[Multiplex PCR for analysis of the Plasmodium falciparum drug resistance molecular markers].
OBJECTIVE
To develop a multiplex PCR protocol for amplification of five Plasmodium falciparum drug resistance related genes, thereby facilitate the rapid and high throughput analysis of the drug resistance molecular markers.


METHODS
Five pairs of primers were designed according to the reference sequences by using Primer Premier 5.0 and Oligo 6.0 software. Drug resistance related genes, including P. falciparum chloroquine resistance transporter (Pfcrt), multi-drug resistance 1 (Pfmdr1), dihydropteroate synthetase (Pfdhps), dihydrofolate reductase (Pfdhfr) and sarco/endoplasmic reticulum Ca2+-ATPase (PfATPase6), were amplified by single-tube multiplex PCR using Hot Start Taq DNA Polymerase among negative controls (P. vivax, P. berghei, P. cynomolgi, Leishmania donovani, Cryptosporidium andersoni), blank control (using H2O as template), as well as P. falciparum laboratory isolates (3D7, Dd2, HB3, FCC1/HN and CMH/YN) and field samples (collected from Yunnan, Hainan of China and Myanmar). After amplification, the PCR products were analyzed by agarose gel electrophoresis. The sequencing results were aligned to the reference sequence using BLAST.


RESULTS
Five expected bands at 315, 437, 514, 594 and 770 bp were obtained with no additional or nonspecific products in P. falciparum laboratory isolates and field samples. The sequencing results were identical with the reference sequence except the polymorphism sites, and exhibited more than 98.5% homology. The multiplex amplification was performed successfully starting from 0.1 ng of DNA template. No band was observed in negative controls and blank control.


CONCLUSIONS
The present study establishes a method to amplify five Plasmodium falciparum drug resistance related genes harboring 21 SNPs by one-tube reaction. The multiplex PCR protocol showing high specificity and sensitivity is more convenient and efficient in analyzing the P. falciparum drug resistance molecular markers as compared with traditional nested PCR.
Zhang
Guo-Qing
GQ
National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai 200025, China.
Tang
Lin-Hua
LH
Guan
Ya-Yi
YY
Zhou
Shui-Sen
SS
Zheng
Bin
B
Huang
Fang
F
Wu
Song
S
Liu
Yan
Y
D000818:Animals; D016054:DNA, Protozoan / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics*; D006801:Humans; D016778:Malaria, Falciparum / Q000469:parasitology; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D015800:Protozoan Proteins / Q000235:genetics
D016054:DNA, Protozoan; D015800:Protozoan Proteins