Unit of Typing & Genetics of Mycobacteria, Laboratory of Molecular Microbiology, Vaccinology, and Biotechnology Development, Institut Pasteur de Tunis, Université de Tunis El Manar, Tunis, Tunisia.
DST/NRF Centre of Excellence for Biomedical Tuberculosis Research, SAMRC Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa.
PLoS One. 2018 Jun 1;13(6):e0197913. doi: 10.1371/journal.pone.0197913. eCollection 2018.
Several technical hurdles and limitations have restricted the use of IS6110 restriction fragment length polymorphism (IS6110 RFLP), the most effective typing method for detecting recent tuberculosis (TB) transmission events. This has prompted us to conceive an alternative modality, IS6110-5'3'FP, a plasmid-based cloning approach coupled to a single PCR amplification of differentially labeled 5' and 3' IS6110 polymorphic ends and their automated fractionation on a capillary sequencer. The potential of IS6110-5'3'FP to be used as an alternative to IS6110 RFLP has been previously demonstrated, yet further technical improvements are still required for optimal discriminatory power and versatility.
Here we introduced critical amendments to the original IS6110-5'3'FP protocol and compared its performance to that of 24-loci multiple interspersed repetitive unit-variable number tandem repeats (MIRU-VNTR), the current standard method for TB transmission analyses.
IS6110-5'3'FP protocol modifications involved: (i) the generation of smaller-sized polymorphic fragments for efficient cloning and PCR amplification, (ii) omission of the plasmid amplification step in E. coli for shorter turnaround times, (iii) the use of more stable fluorophores for increased sensitivity, (iv) automated subtraction of background fluorescent signals, and (v) the automated conversion of fluorescent peaks into binary data.
In doing so, the overall turnaround time of IS6110-5'3'FP was reduced to 4 hours. The new protocol allowed detecting almost all 5' and 3' IS6110 polymorphic fragments of any given strain, including IS6110 high-copy number Beijing strains. IS6110-5'3'FP proved much more discriminative than 24-loci MIRU-VNTR, particularly with strains of the M. tuberculosis lineage 4.
The IS6110-5'3'FP protocol described herein reached the optimal discriminatory potential of IS6110 fingerprinting and proved more accurate than 24-loci MIRU-VNTR in estimating recent TB transmission. The method, which is highly cost-effective, was rendered versatile enough to prompt its evaluation as an automatized solution for a TB integrated molecular surveillance.
几种技术障碍和限制因素限制了 IS6110 限制片段长度多态性（IS6110 RFLP）的使用，该方法是检测近期结核病（TB）传播事件的最有效分型方法。这促使我们构思了一种替代模式，IS6110-5'3'FP，这是一种基于质粒的克隆方法，结合了 5'和 3'IS6110 多态性末端的差异标记的单个 PCR 扩增，并在毛细管测序仪上自动分离。IS6110-5'3'FP 作为 IS6110 RFLP 的替代方法的潜力此前已得到证明，但为了获得最佳的区分能力和多功能性，仍需要进一步的技术改进。
本研究引入了对原始 IS6110-5'3'FP 方案的关键修正，并将其性能与当前用于 TB 传播分析的 24 个位点多位点间隔重复单元-可变数串联重复（MIRU-VNTR）标准方法进行了比较。
IS6110-5'3'FP 方案的修改包括：（i）生成更小的多态性片段，以提高克隆和 PCR 扩增的效率，（ii）省略大肠杆菌中的质粒扩增步骤，以缩短周转时间，（iii）使用更稳定的荧光团以提高灵敏度，（iv）自动扣除背景荧光信号，以及（v）将荧光峰自动转换为二进制数据。
这样，IS6110-5'3'FP 的总周转时间缩短至 4 小时。新方案允许检测到任何给定菌株的几乎所有 5'和 3'IS6110 多态性片段，包括 IS6110 高拷贝数北京株。IS6110-5'3'FP 比 24 个位点 MIRU-VNTR 具有更高的区分能力，特别是对于结核分枝杆菌 4 谱系的菌株。
本文描述的 IS6110-5'3'FP 方案达到了 IS6110 指纹图谱的最佳区分潜力，并被证明比 24 个位点 MIRU-VNTR 更能准确估计近期 TB 传播。该方法具有很高的成本效益，并且足够灵活，可以评估其作为 TB 综合分子监测的自动化解决方案。
