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
| Domain | Bacteria |
|---|---|
| Phylum | Proteobacteria |
| Class | Gammaproteobacteria |
| Order | Vibrionales |
| Family | Vibrionacea |
| Genus | Vibrio |
| Species | Vibrio cholerae |
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. Vibrio Cholerae extraction
3.1 Vibrio Cholerae sensitivity
To lyse bacteria, several disinfection solutions were prepared, such as 2-5% phenol ; 1% sodium hypochlorite ; 4% formaldehyde ; 2% glutaraldehyde ; 70% ethanol ; 70% propanol ; peracetic acid To 2%, to the hydrogen peroxide to 3-6% and to the iodine to 0.16% [19]. As mentioned before, V.cholerae is not supposed to survive in acid mediums [6]. Another possibility to kill it is to create a thermal shock (0°C)[20]
3.2 Lysis buffer
After many intern discussions, DNA amplifications were dropped because the bacteria is overexpressed in the sample. Lysis buffer is used to extract DNA from bacterias. It contains Urea 4M and DNAse inhibitors. Urea 4M allows a lysis efficiency of 90% in less than 10 minutes [21], which is fast and interesting given the fact that V.cholerae is overrepresented in patient’s faeces [3] [5].
3.3 Protocols and Results
Late exponential phase cells of V.Cholera will be harvested and resuspended in 20 mL of 4 M-urea with DNAse inhibitors. The following step is an incubation of 10 min at 24°C, by which time more than 90% lysis occurs. The only difference is that we extracted the DNA from E.Coli instead of V.Cholerae for security measures.
The following protocols (Table 2) has been done in laboratory in order to check if the centrifugation can be removed during the DNA extraction. Two different elution buffers are compared.
| Test A Silica column + TE elution buffer | Test B Silica column + elution with distilled water |
|---|---|
| Insert 20uL of PK in a 1,5mL eppendorf | Insert 20uL of PK in a 1,5mL eppendorf |
| Add 200 uL of sample + 200 uL of PBS 1X | Add 200 uL of sample + 200 uL of PBS 1X |
| Add 200 uL of AL buffer + shake for 15 sec | Add 200 uL of AL buffer + shake for 15 sec |
| Incubate for 20 min at RT | Incubate for 20 min at RT |
| Add 200 uL of 96% ethanol + shake for 15 sec | Add 200 uL of 96% ethanol + shake for 15 sec |
| Drop the mix in the column Elute (using the piston) → Discard the eluant | Drop the mix in the column Elute (using the piston) → Discard the eluant |
| Add 500 uL of AW1 buffer Elute (using the piston) → Discard the eluant | Add 500 uL of AW1 buffer Elute (using the piston) → Discard the eluant |
| Add 500 uL of AW2 buffer Elute (using the piston) → Discard the eluant | Add 500 uL of AW2 buffer Elute (using the piston) → Discard the eluant |
| Add 200 uL of TE buffer | Add 200 uL of distilled water |
| Incubate 1 min Elute (using the piston) | Incubate 1 min Elute (using the piston) |
The use of the piston is the most important step. Indeed, it enables to replace the centrifugation and also to reduce the extraction time. The piston needs to have exactly the same diameter than the column. It is inserted in the column and pushed to exert a pressure on the mix, allowing the liquid to pass through the silica.
Figure 2 : Explanatory scheme of the use of the piston during the experience
NanoDrop measures have been done, giving the following results :
| Test A | Test B |
|---|---|
| 28.8 | 19 |
Finally, extracting genomic DNA without centrifugation is possible. The concentration is higher when the elution is done with the TE buffer. This concentration range allows the pursuit of the experience.
3.4 Preparation of the target
The next step is the cutting of the Vibrio DNA by Alu I: one hour at 37°C in CutSmart Buffer. Then a denaturation is done at 73°C. The target now ready to be detected and the next step, the detector activation, can occur.
Step 2 :
Step 3 :
