One-Step Multiplex Real-Time Fluorescent Quantitative Reverse Transcription PCR for Simultaneous Detection of Four Waterfowl Viruses

Abstract

Duck Tembusu virus (DTMUV), duck hepatitis virus (DHV), Muscovy duck reovirus (MDRV), and Muscovy duck parvovirus (MDPV) represent four emergent infectious diseases impacting waterfowl, which can be challenging to differentiate due to overlapping clinical signs. In response to this, we have developed a one-step multiplex real-time fluorescence quantitative reverse transcription PCR (qRT-PCR) assay, capable of simultaneously detecting DTMUV, DHV, MDRV, and MDPV. This method exhibits high specificity, avoiding cross-reactivity with other viruses such as Fowl adenoviruses (FADV), infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), Haemophilus paragallinarum (Hpg), duck circovirus (DUCV), goose astrovirus (GoAstV), and mycoplasma gallisepticum (MG). The limit of detection (LOD) established for DTMUV, DHV, MDRV, and MDPV was determined to be 27 copies/μL. In the repeatability test, the intra-assay and inter-assay coefficients of variation (CVs) of the recombinant plasmid standard were less than 2%. Utilizing this method, we analyzed 326 clinical specimens sourced from Guangxi over the period spanning October 2021 through December 2023, yielding promising and precise outcomes. The qRT-PCR method established herein exhibits commendable specificity, sensitivity, and repeatability. Furthermore, it boasts a high clinical detection rate, making it a highly effective tool for diagnosing these pathogenic agents in waterfowl.

Keywords: Muscovy duck parvovirus; Muscovy duck reovirus; duck Tembusu virus; duck hepatitis virus; real-time fluorescent quantitative reverse transcription PCR.

Figure 1

Multiplex qRT-PCR standard curve. Quadratic standard curve showed that there was a linear correlation between the logarithm of copy number and Cq value. The concentration range of standard plasmids (pDTMUV, pDHV, pMDRV, and pMDPV) was 2.68 × 10⁷~2.68 × 10⁰ copies/μL (5.36 × 10⁷~5.36 × 10⁰ copies per reaction).

Figure 2

Multiplex qRT-PCR specificity analysis of different virus strains. Standard recombinant plasmids (pDTMUV, pDHV, pMDRV, and pMDPV), DTMUV, DHV, MDRV, MDPV, and other viruses (FADV, IBDV, IBV, ILTV, Hpg, DUCV, GoAstv, MG) were used for specific detection.

Figure 3

Sensitivity analysis of multiplex qRT-PCR. The sensitivity test was carried out with standard recombinant plasmids (pDTMUV, pDHV, pMDRV, and pMDPV). Curve 1–8: 2.68 × 10⁶~2.68 × 10⁻¹ copies/μL (final reaction concentration).