This science is based on the understanding of: genome molecular analysis, the control of the genetic expression, the structure/function relationships of biological macromolecules, their interactions in supra molecular complexes. In order to grasp a profound knowledge on this subject it is necessary to:
Cellular biology (also called Cell biology) and cytology both mean the study of cells. Knowing the composition of cells and how cells work is fundamental to all of the biologial sciences. In particular, by exploring the similarities and differences between cells of different organisms or different tissues, cytology provides inside knowledge based on observations of the cellular systems. These informations are fundamental for the other disciplines, dealing as well at the molecular (such as genetics, biochemistry or molecular biology) or macroscopic level (such as pathology).
The company's succesful performance should be based on the availability of modern laboratories equipped with all the necessary equipment, able to develop products and provide certifications as per market requirements. The company's activity should have a finalized R&D project as starting point, and its results should be in line with the latest technological progress in challenging fields such as biology, medicine, veterinary or food science.
PCR (Polymerase Chain Reaction) is an in vitro methodology that allows the amplification of a nucleic acid sequence. The extremities of the sequences to be amplified have to be defined in order to synthesize the oligonucleotides (primers) which will then be hybridised, and a small quantity of the sequence has to be available for initiating the reaction. The reaction product will be a discrete molecule of dsDNA with extremities corresponding to those of the 5’ of the used primers.Real-time PCR is one of the principal PCR applications. As a matter of fact real-time PCR is also called quantitative PCR or quantitative PCR in real-time (rtq-PCR); it is a method that permits the simultaneous amplification (PCR) and quantification of DNA.
Probes for the specific pathogens are pre-spotted onto the wells (chips). Nucleic acid is extracted and amplified by Multiplex PCR. Biotin-labelled primers are used to mark the amplified products. The denatured samples are then hybridized on the chip for 1 hour at controlled temperature in an oven with a vibrating plate. Hybridization is followed by a wash step and the addition of alkaline phosphatase (AP) conjugated Streptavidin (30 minutes incubation at RT). After another wash step, a chromogen substrate (NBT-BCIP; 15 minutes of incubation in the dark at RT), mediates the colorimetric reaction required for detection. The target sequence becomes visible by the development of a brown-coloured precipitate that forms on the chip in the position of the corresponding probe: each positive result yields a specific pattern on the chip. On average the system detects up to 12.5 pg/ml of the target sequence for each infectious agent.
The DNA test consists in the molecular examination of particular regions of the human genome endowed with highly variable genetic sequences, so specific to give a sequence pattern which is virtually unique for each individual. This is called DNA fingerprint. The PCR-obtained DNA samples are essentially analysed through electrophoretic techniques, which avoids the obstacles and drawback of previous techniques, such as the use of radio-active reagents, long analysis times, and difficult interpretation. Besides, the large amount of markers used and their distribution on the different chromosome loci guarantee the reliability of the results, avoiding those errors that could derive from mutations or chromosome disorders; as a matter of fact, thanks to these improvements, while the first paternity tests were based on max. 4 to 5 sequences, today we normally analyse 10 to 15. Currently, the diffusion of analysis kit that assess arrays of pre-determined genetic markers without needing sophisticated equipment has permitted to several laboratories to include ascertaining paternal lineage among the offered services. However, results reliability is still strictly linked to lab personnel skill and equipment’s quality, which explains why such tests are still quite expensive.
Extraction of DNA is a well-known technique in the scientific field. The technique is based on the demolition of the external membrane by particular substances; the external membrane of the cells and of their nucleus is composed of lipids. It is a delicate operation that requires precision and experience. There exist several commercial kits that allow the DNA extraction from different biological matrices (blood, sputum, urine, etc…)
Direct sample amplification through molecular biology instruments is still the most reliable and fast method for a correct diagnosis that allows to perform the necessary periodical diagnostic verifications on individuals at risk. Today it is possible to detect in short time from a given biological sample (biopsy or pap test) not only the presence of the papilloma virus but also the specific genetic type to which it belongs (e.g. HPV 6, 11, 16, 18, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 54, 56, 58, or other less frequent types). The attribution of the viral type is performed by using commercial kits or by comparison of the obtained sequence with the identified types and subtypes as described in literature. Through this technique it is thus possible to unveil latent infective pathologies or situations in which the presence of infective agents is not manifest; indeed, in all these cases, the use of alternative techniques could have poor diagnostic value.