3.3.2 Total DNA
Extraction from Tissue
Sample
1. Tissue samples in MagNA Lyser Green Beads tubes are first
disrupted and homogenized using the MagNA Lyser Instrument during 50 s at 4000 Â g.
2. DNA extraction is performed with QIAamp DNA Mini Kit
according to manufacturer’s instructions. After washing steps,
the total DNA is eluted in elution buffer or water.
3. Quantify
total
eluted
DNA
concentration
by
spectrophotometry.
3.3.3 Bacterial Load
Quantification by qPCR
1. Bacterial DNA in hamsters total eluted DNA from previous
steps is analyzed by quantitative PCR (qPCR) assay (see Note
11). Primer sequences are provided in Table 2. Even though
qPCR parameters should be adjusted depending on the instrument and reagents, they should include the following steps:
95
C, 5–8 s (denaturation step); 58–62
C, 5–12 s (annealing
step); 72
C, 10–12 s (elongation steps); 40–50 cycles.
2. Genomic DNA extracted from the Leptospira challenge strain
(here LiVV) is used as a positive control. A standard curve
obtained from serial tenfold dilutions of known numbers of
leptospires is used for absolute quantification of Leptospira
genome found in tissue samples from infected animals.
Table 2
Sequence of primers used for qPCR assays
Gene name
Primer sequence (5
0
! 3
0 )
Amplicon size (bp)
Leptospira LipL32
(F) CGGACGGTTTAGTCGATG
(R) GCATAATCGCCGACATTC
228
Hamster β-actin
(F) TCTACAACGAGCTGCG
(R) CAATTTCCCTCTCGGC
357
Hamster GAPDH
(F) CCGAGTATGTTGTGGAGTCTA
(R) GCTGACAATCTTGAGGGA
170
Hamster IL-1β
(F) ATCTTCTGTGACTCCTGG
(R) GGTTTATGTTCTGTCCGT
156
Hamster TNF-α
(F) AACGGCATGTCTCTCAA
(R) AGTCGGTCACCTTTCT
278
Hamster IL-6
(F) AGACAAAGCCAGAGTCATT
(R) TCGGTATGCTAAGGCACAG
252
Hamster IL-10
(F) TGGACAACATACTACTCACTG
(R) GATGTCAAATTCATTCATGGC
308
Hamster MIP-1α/CCL3
(F) CTCCTGCTGCTTCTTCTA
(R) TGGGTTCCTCACTGACTC
210
Hamster IP-10/CXCL10
(F) CTCTACTAAGAGCTGGTCC
(R) CTAACACACTTTAAGGTGGG
150
250
Julie Cagliero et al.
Extraction from Tissue
Sample
1. Tissue samples in MagNA Lyser Green Beads tubes are first
disrupted and homogenized using the MagNA Lyser Instrument during 50 s at 4000 Â g.
2. DNA extraction is performed with QIAamp DNA Mini Kit
according to manufacturer’s instructions. After washing steps,
the total DNA is eluted in elution buffer or water.
3. Quantify
total
eluted
DNA
concentration
by
spectrophotometry.
3.3.3 Bacterial Load
Quantification by qPCR
1. Bacterial DNA in hamsters total eluted DNA from previous
steps is analyzed by quantitative PCR (qPCR) assay (see Note
11). Primer sequences are provided in Table 2. Even though
qPCR parameters should be adjusted depending on the instrument and reagents, they should include the following steps:
95
C, 5–8 s (denaturation step); 58–62
C, 5–12 s (annealing
step); 72
C, 10–12 s (elongation steps); 40–50 cycles.
2. Genomic DNA extracted from the Leptospira challenge strain
(here LiVV) is used as a positive control. A standard curve
obtained from serial tenfold dilutions of known numbers of
leptospires is used for absolute quantification of Leptospira
genome found in tissue samples from infected animals.
Table 2
Sequence of primers used for qPCR assays
Gene name
Primer sequence (5
0
! 3
0 )
Amplicon size (bp)
Leptospira LipL32
(F) CGGACGGTTTAGTCGATG
(R) GCATAATCGCCGACATTC
228
Hamster β-actin
(F) TCTACAACGAGCTGCG
(R) CAATTTCCCTCTCGGC
357
Hamster GAPDH
(F) CCGAGTATGTTGTGGAGTCTA
(R) GCTGACAATCTTGAGGGA
170
Hamster IL-1β
(F) ATCTTCTGTGACTCCTGG
(R) GGTTTATGTTCTGTCCGT
156
Hamster TNF-α
(F) AACGGCATGTCTCTCAA
(R) AGTCGGTCACCTTTCT
278
Hamster IL-6
(F) AGACAAAGCCAGAGTCATT
(R) TCGGTATGCTAAGGCACAG
252
Hamster IL-10
(F) TGGACAACATACTACTCACTG
(R) GATGTCAAATTCATTCATGGC
308
Hamster MIP-1α/CCL3
(F) CTCCTGCTGCTTCTTCTA
(R) TGGGTTCCTCACTGACTC
210
Hamster IP-10/CXCL10
(F) CTCTACTAAGAGCTGGTCC
(R) CTAACACACTTTAAGGTGGG
150
250
Julie Cagliero et al.