›› 2007, Vol. 25 ›› Issue (6): 4-456.

• 论著 • Previous Articles     Next Articles

Multiplex PCR for Analysis of the Plasmodium falciparum Drug Resistance Molecular Markers

ZHANG Guo-qing,TANG Lin-hua,GUAN Ya-yi，ZHOU Shui-sen,ZHENG Bin,HUANG Fang,WU Song,LIU Yan   

  1. National Institute of Parasitic Diseases，Chinese Center for Disease Control and Prevention; Laboratory of Parasite and Vector Biology，Ministry of Health; WHO Collaborating Centre for Malaria，Schistosomiasis and Filariasis，Shanghai 200025，China
  • Received:1900-01-01 Revised:1900-01-01 Online:2007-12-30 Published:2007-12-30
  • Contact: TANG Lin-hua

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Abstract: 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/endo-plasmic reticulum Ca 2+-ATPase（PfATPase6）， were amplified by single-tube multiplex PCR using Hot Start Taq DNA Poly-merase 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. Conclusion 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.

Key words: Plasmodium falciparum, Drug resistance, Single nucleotide polymorphism, Multiplex polymerase chain reaction