Team:Grenoble-Alpes/LabBook
LabBook
Facing back to the Etendard glacier, in between the peaks of Maurienne and the ones of Oisan. Credits: Estelle Vincent
Step 1 : Target Preparation
The first rate-limiting step in the detection of Vibrio Cholerae is the extraction of its DNA. To this aim, the bacteria has been deeply studied, as well as the current DNA extraction techniques.
1. Vibrio Cholerae
1.1 Classification & Generalities
V.Cholerae is a thin gram-negative proteobacterium that has a flagellum which gives it mobility [2]. This bacteria is responsible of cholera disease, causing severe contagious epidemia. This bacteria use to grow in basic conditions (Optimal growth pH : 9) [1] with 1-3% NaCl in liquid or solid mediums [3].
1.2 Growth in laboratory
Aeroanerobic bacteria grown on conventional media. Optimum growth in medium with 1 to 3% NaCl pH 9, in liquid media (Colonies in 3-4 h at the surface) or in solid media (colonies in 8-10 hours). The bacterium can also grow on bile salt media. According to these characteristics, alkaline peptone water pH 8.6 3% NaCl can be used as an enrichment medium as well as an alkaline agar pH 9. Colonies are 2 to 3 mm in diameter and are smooth, Flat and transparent. [3]
1.3 Tanks and contamination
Main V.Cholerae tanks are humans and dirty water, it seems that global warming is creating favorable conditions to this bacillus [4]. Even if there are many kinds of this bacteria, only 2 serogroups are directly responsible of Cholera : O1 and O139.
Human transmission is linked to inappropriate access to clear water. This bacteria can survive more than 15 days in water. Contaminations are also possible with contaminated food like vegetables or fishes, The infectious dose is between 106 and 1011 vibrios ingested [5]. The infectious dose depends on gastric acidity (the lower the acidity, the fewer vibrios required to cause infection)[6]
1.4 Physiopathology & Virulence
Cholera is a very virulent disease that can cause severe acute watery diarrhea. The bacillus can be found in patient’s stools for 1 to 10 days after infection (106 to 108 bacillus/mL [3], [5]) and is disposed of in the environment where it can potentially infect other people. An untreated choleric person would produce 10 - 20 liters of diarrhea a day [7]. The cholera toxin (CTX) is an oligomeric complex made up of six protein subunits responsible to the symptoms. Once inside the cell, the A1 subunit is freed to bind with a human partner protein : Arf6 [8]. This bound exposes its active site, allowing it to ribosylate the Gs alpha subunit of G protein. This results in constant cAMP production, which leads to the secretion of water, sodium, potassium, and bicarbonate into the lumen of the small intestine and rapid dehydration.
A healthy human feces contains 1012 bacterias per grams, more than 400 different species can be found [3]. V.Cholera is more in patient’s feces than other bacterias [3] [9].
The gene encoding the cholera toxin was introduced into V.Cholerae by horizontal gene transfer. Virulent strains of V.Cholerae carry a variant of a bacteriophage called CTXφ[10].
2. DNA Extraction
In order to obtain the target, the vibrio DNA must be extracted.
2.1 Laboratory extraction
Nowadays, DNA preparations are widely used because of their easy using. A lot of suppliers proposes kits (NEB, QIAGEN…). Kits allow to extract 50 ug to 10 mg of DNA thanks to different protocols/materials (Miniprep, Midiprep, Maxiprep, Megaprep and Gigaprep [11]) and different kind of polynucleotides can be extracted : Plasmidic DNA, RNA or even genomic DNA for instance. Protocols can also be more or less fast, depending on the the supplier’s technology.
2.2 Paper-based technology
A famous field of research consists in paper-based extraction technology. It permits low-costs diagnosis. The point is that these technologies permit easier way to perform sample preparation. For example, it is possible to make an automated DNA extraction from the human whole blood in only 7 minutes [12].
Some scientists also developed an automated way to proceed DNA extraction : it combined magnetic beads, paper, stepper actuators and a micro-computer called Arduino. [13]
Thanks to that technology and using 96 well plates, it is possible to target the apicoplast genome for malaria diagnosis [14].
2.3 Isothermal amplification - LAMP
Loop mediated isothermal amplification (LAMP) is an isothermal technique for the amplification of DNA [15] widely used in point-of care diagnosis because it is cheap and simple[16] [17]. The sensitivity can reach 92% [17] making it a serious technology for malaria diagnosis.
Combined with paper-based technologies, it is possible to develop low-coast paperfluidic molecular diagnostic chip that can extract, amplify and detect DNA from clinical samples in less than 1 hour even in resource-limited settings [18].
3.
3.1
3.2
3.3
3.4
Step 2 :
Step 3 :
