland (Chen et al, 2015). It mainly caused short beak, protruding tongues, fragile tibia and pteroid, and growth retardation to commercial ducks. In order to identify the exact pathogen that caused this disease, pathogen isolation and identification and genomic sequence

Methods

Results

Discussion

Conclusion