田鼠巴贝虫感染FTA-环介导等温扩增检测技术的建立

目的 建立敏感、特异、高效的检测田鼠巴贝虫 FTA-环介导等温扩增技术(LAMP)。方法 根据GenBank公布的田鼠巴贝虫基因序列,就细胞色素B基因保守序列设计了多套LAMP引物,利用LAMP RealTime Turbidimeter LA-320 仪筛选最佳引物、反应条件,建立LAMP检测方法,从保存有血液样本的FTA卡片中提取田鼠巴贝虫 DNA进行LAMP,观察检测效果。结果 设计的4组引物做LAMP试验,其中以引物1,最佳反应温度为64 ℃,恒温下作用,敏感性高,可在1 h内完成,其检出限量为0.687 fg/μL,而PCR的最低检测量为0.687 pg/μL,与间日疟原虫、恶性疟原虫、卵形疟原虫、三日疟原虫、诺氏疟原虫、冈地弓形虫、利什曼原虫、冈比亚锥虫均无交叉反应。以建立的FTA-LAMP法检测20份PCR检测阳性的田鼠巴贝虫感染者血样,其阳性符合率100%。结论 成功建立了检测田鼠巴贝虫特异性细胞色素B基因的FTA-LAMP方法,该法特异性强、灵敏度高、简便、快速、适于现场应用。

Development of loop-mediated isothermal amplification (LAMP)assay combined with FTA card for detecting Babesia microti

The aim was to establish sensitive, specific and high-performance method of loop-mediated isothermal amplification (LAMP) combined with Flinders Technology Associates (FTA) card for detection of Babesia microti. According to the published sequences of B. microti in GenBank, many pairs of primers were designed targeting the conserved region of Cytochrome B gene of B. microti. The amplification was monitored by LAMP Real Time Turbidimeter LA-320. Through optimizing the LAMP primers and reaction conditions, a rapid and specific detection of B. microti was established. Meanwhile, the amplified products were colored by Calcein that could be detected with naked eyes, and also detected by agarose gel electrophoresis. The method of LAMP showed a highly efficient amplification for B. microti target gene which performed at 64 ℃ for 1h by the LAMP Real Time Turbidimeter LA-320. The detection limit was 0.687 fg/μL higher than PCR, no cross reaction with Plasmodium vivax, P. falciparum, P. ovale, P. malariae, P. knowlesi, Toxoplasma gondii, Leishmania donovani, and Trypanosoma gambiense. Blood samples of twenty individuals of positive for B. microti detection with PCR were detected by the established LAMP assay, and the coincidence rate was 100%. FTA-LAMP method for testing the specific cytochrome B gene of Babesia microti is successfully established, with high sensitivity, specificity andsimplicity,and is suitable for field detection.