Simultaneous and rapid detection of avian respiratory diseases of small poultry using multiplex reverse transcription-Polymerase Chain Reaction assay

Major viral infections, such as Newcastle disease virus, infectious bronchitis virus, avian influenza virus, and infectious bursal disease virus, inflict significant injury to small poultry and tremendous economic damage to the poultry sector. This research aims to develop a multiplex reverse transcriptase polymerase chain reaction (m-RT-PCR) approach to simultaneously determine these important viral pathogens. The conserved segment of various viral genetic sequences was used to design and synthesize specific primers. Moreover, as positive controls, recombinant vectors were synthesized in this investigation. The d-optimal approach was used to improve PCR conditions in this investigation. Positive controls and clinical samples were used to assess the m-PCR assay's specificity, sensitivity, repeatability, and reproducibility. According to the sensitivity test findings, the m-PCR technique could generate the 8 target genes from viral genomes using 1 × 102. In addition, 8 viral pathogens were detected from the infected samples. The findings also suggest that live animal oral swabs were not significantly different from tissue sampling of a dead animal (P < 0.05), and this kit had a high sensitivity for analyzing both types of samples. The suggested m-PCR test may detect and evaluate viral infection in birds with excellent specificity, sensitivity, and throughput.


INTRODUCTION
Avian influenza virus (AIV) (Wen et al., 2022), Newcastle disease virus (NDV) (Ul-Rahman et al., 2022), infectious bronchitis virus (IBV) (Shariatmadari, 2000;Birhanu et al., 2022), and infectious bursal disease virus (IBDV) (Beiranvand et al., 2022;Hu et al., 2022) are the principal viruses that induce serious financial losses in the small poultry sector (Yao et al., 2019). AIV has been derived from many small poultry species. Small poultry is often the major reservoir host. On the other hand, highly virulent avian influenza has the potential to cause substantial fatality in small poultry (de Camargo et al., 2022). As a result, detecting AIV in small poultry is critical in epidemiological studies (de Camargo et al., 2022). Small poultry is typically regarded as possible reservoirs for NDV, like AIV, which has been detected in Iran occasionally. Furthermore, NDV was common in small poultry across Iran, resulting in significant economic losses (Sabouri et al., 2018). Infectious bursal disease (IBD), commonly known as Gumboro syndrome, is a viral infection in the Avibirnavirus species (family Birnaviridae). While geese, waterfowl, guinea hens, birds, and ostriches can be infected, the symptomatic illness only develops in poultry. Only chicks under the age of 10 wk are often clinically impacted. Clinical indications are frequently absent in older hens (Dey et al., 2019).
Despite substantial immunization, the causal agent of IBDV is an extremely infectious and inflammatory illness of chickens that causes enormous economic losses to the poultry sector (Wang et al., 2021). An avian coronavirus, an enclosed single-stranded RNA virus with a distinctive spike-like protrusion on the outside of its membrane, causes IBV. Mutation in the viral spike molecule results in the emergence of various strains of the virus, which might also differ locally. The pathogen spreads quickly throughout the flock, causing respiratory discomfort (Stevenson-Leggett et al., 2021). In simple infections, fatality is normally minimal; nevertheless, some virus variants have a preference for the renal, which causes death due to renal disease. Consequences, including coinfection with other diseases, may also contribute to increased fatality (Lin and Chen, 2017). IBV is ubiquitous in all nations with a large chicken economy, with infection rates surpassing 100% in most places (Piri Gharaghie et al., 2021).
Given the significant threat posed by these infections to the small poultry sector, quick and simple approaches for identifying these pathogens and applying preventative actions to decrease financial damage as soon as feasible Yang et al., 2017) are critical. Viral diagnostic tools include included virus isolation and characterization, serological diagnosis, immunoelectron spectroscopy, enzyme-linked immune sorbent assay (ELISA), lateral flow assay (LFA), and polymerase chain reaction (PCR). Moreover, the procedure is time demanding, which limits its applicability in immediate rapid diagnosis (Xu et al., 2012). Immunoassaybased approaches, such as ELISA, are commonly utilized. The issue with this technology is that it requires specific antibodies, which are time demanding and tiring to produce (Smith and Dunstan, 1993). Immuno-electron scanning necessitates specialized equipment and a large volume of the virus, making it unsuitable for diagnostic techniques (Mirmajlessi et al., 2015). In contrast to these approaches, PCR is a widely utilized technique in molecular biology (Hao et al., 2016). It can multiply a single copy or a few copies of a given DNA sequence dramatically. Because of its high sensitivity, nonstrict detecting requirements, great specificity, fast response, and reliability, it has been widely employed in diagnostic medical research for a wide range of pathogen identification (Almeida et al., 2017;Diao et al., 2021). M-PCR (m-PCR) relates to PCR amplification that employs 3 or more specific primers in a PCR reaction volume to amplify several genomic sequences at the same time (Chen et al., 2013). M-PCR has unrivaled benefits over uniplex PCR, notably high replication accuracy, time savings, and maximum throughput (Ali et al., 2015;Piri Gharaghie et al., 2021). More notably, this technique can differentiate between many viruses at the same time; it is an efficient way for quick identification of mixed-virus disease in early diagnosis (Moustacas et al., 2013;Cassedy et al., 2021). The goal of this study was to design and develop an m-RT-PCR technology capable of identifying and distinguishing significant serotypes of 4 main small poultry virus infections: AIV, NDV, IBV, and IBDV.

Declaration of Ethics
This experiment was carried out in compliance in accordance with ARRIVE guidelines (https://arrive guidelines.org/arrive-guidelines). Autoclave cotton swabs were used to carefully gather biological specimens from normal poultry. The chickens were not anesthetized before testing, and following the sample, they were monitored for 30 min before being transferred to their cages.

Viral Variants and Growing Conditions
Pathogenic viruses, including AIV, NDV, IBV, and IBDV subtypes, were collected from infected animals ( Table 1). The viruses were stored in the AmitisGen Tech Dev Group and Parsian BioProducts companies.
All medical swab specimens were tested from healthy poultry's cloacae, larynges, and tracheae. Also, the genome of the viruses was prepared from the Razi Serum and Vaccine Institute (Karaj, Iran). The genome of the viruses was stored in AmitisGen Tech Dev Group Parsian BioProducts companies.

Extracting Nucleic Acids
The nucleic acid of the viral pathogens was isolated and diluted in a nuclease-free solution by using the Viral RNA/DNA Extraction Kit (Takara Bio, Japan). The Reverse Transcription Kit was used to convert the RNAs of AIV, NDV, IBV, and IBDV into cDNA (Thermo Scientific, Waltham, Massachusetts, US). Spectrophotometry was used to assess the quantity and quality of each genome (Thermo Scientific). The cDNA was kept at a temperature of À20°C.

Primer Designing for Viral Genomes
The AIV, NDV, IBV, and IBDV strains' wholegenome sequences were obtained from GenBank, and DNAMAN was used to align the conserved domain of viral-specific genes (LynnonBiosoft). Using oligo7 (https://www.oligo.net/), 2 pairs of particular primers for each virus based on the sequence alignment results were designed. Macrogen, Inc. produced the primers indicated in Table 2 (Macrogen, Seoul, South Korea).

Reverse Transcription-PCR Assay
Each reaction mixture had an overall volume of 25 mL, including 2.5 mL 10£ Buffer (Mg2+ free), 4 mL (25 mM) MgCl 2 , 0.75 mL dNTP (10 mM each), 0.25 L (5 U/ mL Taq DNA Polymerase Vazyme, China), 1 mL forward primer, 1 mL reverse primer, 2 mL single-virus vector, and 13.5 mL ddH 2 O. The PCR procedure was performed as follows: Predenaturation at 95°C for 5 min, followed by denatured nucleic acids at 95°C for 60 s, annealing at 55°C for 40 s, elongation at 72°C for 45 s, 35 cycles, and a final extension at 72°C for 10 min. About 1.5% agarose gel electrophoresis was used to evaluate the PCR results. Double distilled water was used as the blank control.

Multiplex Reverse Transcription-PCR Assay: Experimental Design
A D-optimal strategy of 25 trials was used to optimize the m-PCR procedure. Annealing rates (49°C−67°C), Mg2+ ratios (1−6 mM), Taq DNA Polymerase ratios (0.02−0.06 U/L), and dNTP concentrations (0.08−0.48 mM) were all taken into account. In consequence, the data analysis was determined by the intensity of the PCR-produced bands. MODDE 12.1 program was used for all evaluations (Umetrics, Sweden).   Sensitivity and Specificity Analysis of the m-PCR Approach a) Specificity analysis of the m-PCR: Primer blast analysis was used to assess the specificity of m-PCR. The combined vectors were diluted from 1 £ 10 6 to 1 £ 10°copies/mL using a 10-fold gradient diluting procedure to test the m-PCR technique's sensitivity. Also, Escherichia coli, Salmonella, and Clostridium perfringens were used to test the specificity of the m-PCR approach.
b) The m-PCR technique's efficiency: By using a 10-fold gradient dilution method, each one of the recombinant DNA vectors was diluted from 1 £ 10 6 to 1 £ 10 0 copies/mL. After that, m-PCR was carried out to determine limit of detection (LOD). m-RT-PCR reaction was performed for each sample with 3 replications. c) Detection limit, analytic sensitivity, and normal range: Two inactive positive samples of NDV, AIV, IBV, and IBDV viruses at a concentration of 1 £ 10 6 copies/mL were evaluated at the Virology Research Center to verify the technique. Each sample received 3 replications of the m-RT-PCR procedure.
Repeatability and Reproducibility Analysis of the m-PCR a) Evaluation of reproducibility within a single LOT: On the same day, the technique's repeatability was determined. Two users evaluated 20 positive control (1 £ 10 2 copies/mL recombinant plasmids) using kits made by a single LOT. The correlating percentages of different LOTs were evaluated.
b) Evaluation of reproducibility between multiple LOTs: The evaluation of reproducibility was carried out on 2 different LOTs. Kits manufactured in 2 LOTs were used to examine 20 recombinant plasmids. The recombinant plasmid amounts were 1 £ 10 2 copies/mL.

Kit Validation Test
To validate the kits synthesized in this study, a multiplex kit for respiratory diseases (GeneProof, Czech Republic, CAS No. QAV054134) was prepared. In 3 different laboratories from different parts of Tehran, multiplex-PCR test was performed on recombinant plasmids at the concentration of 1 £ 10 2 copies/ mL. The results obtained from the kit synthesized in this study were compared with the results of the standard kit (GeneProof, Czech Republic). The test was repeated 3 times.

Simulation of Coinfection and Identification of Clinical Specimens
The coinfection investigation aimed to see if the m-PCR approach was practicable. In addition, 200 clinical samples (100 tissue specimens and 100 oral swabs) were gathered from small poultry farms and live chicken markets (these specimens were acquired with the permission of the animals' owners, and the animals' suffering was reduced). For PCR amplification, the standard m-PCR technique was applied. Uniplex PCR and conventional or published PCR procedures were used to corroborate the results. The positive PCR amplification products were then sequenced to corroborate the detection rate.

Statistical Analysis
GraphPad Prism 5.0 was used to examine the data and perform statistical tests. A one-way ANOVA was used to compare means, followed by a Tukey−Kramer post hoc test with a 95% confidence interval. Differences were considered significant at P < 0.05.

The m-PCR Technique Was Optimized and Established
With 20 runs completed in one randomly selected LOT, a D-optimal technique was chosen to optimize the m-PCR process. With annealing temperature at 57°C, Mg2+ concentration at 4 mM, Taq DNA Polymerase concentration at 0.05 U/L, and dNTP concentration at 0.32 mM, we reached the final optimal settings, taking into account the economic approach, with the required compromise. We developed the m-PCR technique, which could efficiently generate duplex and triplex genes, using the ultimate ideal primers and settings. The primers' BLAST findings are presented in Table 3. The blast primer results of this study showed that each primer corresponds to different serotypes of the target viruses.

Construction and Identification of Recombinant Plasmids
Virulence genes of AIV, NDV, IBV, and IBDV strains were cloned into the eukaryotic expression vector pcDNA3.1(+), as shown in Figure 1, separately. DNA sequencing revealed that the gene sequences from the 4 recombinant plasmids were identical to the AIV, NDV, IBV, and IBDV strains. BamHI and EcoRV were used to digest the plasmids that had been constructed. The digestion products separated electrophoretically at 520 and 5400 bp for AIV, 470 and 5400 bp for NDV, 840 and 5400 bp for IBV, and 600 and 5400 bp for IBDV strains (Figure 1), indicating that the recombinant plasmid was successfully constructed.

The m-PCR Approach Has a High Level of Specificity
These 8 viruses, as well as E. coli, Salmonella, and C. perfringens, were used to test the specificity of the m-PCR approach. The band for each viral pathogen was apparent for m-PCR evaluation, as seen in Figure 2A, and was comparable to that of uniplex PCR ( Figure 2B). Moreover, despite the presence of other bacterium sequences in the collection pool, only the DNA of these 8 viruses was replicated; no amplification happened with the interfering genomes. The sequencing findings confirmed the multiplex-PCR's high specificity.

The m-PCR Technique's Efficiency
The m-PCR technique's efficiency for each viral sample was assayed by positive control (Recombinant plasmids). According to the results of Figure 3, the minimum detectable amount of virus in this kit is 100 (1 £ 10 2 ) copies. Therefore, the performance of this kit is equal to 100 copies of the number. In the next step, the kit was tested for verification with inactive virus samples.

Sensitivity of the m-PCR Method
The lowest detectable concentration of viral genome is measured in this assay. The results demonstrated that this kit can detect the viral genome when the virus concentration in the sample is 100 copies per microliter. This kit's LOD value was 100 (1 £ 10 2 copies/mL) copies per microliter (Table 4). Table 5 shows the findings of the m-PCR product's reproducibility. Multiplex-PCR was carried out using recombinant plasmids at ratios of 1 £ 10 2 copies/mL, showing the validity of the suggested approach. Two users repeated the test 20 times using kits made by a LOT and there was 100% concordance between the LOTs (Table 5). The investigation was also carried out 20 times by one user, using kits from 2 different LOTs. There was 100% agreement between the LOTs in the results of this test (Table S1).

Result of Kit Validation
Multiplex-PCR tests were done in 3 different laboratories from different geographical location of Tehran to validate the kits manufactured in this work, and the findings obtained from the kits are 100% compatible with the results of the reference kit (GeneProof). Table 6 is used to display the information.

Model for Coinfection and Identification of Clinical Samples
Infections of various combinations of viruses were simulated at the same dose (1 £ 10 2 copies/mL). In addition, the developed m-PCR technology and uniplex PCR were used to investigate a total of 20 clinical specimens. Sixteen specimens tested positive for AIV, NDV, IBV, and IBDV. Six specimens were positive for AIV, 5 samples were positive for NDV, 3 samples were positive for IBV, and 2 samples were positive for IBDV. Table 7 shows the positive rate of each virus. Four of the positive samples had both NDV and AIV infections (Table 7). In addition, various experiments corroborated these findings. The results also revealed that sampling using live animal oral swabs was not substantially different from tissue sampling of a dead animal (P < 0.05), and this kit had a high sensitivity for evaluating both types of samples.

ACKNOWLEDGMENTS
The design of the m-PCR method was supported by the Biotechnology Research Center, Microbial Biotechnology Laboratory, Parsian BioProducts (PBP) Shahrekord, Iran and of AmitisGen Tech Dev Group company, Tehran, Iran; (2021Bio3P0908).
Data Availability Statement: All data generated or analyzed during this study are included in this article and its additional files. The datasets generated and/or analyzed during the current study are available in the [Figshare] repository [10.6084/m9.figshare.20092625].
Author Contributions: TPG and GhGh designed the study and approved the manuscript. NTL, TPG, MAS, and GhGh developed the multiplex-PCR method, analyzed data, and drafted the manuscript. NTL and TPG collected clinical samples. RDCP, FH and RYCA revised the manuscript. All authors read and approved the final manuscript.
Ethics Approval and Consent to Participate: This study was performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Ministry of BIO3P and VetCareGen company of AmitisGen Tech Dev Group, Tehran, Iran.
The protocol of the current study was reviewed and approved by the Institutional Animal Care and Use Committee of VetCareGen company of AmitisGen Tech Dev Group, Tehran, Iran (approval no. VetCareGen 2021−0908). Consent for Publication: Not applicable.