Concepto Azul S.A. counts with a team of researchers in charge with the development, evaluation, testing and cross-validation of molecular diagnostic kits to be used for the detection of the most important pathogen agents affecting marine species and particularly cultured shrimp (L. vannamei and P. monodon)  or fishes (Oreochromis sp., Onchorynchus sp.,. catfish). Tests and diagnostic kits are, in a similar way, designed for the detection of pathogen agents affecting the principal agricultural crops (rice and sugar cane).
Eventually, in the field of quality control of export products, Concepto Azul has developed molecular diagnostic kits specific to human pathogens such as Vibrio cholerae, V. parahaemolyticus, Salmonella sp., Escherichia coli.
  
 

The kits designed by Concepto are continuously assessed and comparatively tested with the latest kits available on the market hence guaranteeing their competitiveness. They provide significant advantages in terms of sensitivity, specificity, reliability as well as from the economical standpoint.

  
 

Concepto Azul’s team offers personalised and highly specialised services in order to effectively respond to its client’s needs and expectations. Many options are made available to Concepto Azul’s clients ranging from scientific and technical advisory for the management of molecular proofs, technical training for professionals, and support for the development of investigation programs to the whole implementation and management of molecular biology laboratories.

  
 

Short description of various pathogen agents that affect Litopenaeus vannamei cultures.

  • WSSV ‘White Spot Syndrome Virus’: a virus that causes heavy losses, devastating until 100% of the whole culture stock in a week.
  • MBV ‘Monodon Baculovirus’: a virus affecting shrimp hepathopancreas, causing losses, growth delays and eventually a great disparity in animal size when harvested.
  • HPV “Hepatopancreatic Parvovirus”: a virus affecting the hepathopancreas, causing a delay in individuals’ growth and eventually a great disparity in animal size when harvested.
  • YHV/GAV “Yellow head virus/Gill associated Virus”: ARN viruses causing significant death rates in Penaeus monodon cultures.
  • IHHNV “Infectious Hypodermal and Haematopoietic Necrosis Virus”: a Densovirus causing deformation, growth retardation and a characteristic disparity at harvest time. IHHNV causes losses in Litopenaeus stylirostris cultures as well.
  • TSV “Taura Syndrome Virus” is a virus that causes massive losses in Litopenaeus vannamei.
  • Intracellular bacteria: since 1989 a specific strain of intracellular bacteria (proteobacteria) that causes NHP ‘Necrotizing Hepato pancreatitis’ is associated to a rickettsia type of bacteria that is a gram-negative bacillus, obligate intra cellular.
  • Extracellular bacteria: the main causes of mortality are Vibrios spp. (proteobacteria)
  
 

Short description of pathogen agents affecting fish culture

  • Columnare or “Silla de montar” name given in view of discolorations observed on the basis of the dorsal spine. Flavobacterium columnare,
  • Estreptococosis, Streptococcus agalactiae and to a lesser extent S. iniae, in tilapia, in rodaballo a Streptococcus parauberis, in rainbow trout Streptococcus constellatus (Lactococcus garvieae). The Estreptococosis can provoque heavy losses, more than 50% of the stock.
  • Blood poisoning Aeromonas hydrophila. Bacteria that are morphologically assimilated to the Enterobacteriaceae family cause severe ulcerations and necrosis lesions of fishes’ skin.
  
  Short description of molecular tests:  
 

- PCR (Polymerase Chain Reaction): “in vitro” DNA replication based technique consists of the amplification (or synthesis) of a genomic region (for instance a sequence of the virus gene).  This process is achieved using a termal cycler, which undertake cycles at various temperatures, necessary to complete the different stages of the synthesis reaction:
1) The denaturalization of DNA (that is to say the separation of DNA chains),
2) Hybridising (or the union by complementarities of nitrogen bases between DNA chains) of primers (small sequences of synthetic DNA) that are specific to the genome region to be synthesized, and
3) Polymerization (or synthesis) of the specific fragment of the genomic region to be detected. This process is reproduced various times (between 30 and 40 times) in order to obtain the production of millions of copies of the sequence targeted.
This fragment can be observed in an agarose gel by electrophoresis, after a stained with ethidium bromide, and illumination with ultraviolet light. The observation of a band with the expected size indicates that the result is positive.
A derived technique, namely the RT-PCR (Reverse transcription-PCR) is used in order to identify pathogen agents with a DNA genome. A first step preliminary to the classic PCR consists in converting a segment or the totality of the viral genome to complementary DNA (ADNc) which is later used as sample in the PCR reaction.

- LAMP (Loop-mediated Isothermal Amplification) is an innovative technique based on the amplification of DNA sequences that does not require the alternate temperature cycles used in PCR. The technique uses a set of 4 primers in a single reaction, thus incrementing the level of specificity; DNA synthesis is achieved at a stable temperature (isothermal reaction). The result is observed through electrophoresis and a positive result can be observed by turbidity variations in the reaction tube or through a measurable fluorescent agent incorporated in the reaction.

- Real-Time PCR is a PCR technique that enables the quantification (in other words the determination of a number of copies) of the DNA genome sequence in the analysed sample. Thermociclador is associated to an optic module that analyzes the PCR reaction process in real time and transmits data to a central unit that displays the quantity of synthesised sequences. Eventually, this technique does not require a posterior analysis through electrophoresis. The reaction is monitored through the use of fluorescent agents that emit fluorescence proportionally with the quantity of sequences that are produced. This technique enables the collection of data that provide the initial quantity of the amplified sequences. Otherwise Real-Time PCR technique is currently used to evaluate genes’ expression (for instance, immune genes involved in pathogen resistance), hence enabling the selection of animals with molecular markers to be used in genetic improvement programs.

  
 


 
     
 
 
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