Biomedical Engineering Reference
In-Depth Information
14
Detection and Quantitation of Circulating Plasmodium
falciparum DNA by Polymerase Chain Reaction
Shira Gal and James S. Wainscoat
Summary
This chapter describes the application of polymerase chain reaction (PCR) for the
detection and quantitation of
Plasmodium falciparum
DNA in the plasma of malaria-
infected individuals. The procedure includes the following protocols: plasma sample prepa-
ration, DNA extraction, detection of
P. falciparum
DNA in the plasma by nested PCR, and
quantitation of
P. falciparum
DNA in the plasma by real-time PCR technology.
Key Words:
Plasmodium falciparum
; malaria; plasma; PCR; real-time PCR.
1. Introduction
The standard method for the diagnosis of malaria is the preparation and
microscope examination of stained blood smears
(
1
)
. However, in recent years,
polymerase chain reaction (PCR) has been introduced for the detection of DNA
from the
Plasmodium
parasite. The major advantage of sensitive PCR tech-
niques is the ability to detect a low level of parasitemia. Detection of as few as
one parasite per microliter of blood have been reported
(
2-4
)
. Other applica-
tions of PCR in malaria studies include the detection of mixed infections
(
3
,
5
)
and the identification of different genotypes and genetic markers of virulence
(
6
)
. The introduction of real-time PCR technology has enabled a quantitative
method for the measurement of parasite burden. Real-time PCR was used for
several purposes: monitoring the levels of
P. falciparum
in the blood of malaria-
infected individuals
(
7
)
, the quantitation of malaria liver-stage parasites in mice
(
8
,
9
)
, and the detection of malaria parasites in blood of infected people
(
10
)
.
The following protocol describes the application of PCR and real-time PCR
for the detection and quantitation of circulating
P. falciparum
DNA in the
plasma of malaria patients.
