Dong Hee-Jin, Cho Ae-Ri, Hahn Tae-Wook, Cho Seongbeom
BK21 PLUS Program for Creative Veterinary Science Research, Research Institute for Veterinary Science and College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea.
J Vet Sci. 2014;15(2):317-25. doi: 10.4142/jvs.2014.15.2.317. Epub 2014 Mar 21.
A multiplex loop-mediated isothermal amplification (mLAMP) assay was developed for simultaneous detection of the stx1 and stx2 genes and applied for detection of shiga toxin-producing Escherichia coli (STEC) in cattle farm samples. Two target genes were distinguished based on Tm values of 85.03 ± 0.54°C for stx1 and 87.47 ± 0.35°C for stx2. The mLAMP assay was specific (100% inclusivity and exclusivity), sensitive (with a detection limit as low as 10 fg/μL), and quantifiable (R² = 0.9313). The efficacy and sensitivity were measured to evaluate applicability of the mLAMP assay to cattle farm samples. A total of 12 (12/253; 4.7%) and 17 (17/253; 6.7%) STEC O157, and 11 (11/236; 4.7%) non-O157 STEC strains were isolated from cattle farm samples by conventional selective culture, immunomagnetic separation, and PCR-based culture methods, respectively. The coinciding multiplex PCR and mLAMP results for the types of shiga toxin revealed the value of the mLAMP assay in terms of accuracy and rapidity for characterizing shiga toxin genes. Furthermore, the high detection rate of specific genes from enrichment broth samples indicates the potential utility of this assay as a screening method for detecting STEC in cattle farm samples.
开发了一种多重环介导等温扩增（mLAMP）检测方法，用于同时检测stx1和stx2基因，并应用于检测奶牛场样本中产志贺毒素大肠杆菌（STEC）。根据stx1的解链温度为85.03±0.54°C和stx2的解链温度为87.47±0.35°C来区分两个靶基因。mLAMP检测方法具有特异性（包含率和排他率均为100%）、灵敏性（检测限低至10 fg/μL）和可定量性（R² = 0.9313）。通过测量该mLAMP检测方法的有效性和灵敏性来评估其对奶牛场样本的适用性。分别采用传统选择性培养、免疫磁珠分离和基于PCR的培养方法，从奶牛场样本中分离出12株（12/253；4.7%）STEC O157和17株（17/253；6.7%）STEC O157，以及11株（11/236；4.7%）非O157 STEC菌株。志贺毒素类型的多重PCR和mLAMP结果一致，表明mLAMP检测方法在鉴定志贺毒素基因的准确性和快速性方面具有价值。此外，从富集肉汤样本中特定基因的高检出率表明该检测方法作为奶牛场样本中检测STEC的筛查方法具有潜在用途。
