and elute the DNA in 100 μl elution buffer. Determine the
DNA concentration using a spectrophotometer at 260 nm (see
Note 21).
4. Dilute Leptospira genomic DNA to 10,080 pg/μl. Prepare
tenfold serial dilutions of the DNA for the standard curve
(1 Â 10
7 to 1 Â 10
0 genomic equivalents (Geq)/5 μl) (see
Note 22).
5. Prepare the following PCR mix using Bio-Rad iTaq Universal
Probes Supermix.
Reagent
1Â reaction
Master mix 2Â
10 μl
Primer 1, 250 nM (lipL32-45F)
0.5 μl
Primer 2, 250 nM (lipL32-268R)
0.5 μl
Probe, 150 nM (lipL32-189P)
0.6 μl
Water
3.4 μl
DNA
5 μl
Final volume
20 μl
6. Run the following program on a quantitative thermal cycler:
95
C for 3 min followed by 40 cycles of amplification (95
C
for 15 s and 60
C for 1 min). Process all samples and standards
in duplicates.
7. Generate a standard curve, using the thermal cycler software.
Plot Ct (cycle threshold) against the number of Geq/5 μl.
8. Determine the number of Geq/5 μl in the samples by using the
standard curve generated by the thermal cycler software and
threshold cycle obtained for each sample. A C t ! 36 is considered as no amplification.
9. Calculate the number of Geq per gram of tissue or μl of the
blood using the formula Geq/g ¼ Geq/5 μl  elution factor  volume factor (see Note 23).
4 Notes
1. Purified Leptospira protein for vaccine testing is prepared by
recombinant DNA technology. The selected Leptospira gene is
cloned and expressed as a recombinant protein in E. coli. Antigen preparations should be in PBS, sterile, and free of urea,
acetic acid, endotoxin (LPS), and other toxic solvents.
2. Complete Freund’s adjuvant (CFA) is extensively used and
considered to be the most effective adjuvant for consistently
Leptospirosis Vaccine Studies in Hamsters
265