A snapshot on molecular technologies for diagnosing FAdV infections

Abstract

Fowl adenoviruses (FAdV) are prevalent in chickens worldwide, responsible for several poultry diseases, including inclusion body hepatitis (IBH), hepatitis-hydropericardium syndrome (HHS), and gizzard erosion (GE), which result in significant economic losses in the poultry industry. Consequently, detection and efficient identification of FAdV serotypes are becoming extremely urgent to monitor outbreaks and develop vaccination strategies. Conventional PCR (cPCR) tests, combined with Restriction Fragment Length Polymorphism (RFLP) or sequencing, were developed for FAdV diagnosis. Although these molecular tests have considerably improved the accuracy of FAdV diagnosis compared with conventional methods, certain drawbacks remain unresolved, including lack of sensitivity and post-PCR analysis. Subsequently, advanced molecular technologies such as real-time PCR (qPCR), Loop Isothermal Amplification (LAMP), Cross-Priming Amplification (CPA), Recombinase Polymerase Amplification (RPA), Digital Droplet Polymerase Chain Reaction (ddPCR), Dot Blot Assay Combined with cPCR, Nanoparticle-Assisted PCR (nano-PCR), PCR-Refractory Quantitative Amplification (ARMS-qPCR), CRISPR/Cas13a Technology, and High-Resolution Melting Curve (HRM), have been developed to improve FAdV diagnosis.

Keywords: CRISPR/Cas13; HRM; fowl adenovirus; genotyping; isothermal amplification; molecular diagnosis; polymerase chain reaction; real-time PCR.

Figure 1

(A) Position of the LAMP primers on the FAdV target gene. (B) Schematic representation of the Loop-Mediated Isothermal Amplification (LAMP) technique. (C) Methods used to detect the LAMP product, including End-point detection (using electrophoresis, addition of fluorescent substrates, turbidity measurement, or lateral flow dipstick or (real-time detection)). The gel image presented in this figure are adapted from previous work (81) and are included to ensure the figure is comprehensive and meaningful.

Figure 2

Schematic of single cross-priming amplification technology. (A) Primer design site. (B) Generation of template with cross-primer sites.

Figure 3

(A) RPA primer design. (B) Schematic of the recombinase polymerase amplification technique workflow and product detection by Electrophoresis. The gel images presented in this figure are adapted from previous work (101) and are included to ensure the figure is comprehensive and meaningful.

Figure 4

Workflow for detecting vaccine contamination by FAdV-4 using Digital Droplet PCR.

Figure 5

Diagram illustrating the use of ARMS-qPCR to discriminate between the CELO strain and the PA7127 challenge strain.

Figure 6

CRISPR-Cas13a DNA combined with lateral flow strips for FAdV-4 detection workflow. (A) Recombinase Polymerase Amplification (RPA) performed at a single temperature of 37°C. (B) Generation of single-stranded DNA by T7 Polymerase. (C) Target sequences complementary to cRNA bind to the CRISPR/Cas13a system. Once the target sequence is present within the system, the non-specific RNA cleavage activity of Cas13a is activated and the reporter RNA is cleaved, resulting in the activation of the fluorescence signal. (D) Lateral flow-based detection can be read from strips with a colored positive/negative band using a FAM/Biotin.

Figure 7

Overview of techniques used for FAdVs genotyping. (a) REA digesting the whole genome using BamH1 and HindIII enzymes. (b.1) PCR/RFLP digesting the PCR product derived from HexonA/HexonB primers with restriction enzymes. (b.2) Sequencing of PCR product followed by sequence analysis using BLAST and alignment with other FAdVs sequences on GenBank. (c.1) Specific qPCR test using a specific primer of serotype (e.g., FAdV4 F/FAdV4 R specific for FAdV-4). (c.2) Analysis of High-Resolution Melting (HRM) curve derived from the Hex L1 s/Hex L1 as primer. The gel images presented in this figure are adapted from previous work (43) and are included to ensure the figure is comprehensive and meaningful.