Molecular - pcr
PCR or Polymerase Chain Reaction is a rapid and reliable method to detect the genetic material of pathogens (bacteria, viruses and parasites) already at an early stage of the disease. The main advantage of PCR is that the result is known faster than bacterial or viral culture. Also, a positive sample will be detected days faster by PCR than by serology, which detects antibodies in the blood.
PCR techniques offered by Poulpharm are:
- Conventional PCR
- Qualitative real-time PCR
- Quantitative real-time PCR
- Sequencing
Poulpharm offers besides real-time PCR techniques for the most common pathogens, also in-house developed PCR assays for relative quantification of Eimeria spp. in chickens and turkeys, and determination of Histomonas meleagridis in various matrices.
For relative quantification of Eimeria spp. we offer two PCR assays:
- qPCR Cox differentiation: This analysis determines the relative quantification of the different Eimeria spp. for chicken or turkey. The complete process of this analysis takes at least a week and is usually applied to inocula. Detection limit of this qPCR is 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.
CONVENTIONAL PCR
In conventional PCR the genetic material is multiplied and made visible by means of a gel. On the basis of an added base pair ladder, the size of the genetic material can be determined. With this method, bacteria can also be characterized by means of REP-PCRs and wild type strains can be distinguished from vaccine strains.
QUALITATIVE REAL-TIME PCR
Qualitative real-time PCR examines the presence of genetic material from the pathogen. The results of this PCR are represented by a numerical value or a treshold cycle value (Ct value). The smaller this value, the more the pathogen is present.
If inhibition occurs during the PCR due to the presence of PCR inhibitors, the presence of the pathogen cannot be demonstrated. In this case, a second PCR is used, but if with the same result, a new sampling is indicated.
QUANTITATIVE REAL-TIME PCR
Quantitative real-time PCR, like qualitative real-time PCR, verifies the presence of genetic material from the pathogen, but determines the exact amount through the use of a standard curve. The results of this PCR are represented by number of copies per milliliter (copies/mL).
Sequencing
Sequencing involves determining the sequence of the genetic material of bacteria, viruses or parasites. By determining sequences, wild type strains can be distinguished from vaccine strains or certain mutations in the genetic material can be detected. In addition, phylogenetic profiles can be drawn up by means of sequencing.
Conventional pcr
In conventional PCR the genetic material is multiplied and made visible by means of a gel. On the basis of an added base pair ladder, the size of the genetic material can be determined. With this method, bacteria can also be characterized by means of REP-PCRs and wild type strains can be distinguished from vaccine strains.
Qualitative real-time PCR
Qualitative real-time PCR examines the presence of genetic material from the pathogen. The results of this PCR are represented by a numerical value or a treshold cycle value (Ct value). The smaller this value, the more the pathogen is present.
If inhibition occurs during the PCR due to the presence of PCR inhibitors, the presence of the pathogen cannot be demonstrated. In this case, a second PCR is used, but if with the same result, a new sampling is indicated.
quantitative real-time pcr
Quantitative real-time PCR, like qualitative real-time PCR, verifies the presence of genetic material from the pathogen, but determines the exact amount through the use of a standard curve. The results of this PCR are represented by number of copies per milliliter (copies/mL).
Sequencing
Sequencing involves determining the sequence of the genetic material of bacteria, viruses or parasites. By determining sequences, wild type strains can be distinguished from vaccine strains or certain mutations in the genetic material can be detected. In addition, phylogenetic profiles can be drawn up by means of sequencing.