Conventional PCR & Capillary Gel Electrophoresis

Conventional PCR amplifies genetic material, which is then visualized using gel electrophoresis. By comparing the amplified fragments with a base-pair ladder, the size of the genetic material can be determined. This technique also enables bacterial characterization through REP-PCR and allows differentiation between wild-type and vaccine strains.

Qualitative/quantitative real-time PCR (qPCR):

Real-time PCR (qPCR) is used to detect the presence of specific pathogens with high sensitivity and speed. Results are expressed as Ct (cycle threshold) values, providing semi-quantitative insight into pathogen load — the lower the Ct value, the higher the pathogen concentration.

To ensure reliable results, inhibition controls are included during analysis. In cases where PCR inhibitors are present, additional testing on diluted samples may be performed.

qPCR can also support:

· Differentiation between vaccine and field strains

· Pathotyping analyses

· Quantification of pathogen load through standard curve analysis

At Poulpharm, we have developed several specialized in-house assays, including:

– qPCR Cox differentiation: Determines the relative quantification of different Eimeria species in chickens and turkeys. Commonly applied to inocula, with a detection limit of 40 oocysts per gram of feces.

– Multiplex qPCR Cox differentiation: This analysis is also used to determine the relative quantification of the various Eimeria spp. for chicken. Results are known

the day after receipt of sample and can be performed directly on fecal material. Detection limit of this qPCR depends on the Eimeria spp. and varies between 40 to 1000 oocysts per gram faeces.

At Poulpharm we are also BELAC accredited for the following tests at the molecular department:

– Mycoplasma gallisepticumqPCR

– Salmonella serotyping

– Mycobacterium avium subspecies paratuberculosis (MAP) qPCR

– Bovine viral diarrhoea (BVD) qPCR

To quantify the presence of a specific pathogen (gene copy numbers/mL) a standard curve needs to be added in the reaction. Then we talk about quantitative real-time PCR. This is an indirect quantitative method.

1. Digital droplet PCR

Digital droplet PCR provides highly accurate and direct quantification of pathogen load by partitioning genetic material into thousands of droplets. This advanced technology enables precise determination of gene copy numbers per mL.

Poulpharm utilizes state-of-the-art ddPCR technology and offers customized assay development tailored to specific research or diagnostic needs.

Sanger Sequencing

Sanger sequencing is used to determine the sequence of targeted regions within the genome of bacteria, viruses, or parasites. This method supports:

– Differentiation between vaccine and field strains

– Genotyping and phylogenetic analysis

– Detailed pathogen characterization

Whole genome sequencing

We offer in-house Whole Genome Sequencing (WGS) using advanced Oxford Nanopore Technologies platforms.

WGS enables complete genomic characterization of pathogens, providing a far more detailed level of identification and differentiation compared to targeted sequencing methods such as Sanger sequencing. This technology is particularly valuable when:

· Higher-resolution differentiation is required

· Conventional methods fail to identify pathogens

· Multiple pathogens may be present in a sample

WGS can achieve differentiation down to nucleotide level, making it a powerful tool for epidemiological studies, strain tracking, and advanced pathogen characterization.

Be aware that the quality of WGS results highly depends on the quality of the samples that are collected. For more information on what we are able to offer you at Poulpharm, contact our experts.