Polymerase Chain Reaction and Dot-Blot Hybridization for Leptospira Detection in Water Samples

This protocol is designed to identify the presence of Leptospira DNA water samples. The Dot-Blot technique is utilized for these particles, which enables accurate detection and analysis of the DNA. The Dot-Blot is an easy technique, and may help to answer key questions about environmental distribution of Leptospira.

This method has a potential to assess huge water bodies and aid in disease surveillance. To begin, take 1.5 millimeter micro centrifuge tubes containing the water samples. Centrifuge the samples at 8, 000G for 10 minutes at four degrees Celsius.

After extracting the DNA from the samples, perform the specific PCR according to the Dot-Blot probe used. Then place 10 microliters of each PCR product to be blotted in a separate well of a 96 well plate. Add 40 microliters of Tris-EDTA buffer to each well and mix by pipetting.

Take a nylon membrane, and with scissors, notch one edge of the membrane to indicate the right orientation. Moisten the membrane and filter paper with 10 times SSPE. To assemble the Dot-Blot chamber, first, place the filter paper, followed by the membrane and the plastic seal.

Then secure the cover with screws in a crosswise manner. Now, connect the chamber to the vacuum. Add 100 microliters of Tris-EDTA buffer to each well.

Maintain the vacuum for one minute, then stop it. Afterwards, turn on the vacuum at low speed and load 50 microliters of each sample into the corresponding well on the membrane of the Dot-Blot apparatus. Allow the vacuum to dry the membrane.

Then, place 100 microliters of TE buffer into each well under a continuous vacuum and allow it to dry. To begin, take out the nylon membrane from the Dot-Blot apparatus after transferring the amplified DNA. Incubate the membrane with 0.4 molar sodium hydroxide for 10 minutes at room temperature.

Equilibrate the membrane with 10 times SSPE for 10 minutes at room temperature. Then, dry the membrane at 80 degrees Celsius for two hours. Now, transilluminate the membrane with 120 millijoules of ultraviolet light for three minutes.

After that, wash the membrane with two times SSPE for 10 minutes. Carefully fold the membrane and insert it into the hybridization tube. Next, add 10 milliliters of the pre-hybridization solution to the tube and incubate at 42 degrees Celsius overnight.

For probe hybridization, remove five milliliters of the pre-hybridization solution from the hybridization tube. Then, add 15 microliters of the labeled probe to the hybridization tube. Rinse the tip in the solution to ensure the labeled probe is thoroughly instilled into the dilution.

Incubate at 42 degrees Celsius overnight. To begin, transfer the PCR-amplified samples to a nylon membrane, and hybridize them with the probe. Wash the membrane twice with two times SSPE mixed with 0.1%SDS at room temperature for 10 minutes.

Then, incubate the membrane with five times SSPE mixed with 0.1%SDS at the probe's annealing temperature for 15 minutes. At the end of the incubation, wash the membrane with buffer one and two for five and 30 minutes respectively. After washing the membrane with buffer two once, add 15 microliters of anti-digoxigenin antibody and incubate for 45 minutes.

Wash the membrane twice in buffer one at room temperature for 15 minutes. Wash once in buffer three at room temperature for five minutes. Then, add CSPD ready to use and allow it to remain for five minutes.

After that, place the membrane in a transparent plastic bag, and incubate the membrane in a water bath at 37 degrees Celsius for 15 minutes. Afterwards, dry the plastic bag and fix it with scotch tape on an already used radiographic plate. For detection, carefully place the fixed membrane facing a new radiographic plate in the dark, and insert it into a radiographic cassette.

Register the time of exposure. The dot A7 shows that Leptospiral DNA was present on the surface of pond three. The probe's signal can be easily detected down to 3.91 times 10 to the negative first nanograms of Leptospira DNA mix.