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Leptospira PCR 199
6. This method can be used for small vol of relatively “clean” samples, which do
not require concentration and do not contain large amounts of substances, such
as polysaccharides that may interfere with the amplification reaction. This may
include small water or urine samples, cerebrospinal fluid (CSF) samples, and
aqueous humor samples.
7. At this point the Sample Concentration Method (see Subheading 3.1.2.) is completed
as for the Simple Boiling Method (see Subheading 3.1.1.), but the CTAB
method (see Subheading 3.1.3.) may be used to prepare the sample if required
(see Note 4).
8. At this point, if a layer of CTAB-carbohydrate is still visible at the interface,
additional CTAB can be added to the supernatant, and the extraction can be
repeated until no carbohydrate layer is visible.
9. Getting accurate results from PCR requires a high degree of cleanliness. We use
a clean UV hood for preparing the PCR in an area of the laboratory separate from
that used for sample preparation. The area used for preparation of the PCR should
be thoroughly cleaned with bleach before use, and filter-tipped pipets should be
used in order to prevent cross-contamination of samples. Adequate use of both
negative and positive controls will also insure the accuracy of the results.
10. The master mixture should be made fresh as required and not prepared in advance.
While this amount of master mixture is sufficient for 11 samples, it is advisable to
make more master mixture than needed as this will allow for small pipeting mistakes.
Additionally, by preparing fresh master mixture for every run, the risk of
contamination is lessened.
11. Thin-walled microcentrifuge tubes designed for PCR allow faster heat transfer,
which allows shorter PCR cycles to be used.
12. This prevents evaporation of the solution during heating. Fresh molecular biology-grade
mineral oil should be used, as old mineral oil that has been damaged
by UV radiation can inhibit PCR (9). Other substances such as wax beads can
also be used.
13. We use an MJ thermal cycler (MJ Research, Waltham, MA, USA), but any thermal
cycler can be used. Other thermal cyclers can allow different (and in some
cases faster) amplification conditions.
References
1. Faine, S. (1994) Leptospira and Leptospirosis. CRC Press, Boca Raton.
2. Kmety, E., and Dikken, H. (1993) Classification of the Species Leptospira
interrogans and history of its serovars. University Press, Groeningen, The
Netherlands.
3. Zuerner, R. L., Alt, D., and Bolin, C. A. (1995) IS1533-based PCR assay for identification
of Leptospira interrogans sensu lato serovars. J. Clin. Microbiol. 33,
3284–3289.
4. Masri, S. A, Nguyen, S., Gale, P., Howard, C. J., and Jung, S. (1997) A polymerase
chain reaction assay for the detection of Leptospira spp. in bovine semen.
Can. J. Vet. Res. 61, 15–20.