Sequencing of bacterial microflora, translocating in peripheral blood. Our experience with HIV infected patients, I am junior Marti, and I work as assistant professor at the Clinic of Infectious Diseases and Tropical Medicine at the University of Milan in Italy. And we will now show you experiments on how to detect and sequence bacterial DNA from peripheral blood of HIV infected patients.
The healthy gastrointestinal tract is colonized by a large variety of commensal microbes that influence the development of the humeral and cellular cusal immune system. Microbiota is shielded from the immune system via a strong mucosal barrier following several clinical conditions such as infections, antibiotic treatment, postoperative complications, as well as in patients with carcinoma, pancreatitis, and malnutrition. An alteration of the gastrointestinal tract occurs.
HIV infection causes a breach of the gastrointestinal barrier with progressive failure of mucosal immunity and leakage into the systemic circulation of bacterial bioproducts, such as lipopolysaccharide and bacterial DNA fragments, which contribute to systemic immune activation, demonstrating the experimental phases of the detection and sequencing of bacterial DNA. Present in peripheral blood of HIV infected patients will be Camilla ti, an infectious disease fellow Esther, me, a PhD student and ju MariaI, a postdoctoral fellow all attending the clinic of Infectious Diseases and the tropical medicine at the University of Milan. Our experiment aims to identify bacterial species and peripheral blood samples from HIV infected patients.
We're going to use whole blood from both HIV infected and healthy subjects, given that healthy individuals present normal intestinal homeostasis. We do not expect to see translocating microflora in these patients. Whole blood will be collected from patients followed by plasma separation.
DNA will be then extracted in order to perform a broad range PCR reaction for the PCIC 16 s ribosomal gene. Following PCR product, purification, cloning and sequencing analysis will be performed. The first step of our experiment is blood collection by venipuncture, followed by plasma separation.
Whole blood, approximately nine mils is drawn into EDTA containing tubes. Tubes are placed in a centrifuge at 2000 RPM for 10 minutes. At room temperature centrifugation allows for plasma separation.
DNA will now be extracted from our plasma samples Adequately disinfect hood, peppes, and material needed for the experiment in order to guarantee sterility position all material under UV lights for at least 30 minutes. Turn off UV lights, wipe gloves with a disinfectant, soak a few paper towels in ethanol, and place them under the hood every time a tip is discarded. During the DNA extraction procedure, wipe the pipet on the wet paper towels.
You are now ready for DNA extraction. DNA is extracted using a commercial kit, following manufacturing instructions. In brief, 350 microliters of plasma is placed in a two mil eend orph tube.
Ultra pure water is used as negative control. 10 microliters of lysosome at a final concentration of one mg per mil is added to the samples. Samples are then incubated for 30 minutes.
At 37 degrees Celsius solution A, chloroform and solution B are then added to the samples according to instructions following centrifugation. Three distinct phases will appear. The supernatant is collected and added to ethanol fault, allowing for DNA precipitation, DNA concentration is read with a spectrophotometer.
The next step is a broad range of PCR reaction amplifying for the PRIC 16 s ribosomal gene PCR is performed in a dedicated room. A PCR mix Is made using these reagents. The PCR mix is filtered for 30 minutes at 500 G at four degrees Celsius in order to avoid possible contamination by bacterial tap polymerase.
The PCR mix is then divided into aliquots. According to the number of samples which need to be amplified. Remember to consider aliquots for negative and positive reaction controls.
THERMOCYCLING is programmed at the following conditions, 94 degrees Celsius for five minutes. 94 degrees Celsius, 55 degrees Celsius, 72 degrees Celsius for one minute for 40 cycles, 72 degrees Celsius for 10 minutes. Average duration is three hours.
PCR products are visualized on a 2%AGROS gel. This is a 2%agros gel showing PCR products. The first lane contains a 100 base pair ladder.
The second lane, A PCR positive control. The third lane contains a negative PCR control. The fourth lane shows samples from an HIV positive patient and the fifth lane contains water lane.
Six shows samples from a healthy individual and a negative PCR reaction. Lane seven contains water. PCR IFICATION is the following step.
PCR products are purified using a commercial kit, following manufacturer's instructions. Many columns containing filters are used for this step. PCR results are recorded on a working sheet.
We are now going to proceed with cloning by using the blue white screen. A molecular technique that allows for the detection of successful DNA lation into a vector. The vector is transformed into competent cells.Bacteria.
The competent cells are grown in the presence of xal. If the lation is successful, the bacterial colony will be white. If not the colony will be blue.
Xal is used to indicate whether a cell expresses the beta galacto aase enzyme and to visualize the transformation of e coli colonies, IPTG induces the expression of beta galacto. Aase Xal and IPTG are swirled onto ampicillin containing plates. A cloning mix containing PCR products, SALT and a cloning vector is prepared following manufacturer's instructions.
E coli cells are thought on ice. The cloning mix is then added to the cells, which are later following thermal shock plates are incubated overnight at 37 degrees Celsius. Following incubation, white and blue colonies develop on plates.
White colonies are positive for PCR products. Insertion and blue colonies are negative for PCR product insertion. 10 white colonies and one blue colony are selected.
Each colony is placed into two mils of liquid LB growth. Medium with the addition of ampicillin colonies are incubated overnight at 37 degrees Celsius. Following incubation colonies O grown overnight are used for plasmid extraction with a commercial mini prep kit.
Mini columns containing filters are used for the step Following plasmid extraction plasmid are visualized on a 0.7%AROS gel. This is a 0.7%AROS gel showing mini prep products. The first lane contains a one kilo base ladder.
The second lane contains the blue control colony. Lanes three through 12 contain white colonies. Results are recorded with a working sheet sequencing Step follows, plasmids are amplified.
A mix is made using these reagents. The PCR mix is divided into aliquots according to the number of samples which need to be amplified. Thermocycling is programmed at the following conditions, 96 degrees Celsius for one minute, 96 degrees Celsius for 10 seconds, 50 degrees Celsius for five seconds, 60 degrees Celsius for four minutes.
For 25 cycles. Average duration is approximately one hour. Following amplification, PCR products are purified using filter containing columns.
Purified products are loaded on a plate, which is then placed into a sequencer. Once a sequenced sequence is obtained, it is launched in blast and bacterial species from each colony are identified. Colony displaying a 98%to 100%homology sequence are considered positive.
This is an example of bacterial sequencing in plasma from one HIV positive individual. Our results show that microbial translocation in HIV disease involves a polymicrobial flora. We have hereby shown that HIV infected patients present a high polymicrobial microbic microflora in the systemic circulation.
Bacterial DNA in peripheral blood was under detection limit for our HIV negative healthy individuals possibly reflecting the integrity of the gastrointestinal tract. Our research is now focused on understanding and investigating the possible role of microflora in peripheral blood, in conditioning, HIV Immunopathogenesis, and possibly the response to therapy.
