Liu C, Jiang D N, Xiang G M, Luo F K, Liu L L, Yu J C, Pu X Y
Department of Clinical Laboratory, Xinqiao Hospital, Third Military Medical University, Chongqing, China.
Genet Mol Res. 2013 Sep 3;12(3):3296-304. doi: 10.4238/2013.September.3.6.
We used a newly developed electrochemical method, real-time resistance measurement, based on loop-mediated isothermal amplification (LAMP), with real-time resistance monitoring and derivative analysis. DNA extracted from specimens was amplified through LAMP reaction. The 2 products of LAMP, DNA and pyrophosphate, both are negative ions; they combine with positive dye (crystal violet) and positive ions (Mg(2+)), which leads to an increase in the resistivity of the reaction liquid. The changes of resistivity were measured in real-time with a specially designed resistance electrode, to detect Clostridium difficile DNA. We found that electrochemical detection of C. difficile could be completed in 0.5-1 h, with a detection limit of 10(2) CFU/mL, with high accuracy (95.0%), sensitivity (91.1%), and specificity (97.3%) compared to PCR methods. C. difficile is commonly associated with antibiotic-induced diarrhea. Due to the difficulty in performing anaerobic culture and cytotoxicity neutralization assays, a simple, rapid, sensitive, and accurate method is preferred. We conclude that real-time resistance measurement is a rapid, sensitive, and stable method for the diagnosis of C. difficile infection that could be applied to gene chips and pocket instruments.
我们采用了一种新开发的基于环介导等温扩增（LAMP）的电化学方法——实时电阻测量法，并进行实时电阻监测和导数分析。从标本中提取的DNA通过LAMP反应进行扩增。LAMP的两种产物，DNA和焦磷酸，均为负离子；它们与正性染料（结晶紫）和正离子（Mg(2+)）结合，导致反应液的电阻率增加。用专门设计的电阻电极实时测量电阻率变化，以检测艰难梭菌DNA。我们发现，艰难梭菌的电化学检测可在0.5 - 1小时内完成，检测限为10(2) CFU/mL，与PCR方法相比，具有较高的准确度（95.0%）、灵敏度（91.1%）和特异性（97.3%）。艰难梭菌通常与抗生素相关性腹泻有关。由于厌氧培养和细胞毒性中和试验操作困难，因此更倾向于采用一种简单、快速、灵敏且准确的方法。我们得出结论，实时电阻测量是一种用于诊断艰难梭菌感染的快速、灵敏且稳定的方法，可应用于基因芯片和便携式仪器。
