Detection and Quantitation of Circulating Plasmodium falciparum DNA by Polymerase Chain Reaction - Clinical Applications of PCR - page 158

3.3. Detection of P. falciparum Parasite Using a Nested PCR Assay
Described as follows is a nested PCR assay for the detection of P. falciparum
DNA in the plasma of malaria-infected individuals ( 11 ) . The PCR assay was
developed by Snounou et al. ( 2 , 3 ) for the amplification of the small sub-unit
rRNA ( ssrRNA ) gene of P. falciparum DNA. The first amplification round is
genus-specific, and thus will amplify all species of the Plasmodium parasite,
whereas the second amplification round is species-specific ( see Notes 1 and 2 ).

1. Perform PCR in a total volume of 50 µ L and include the extracted plasma DNA

L), 1X Taq polymerase buffer, 3 m M of MgCl 2 , dNTP mix (500 n M ), forward
and reverse primer (50 pmol of each primer), 2.5 U of Taq polymerase.

(5 µ

2. Suggested thermal profile:

95°C 5 min

35 cycles of: 94°C 30 s

58°C 1 min

72°C 1 min

72°C 5 min

3. For the nested PCR reaction, use 2 L of the first-round amplification and follow

the same thermal profile ( see N ote 3 ).

µ

4. Primer sequences are as follows:

a. First amplification Forward: 5' TTA AAA TTG TTG CAG TTA AAA CG 3'.
b. First amplification Reverse: 5' CCT GTT GTT GCC TTA AAC TTC 3'.
c. Second amplification Forward ( P. falciparum ): 5' TTA AAC TGG TTT GGG
AAA ACC AAA TAT AT T 3'.
d. Second amplification Reverse ( P. falciparum ): 5' ACA CAA TGA ACT CAA
TCA TGA CTA CCC GTC 3'.

5. An amplification reaction without DNA template should be included in each PCR

experiment to exclude false-positives resulting from contamination of reagents.
A malaria-positive sample should be included as a positive control. In addition to
PCR amplification of the ssrRNA gene, all DNA samples should be subjected to
PCR amplification of a human gene in order to confirm the presence of intact
DNA in the samples.

6. The presence of the second round amplification product (205 bp) can be detected

by ethidium bromide staining following agarose gel electrophoresis ( Fig. 1 ).

3.4. Quantitation of P. falciparum DNA in Plasma by Real-Time PCR
Described as follows is the protocol for the quantitation of parasite DNA
from plasma ( see Note 2 ) using a 5700 Sequence Detection System (Applied
Biosystems). The procedure can be performed with an equivalent instrument
using the appropriate reagents.

3.4.1. Preparation of the Reagents
1. Standard curve: for absolute quantitation of parasite DNA, a standard curve

should be constructed using serial dilutions of known concentrations of parasite