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目的：构建定量逆转录聚合酶链反应（ＲＴＰＣＲ）的内标准ＤＮＡ模板和ＲＮＡ模板，定量检测多药耐药相关蛋白（ＭＲＰ）基因表达的ｍＲＮＡ。方法和结果：利用现有的ＭＲＰ全基因质粒和分子克隆技术经２次克隆将庚型肝炎病毒的一段２３８ｂｐ的核苷酸序列插入ＭＲＰ２９２ｂｐ的目的ｃＤＮＡ片段，构建了插入突变型ＭＲＰｃＤＮＡ重组体作为定量ＰＣＲ的ＤＮＡ竞争模板；再经第３次克隆构建了插入突变型ＭＲＰＲＮＡ重组体，最后经体外转录出５３０ｎｔ的正链ＲＮＡ作为逆转录的ＲＮＡ竞争模板。结论：所建立的ＲＴＰＣＲ技术简便、快速、灵敏，可检出１ｆｇ水平的ｍＲＮＡ量。用ＤＮＡ竞争模板定量检测比用ＲＮＡ竞争模板更简便、经济、可靠。
OBJECTIVE: To construct an internal standard DNA template and RNA template for quantitative reverse transcription polymerase chain reaction (RT-PCR) and quantitatively detect mRNA expression of multidrug resistance-associated protein (MRP). METHODS AND RESULTS: A 238 bp nucleotide sequence of hepatitis G virus was inserted into the MRP 292 bp target cDNA fragment using the existing MRP whole-gene plasmid and molecular cloning technique, and an insertion mutant MRP cDNA recombinant was constructed. As a DNA competition template for quantitative PCR, the insert-mutant MRP-RNA recombinant was constructed by the third cloning, and finally 530 nt positive strand RNA was transcribed in vitro as a reverse-transcription RNA competition template. Conclusion: The established RT-PCR technique is simple, rapid, and sensitive, and detects 1fg mRNA levels. It is more convenient, economical and reliable to quantify DNA template with competitive template than to use RNA template.