Development of a One-Step Real-Time TaqMan Reverse Transcription Polymerase Chain Reaction (RT-PCR) Assay for the Detection of the Novel Variant Infectious Bursal Disease Virus (nVarIBDV) Circulating in China

Abstract

The novel variant IBDV (nVarIBDV, especially genotype A2dB1) mainly affects broilers in China. It causes an infection characterized by the atrophy of the bursa, a decrease in the level of lymphocytes, proliferation of fibrous tissue around the follicle, and severe atrophy of the follicle in the bursa. Poultry vaccinated with live IBDV vaccines do not have the challenge present with bursa atrophy, which is misdiagnosed for nVarIBDV because of the lack of other gross clinical symptoms. The present study sought to explore the potential and reliability of the real-time TaqMan analysis method for the detection and discrimination of the nVarIBDV genotype from that of the non-nVarIBDV, especially in live vaccine strains. This method will help monitor vaccinated poultry to control and manage infection with the nVarIBDV IBDVs. The nucleotide polymorphism in the 5’-UTR region and the vp5/vp2 overlapping region of the segment A sequences of IBDV were used to establish a one-step real-time TaqMan reverse transcription polymerase chain reaction (RT-PCR) method in this study. The results showed that the method accurately distinguished the nVarIBDV and non-nVarIBDV strains (especially live vaccine strains), and there were no cross-reactions with the infectious bronchitis virus (IBV), Newcastle disease virus (NDV), avian influenza virus (AIV), infectious laryngotracheitis virus (ILTV), fowlpox virus (FPV), Mycoplasma gallisepticum (M. gallisepticum), Mycoplasma synoviae (M. synoviae), and IBDV-negative field samples. The method showed a linear dynamic range between 102 and 107 DNA copies/reaction, with an average R2 of 0.99 and an efficiency of 93% for nVarIBDV and an average R2 of 1.00 and an efficiency of 94% for non-nVarIBDV. The method was also used for the detection of 84 clinical bursae of chickens vaccinated with the live vaccine. The results showed that this method accurately distinguished the nVarIBDV and non-nVarIBDV strains (vaccine strains), compared with a strategy based on the sequence analysis of HVRs at the vp2 gene or the reverse transcription PCR (RT-PCR) for the vp5 gene. These findings showed that this one-step real-time TaqMan RT-PCR method provides a rapid, sensitive, specific, and simple approach for detection of infections caused by nVarIBDV and is a useful clinical diagnostic tool for identifying and distinguishing nVarIBDV from non-nVarIBDV, especially live vaccine strains.

Establishment and Application of a Quadruplex Real-Time Reverse-Transcription Polymerase Chain Reaction Assay for Differentiation of Porcine Reproductive and Respiratory Syndrome Virus, Porcine Circovirus Type 2, Porcine Circovirus Type 3, and Streptococcus suis

“…The quadruplex real-time quantitative PCR test established in this study comprehensively and simultaneously detects multiple PRDC pathogens, rapidly confirming their presence in samples. Moreover, the quadruplex PCR method, detecting four common pathogens in a single reaction, substantially reduces costs compared to individual PCRs or uniplex real-time PCR, thus enhancing overall work efficiency [ 30 ]. This has important implications for the prevention and control of these four diseases in clinical practice.…”

Advancing IBDV diagnostics: a one-step multiplex real-time qRT-PCR for discriminating between vvIBDV and non-vvIBDV viruses, including the newly emerged IBDV variant

“…These include nanoparticle-assisted PCR, SYBR green, and TaqMan-based real-time RT-PCRs (RT-qPCR) ( 18 – 21 ). Including a recent study developed a real-time one-step qRT-PCR assay, this assay can distinguish the nVarIBDV from other non-nVarIBDV strains based on nucleotide polymorphisms in the 5’-UTR and the vp5/vp2 overlapping region of the segment A sequences ( 22 ). Despite the widespread use of the nVarIBDV in several countries, including China, Japan, South Korea, Malaysia, and Egypt ( 6 , 23 – 26 ) the vvIBDV remains a significant threat to poultry production ( 25 , 27 ) due to its high replication efficiency and rapid spread ( 4 ) and its rapid accurate differentiation from classical strains ultimately required to assess the vaccination efficiency.…”

The booster immunization using commercial vaccines effectively protect chickens against novel variants of infectious bursal disease virus (genotype A2dB1)

“…Viral RNA extraction was performed using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. For specific detection of the nVarIBDV FJ2019-01 or vaccine strain, the one-step real-time TaqMan RT-PCR assay was performed on a LightCycler96 (Roche, Basel, Switzerland) as described previously ( Wang et al, 2023 ). Briefly, the 20 μL reaction contained 5 μL Fast 1-Step Mix (4 ×) (ThermoFisher, Lithuania, Vilnius), 0.5 μL forward or reverse primer (10 μM; v-F [5′-CCT CCT TCT AYA RYG CTR TCA T-3′], and v-R [5′-CGT ATG AAC GGA ACA ATC TG-3′]), 0.25 μL probe (10 μM; vac-P [5′-FAM-AGT AGA GAT CAG ACA AA-MGB-3′] and nVar-P [5′-VIC-TAG AGA TCA GAC GAA CG-MGB-3′]), 5 μL template RNA or plasmid DNA, and 8.5 μL deionized distilled water.…”