PCR Detection of Microbial Pathogens PCR Detection of Microbial ...

Leptospira PCR 195
Table 1
Oligonucleotide Primers for PCR of Pathogenic Leptospira
Name and Positiona Nucleotide sequence, 5' to 3'
Outer primer set
428–450 5'-AGGGAAAAATAAGCAGCGATGTG-3'
981–999cb 5'-ATTCCACTCCATGTCAAGCC-3'
Product size: 571 bp
Nested primer Set
552–570 5'-GAAAACTGCGGGCTCAAAC-3'
925–940c b 5'-GCTCCACCGCTTGTGC-3'
Product size: 370 bp
a Corresponds to the L. interrogans sensu stricto 16S gene sequence (Accession no. X17547)
b Primers labeled “c” are complementary to those in the sequence.
2.2. PCR Amplification
1. Sterile double distilled H 2O.
2. Taq DNA Polymerase (see Note 4).
3. PCR buffer (10X): 200 mM Tris-HCl, pH 8.4, 500 mM KCl, 15 mM MgCl 2 , or as
supplied by manufacturer with Taq DNA polymerase (see Note 5).
4. dNTP mixture (dATP, dTTP, dCTP, and dGTP): 1.3 mM each.
5. Oligonucleotide primers (see Table 1), concentration adjusted to 50 pmol/µL.
6. Sterile mineral water.
7. Thermal cycler.
8. Agarose (molecular biology-grade).
9. Tris-Borate-EDTA buffer (TBE): To make a stock solution of 5X TBE, dissolve
54 g of Tris-base and 27.5 g of boric acid in about 750 mL of purified water, add
20 mL of 0.5 M EDTA, pH 8.0, and adjust the vol of the solution to 1 L with
purified water. Dilution of the buffer 1 in 10 with purified water will give a final
0.5X running buffer.
10. Ethidium bromide solution: 0.5 µg/mL in 0.5X TBE buffer.
11. Loading buffer: 20% Ficoll ® 400, 0.1 M Na 2 EDTA pH 8.0, 1.0% sodium dodecyl
sulfate (SDS), 0.25% bromophenol blue.
3. Methods
3.1. Sample Preparation
3.1.1. Simple Boiling Method (see Note 6)
1. Boil the sample for 10 min in a microcentrifuge tube.
2. Centrifuge at 14,000g for 5 min.
3. Transfer supernatant to a fresh tube and store at 20°C.