海南恶性疟原虫分离株Pfcrt和Pfmdr1基因点突变的研究

The Point Mutations in Pfcrt and Pfmdr1 Genes in Plasmodium falciparum Isolated from Hainan Province

目的分析海南省恶性疟原虫分离株氯喹抗性转运蛋白编码基因(Pfcrt)及其P-糖蛋白同系物1(Pgh1)编码基因(Pfmdr1)的点突变特征,为探讨抗性分子标记用于氯喹抗性监测提供参考。方法采用巢式PCR(nested-PCR)和限制性酶切片段长度多态性(RFLP)方法,检测Pfcrt基因编码第76位氨基酸的密码子和Pfmdr1基因编码第86、1246位氨基酸的密码子发生点突变情况。按世界卫生组织(WHO)推荐的体外微量法测定恶性疟原虫对氯喹的敏感性。结果检测的36份血样中,28份成功扩增Pfcrt基因,导致第76位氨基酸由赖氨酸(K)变为苏氨酸(T)的突变型占64.3%,混合型占14.3%,野生型占21.4%;29份成功扩增Pfmdr1基因,导致第86位氨基酸由天冬酰胺(N)变为酪氨酸(Y)的突变型占3.4%,混合型占6.9%,野生型占89.7%。未发现编码第1246位氨基酸的密码子发生点突变。体外氯喹敏感试验结果显示,72.2%(26/36)分离株存在抗性。治疗前恶性疟原虫Pfcrt76T突变发生率在体外微量测定法显示的氯喹抗性与敏感株中的差异有统计学意义(P<0.05),而Pfmdr1点突变发生率在氯喹抗性与敏感株中的差异无统计学意义(P>0.05)。结论恶性疟原虫Pfcrt基因76T可以作为监测氯喹抗性的一个分子标记。

Objective To evaluate the point mutations in Pfcrt and Pfmdr1 genes in Plasmodium falciparum isolated from Hainan Province. Methods Nested polymerase chain reaction and restriction fragment length polymorphism were used to detect the point mutations at codon 76 of Pfcrt and at codon 86, 1246 of Pfmdr1 in P. falciparum isolates. Chloroquine resistance was measured by the in vitro microtest recommended by WHO. Results In 36 samples tested, 28 were successfully amplified for Pfcrt, 64.3% of them carried mutant allele at codon 76, 21.4% with wild allele K76 and 14.3% with mixed allele mutation. While for Pfmdr1, 3.4% isolates displayed the 86Y mutation, 89.7% with wild allele N86 and 6.9% with the mixed alleles in 29 isolates which were amplified successfully for N86Y. No point mutation in Pfmdr1 at codon 1246 was found in 13 isolates from the total 36 samples. By the in vitro test, 72.2% (26/36) showed resistance to chloroquine. The 76T and 86Y mutant alleles were present in both in vitro susceptible and resistant isolates. There was a significant difference between susceptible and resistant isolates carrying 76T mutant codon(P<0.05), but no significant difference was found in Pfmdr1(P>0.05). Conclusion There is a significant difference of the 76T prevalence in Pfcrt gene between the susceptible isolate and resistant one of P. falciparum to chloroquine in vitro. The Pfcrt 76T may be used as a predictive marker for chloroquine resistance surveillance.