摘要
目的应用一种敏感快速的多重聚合酶链反应（ＰＣＲ），检测耐甲氧西林的金黄色葡萄球菌（ＭＲＳＡ）和凝固酶阴性葡萄球菌（ＭＲＣＮＳ）。方法临床分离的北京地区金黄色葡萄球菌１２３株，ＭＲＣＮＳ１２２株，用溶壁素和蛋白酶Ｋ制备模板ＤＮＡ，设计葡萄球菌甲氧西林耐药的决定基因，金黄色葡萄球菌独有的一个辅助基因和细菌中均有的１６ＳｒＲＮＡ基因引物，通过多重ＰＣＲ技术对标本进行扩增。结果１２３株金黄色葡萄球菌的ｆｅｍＡ基因１００％（１２３／１２３）阳性，ｍｅｃＡ基因阳性的占１８．７％（２３／１２３），１２２株ＭＲＣＮＳ的ｆｅｍＡ１００％（１２２／１２２）阴性，ｍｅｃＡ阳性的占２４．６％（３０／１２２）。１６ＳｒＲＮＡ基因片断在多重ＰＣＲ中作为内部参照避免了假阴性结果的出现。结论建立的多重ＰＣＲ技术检测ＭＲＳＡ和ＭＲＣＮＳ具有敏感、快速、特异的特点，是一种可靠的实验诊断手段。
Objective To establish a sensitive and speedy multiple polymerase chain reaction
(multiplex PCR) technique for methicillin resistant staphylococcus aureus and methicillin
resistant coagulase negative staphylococci detection. Methods 123 strains staphylococcus
aureus and 122 strains coagulase negative staphylococci were detected for mecA, femA and
16S rRNA genes. Template DNA of bacteria was prepared by Lysostaphine and Proteinase K.
By using a multiplex PCR strategy, 392 bp and 686 bp regions of the mecA and femA genes,
were coamplified to identify susceptible (lacking mecA) and resistant (mecA+) staphylococci and
to differentiate staphylococcus aureus (femA+) from coagulasenegative staphylococci (laking
femA). The 876 bp region of the 16S rRNA gene was used as a PCR internal control. Results The
femA gene positive rate of 123 staphylococcus aureus and 122 coagulasenegative
staphylococcus was 100%(123/123) and 0%(0/122) respectively. The mecA gene positive rate of
staphylococcus aureus and coagulasenegative staphylococcus was 18.7% (23/123) and 24.6%
(30/122). Conclusion The multiplex polymerase chain reaction is a specific, sensitive and fast
method for MRSA and MRCNS detection.
关键词
聚合酶链反应
MRCNS
MRSA
Polymerase chain reactionStaphylococcus,
aureusMethicillin resistanceCoagulase
