Anapa Biotech A/S, Hørsholm, Denmark.
Department of Clinical Microbiology, Copenhagen University Hospital Hvidovre, Denmark.
PLoS One. 2018 Jun 26;13(6):e0199766. doi: 10.1371/journal.pone.0199766. eCollection 2018.
Sensitive, probe-based detection of multiple DNA targets is limited by the competitive reannealing of the antiparallel duplex DNA helix with the complementary DNA strand. To address this, we developed Cliffhanger primers, which create single-stranded DNA overhangs on PCR amplicons while simultaneously increasing the multiplex PCR efficacy and allowing PCR amplification using crude lysates of human faecal samples. A multiplex PCR that targeted eight genes from diarrhoeagenic Escherichia coli plus an internal control was performed and compared to a routine method that consisted of culture followed by multiplex PCR with fragment length separation. A total of 2515 clinical faecal samples from patients with diarrhoea were tested using both methods, and there was a significant increase in clinical sensitivity and negative predictive value with the Cliffhanger method for seven out of eight genes. All Cliffhanger-only positive samples were confirmed by Sanger sequencing of the PCR amplicon. Notably, the Cliffhanger method reduced the total sample turn-around time in the laboratory from 20 hours to 6 hours. Hence, use of Cliffhanger primers increased assay robustness, decreased turn-around time and increased PCR efficacy. This increased the overall clinical sensitivity without the loss of specificity for a heavily multiplexed PCR assay.
基于探针的敏感检测方法受到互补 DNA 链与反平行双链 DNA 螺旋竞争复性的限制。为了解决这个问题，我们开发了 Cliffhanger 引物，该引物在 PCR 扩增子上产生单链 DNA 突出端，同时提高了多重 PCR 的效率，并允许使用人类粪便样本的粗裂解物进行 PCR 扩增。我们进行了靶向致泻性大肠杆菌 8 个基因的多重 PCR，并与包括培养和片段长度分离的多重 PCR 的常规方法进行了比较。使用这两种方法检测了 2515 例腹泻患者的临床粪便样本，对于 8 个基因中的 7 个基因，Cliffhanger 方法显著提高了临床敏感性和阴性预测值。所有 Cliffhanger 阳性样本均通过 PCR 扩增子的 Sanger 测序得到确认。值得注意的是，Cliffhanger 方法将实验室的总样本周转时间从 20 小时缩短至 6 小时。因此，使用 Cliffhanger 引物提高了检测的稳健性，缩短了周转时间，并提高了 PCR 效率。在不损失特异性的情况下，这提高了整体临床敏感性。
