Biomedical Engineering Reference
In-Depth Information
Fig. 2. An example of a standard curve for the real-time polymerase chain reaction
assay of the
small sub-unit rRNA
(
ssrRNA
) gene of
Plasmodium falciparum
. Standard
curves constructed by plotting the threshold
cycle value (
y
-axis) against the input
concentration of each dilution (
x
-axis) from 400,000 to 4 genome equivalents in
logarithmic scale.
4. Notes
1.
By using different second round amplification primers, it is possible to detect the
other species of the
Plasmodium
parasite.
2.
The nested PCR assay for the detection of
P. falciparum
DNA as well as the
TaqMan assay can be performed on DNA extracted from the blood of malaria
patients. DNA may be extracted using the Qiagen QIAamp blood mini kit for
small volumes of blood or any other DNA extraction kit. It is sometimes recom-
mended to extract parasite DNA from the red cells.
3.
PCR conditions suggested under
Subheading 3.3.
may need adjustment—i.e.,
reagent concentration and thermal profile may be different when using different
PCR kits and equipment.
4.
Because 4
L of sample/standard is used for each reaction, the quantities of the
standards are 400,000, 40,000, 4000, 400, 40, and 4 genome equivalents.
µ
5.
Cycle number may be increased to 45 if the samples have very low parasitemia.
References
1. Makler, M. T., Palmer, C. J., and Ager, A. L. (1998) A review of practical tech-
niques for the diagnosis of malaria.
Ann. Trop. Med. Parasitol.
92,
419-433.
2. Snounou, G., Viriyakosol, S., Jarra, W., Thaithong, S., and Brown, K. N. (1993)
Identification of the four human malaria parasite species in field samples by the
polymerase chain reaction and detection of a high prevalence of mixed infections.
Mol. Biochem. Parasitol.
58,
283-292.
