利用环介导等温扩增法快速检测携带bla(VIM-2)、bla(IMP-1)和bla(OXA-23)基因的铜绿假单胞菌和鲍曼不动杆菌。
Rapid detection of Pseudomonas aeruginosa and Acinetobacter baumannii Harboring bla(VIM-2), bla(IMP-1) and bla(OXA-23) genes by using loop-mediated isothermal amplification methods.
作者信息
Kim Hye Jin, Kim Hyung Sun, Lee Jae Myun, Yoon Sang Sun, Yong Dongeun
机构信息
Department of Microbiology and Immunology, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea.
Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, Korea.
出版信息
Ann Lab Med. 2016 Jan;36(1):15-22. doi: 10.3343/alm.2016.36.1.15.
BACKGROUND
Carbapenem-resistant Pseudomonas aeruginosa (CRPA) and Acinetobacter baumannii (CRAB) are the leading causes of nosocomial infections. A rapid and sensitive test to detect CRPA and CRAB is required for appropriate antibiotic treatment. We optimized a loop-mediated isothermal amplification (LAMP) assay to detect the presence of bla(VIM-2), bla(IMP-1), and bla(OXA-23), which are critical components for carbapenem resistance.
METHODS
Two sets of primers, inner and outer primers, were manually designed as previously described. The LAMP buffer was optimized (at 2mM MgSO₄) by testing different concentrations of MgSO₄. The optimal reaction temperature and incubation time were determined by using a gradient thermocycler. Then, the optimized bla(VIM-2), bla(IMP-1), and bla(OXA-23) LAMP reactions were evaluated by using 120 P. aeruginosa and 99 A. baumannii clinical isolates.
RESULTS
Only one strain of the 100 CRPA isolates harbored bla(IMP-1), whereas none of them harbored bla(VIM-2). These results indicate that the acquisition of bla(VIM-2) or bla(IMP-1) may not play a major role in carbapenem resistance in Korea. Fifty two strains of the 75 CRAB isolates contained bla(OXA-23), but none contained bla(VIM-2) and bla(IMP-1) alleles.
CONCLUSIONS
Our results demonstrate the usefulness of LAMP for the diagnosis of CRPA and CRAB.
背景
耐碳青霉烯类铜绿假单胞菌（CRPA）和鲍曼不动杆菌（CRAB）是医院感染的主要原因。为了进行适当的抗生素治疗，需要一种快速灵敏的检测方法来检测CRPA和CRAB。我们优化了一种环介导等温扩增（LAMP）检测方法，以检测bla(VIM-2)、bla(IMP-1)和bla(OXA-23)的存在，这些是耐碳青霉烯类的关键成分。
方法
如前所述手动设计两组引物，即内引物和外引物。通过测试不同浓度的MgSO₄对LAMP缓冲液进行优化（2mM MgSO₄）。使用梯度热循环仪确定最佳反应温度和孵育时间。然后，使用120株铜绿假单胞菌和99株鲍曼不动杆菌临床分离株评估优化后的bla(VIM-2)、bla(IMP-1)和bla(OXA-23) LAMP反应。
结果
100株CRPA分离株中只有1株携带bla(IMP-1)，而它们均不携带bla(VIM-2)。这些结果表明，bla(VIM-2)或bla(IMP-1)的获得在韩国耐碳青霉烯类中可能不发挥主要作用。75株CRAB分离株中有52株含有bla(OXA-23)，但均不含有bla(VIM-2)和bla(IMP-1)等位基因。
结论
我们的结果证明了LAMP在CRPA和CRAB诊断中的实用性。
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