PDF(Pubmed)
Abstract：
Detection of bacterial RNA by nucleic acid amplification tests (NAATs), such as reverse transcription PCR (RT-PCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP), offers distinct advantages over DNA-based methods. However, such assays also present challenges in ascertaining positive and internal control material that can reliably monitor success over all phases of testing (bacterial lysis, nucleic acid recovery, reverse transcription, amplification, and signal detection): since they are unable to distinguish between amplification of bacterial RNA transcripts and the DNA templates that encode them, using intact organisms as controls can inform cell lysis but not successful detection of RNA. We developed a control strategy for RNA-based bacterial NAATs that allows ready discrimination of RNA from DNA templates using self-splicing bacterial introns, such that those nucleic acids ultimately encode different sequences. We engineered two vectors encoding synthetic transgenes based on this principle, one that is active in the Gram-negative bacterium Escherichia coli and one that functions in both E. coli and the Gram-positive organism Staphylococcus aureus. We subsequently designed RT-LAMP assays that either target RNA and DNA from transgenic organisms or target RNA exclusively and demonstrated the specificity of amplification using purified nucleic acids. Using multiplex fluorescent RT-LAMP of heat-lysed specimens, we showed the practicality of deploying such transgenic organisms as an internal control to ascertain sample integrity and assay performance during clinical diagnostic testing. Our approach has broad utility for RNA-based bacterial NAATs, especially point-of-care assays and other applications where nucleic acids are nonspecifically liberated for testing.
摘要：
通过核酸扩增试验（NAAT）检测细菌RNA，如逆转录PCR（RT-PCR）和逆转录环介导等温扩增（RT-LAMP），与基于DNA的方法相比，它具有明显的优势。然而,这样的测定也提出了挑战，在确定阳性和内部控制材料，可以可靠地监测成功的所有阶段的测试（细菌裂解，核酸回收,逆转录,扩增,和信号检测）：由于它们无法区分细菌RNA转录本的扩增和编码它们的DNA模板，使用完整的生物体作为对照可以告知细胞裂解，但不能成功检测RNA。我们开发了一种基于RNA的细菌NAAT的控制策略，可以使用自剪接细菌内含子从DNA模板中轻松区分RNA，这些核酸最终编码不同的序列。基于这个原理，我们设计了两个编码合成转基因的载体，一种在革兰氏阴性菌大肠杆菌中具有活性，一种在大肠杆菌和革兰氏阳性菌金黄色葡萄球菌中均起作用。我们随后设计了RT-LAMP测定,其靶向来自转基因生物的RNA和DNA或专门靶向RNA,并证明了使用纯化的核酸扩增的特异性。使用热裂解标本的多重荧光RT-LAMP，我们展示了在临床诊断测试过程中部署此类转基因生物作为内部对照以确定样品完整性和测定性能的实用性。我们的方法对基于RNA的细菌NAAT具有广泛的实用性，特别是即时测定和其他应用，其中核酸被非特异性释放用于测试。
