Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 East California Boulevard, Pasadena, California 91125, United States.
Anal Chem. 2013 Feb 5;85(3):1540-6. doi: 10.1021/ac3037206. Epub 2013 Jan 16.
Here we used a SlipChip microfluidic device to evaluate the performance of digital reverse transcription-loop-mediated isothermal amplification (dRT-LAMP) for quantification of HIV viral RNA. Tests are needed for monitoring HIV viral load to control the emergence of drug resistance and to diagnose acute HIV infections. In resource-limited settings, in vitro measurement of HIV viral load in a simple format is especially needed, and single-molecule counting using a digital format could provide a potential solution. We showed here that when one-step dRT-LAMP is used for quantification of HIV RNA, the digital count is lower than expected and is limited by the yield of desired cDNA. We were able to overcome the limitations by developing a microfluidic protocol to manipulate many single molecules in parallel through a two-step digital process. In the first step we compartmentalize the individual RNA molecules (based on Poisson statistics) and perform reverse transcription on each RNA molecule independently to produce DNA. In the second step, we perform the LAMP amplification on all individual DNA molecules in parallel. Using this new protocol, we increased the absolute efficiency (the ratio between the concentration calculated from the actual count and the expected concentration) of dRT-LAMP 10-fold, from ∼2% to ∼23%, by (i) using a more efficient reverse transcriptase, (ii) introducing RNase H to break up the DNA:RNA hybrid, and (iii) adding only the BIP primer during the RT step. We also used this two-step method to quantify HIV RNA purified from four patient samples and found that in some cases, the quantification results were highly sensitive to the sequence of the patient's HIV RNA. We learned the following three lessons from this work: (i) digital amplification technologies, including dLAMP and dPCR, may give adequate dilution curves and yet have low efficiency, thereby providing quantification values that underestimate the true concentration. Careful validation is essential before a method is considered to provide absolute quantification; (ii) the sensitivity of dLAMP to the sequence of the target nucleic acid necessitates additional validation with patient samples carrying the full spectrum of mutations; (iii) for multistep digital amplification chemistries, such as a combination of reverse transcription with amplification, microfluidic devices may be used to decouple these steps from one another and to perform them under different, individually optimized conditions for improved efficiency.
在这里，我们使用 SlipChip 微流控装置来评估数字逆转录环介导等温扩增（dRT-LAMP）在定量 HIV 病毒 RNA 方面的性能。需要进行测试以监测 HIV 病毒载量，以控制耐药性的出现并诊断急性 HIV 感染。在资源有限的环境中，特别需要以简单的格式进行 HIV 病毒载量的体外测量，而使用数字格式进行单分子计数可能是一种潜在的解决方案。我们在这里表明，当一步法 dRT-LAMP 用于定量 HIV RNA 时，数字计数低于预期，并且受到所需 cDNA 产量的限制。我们通过开发一种微流控方案来克服这些限制，该方案能够通过两步数字过程并行处理许多单个分子。在第一步中，我们将单个 RNA 分子进行分区（基于泊松统计），并对每个 RNA 分子进行独立的逆转录以产生 DNA。在第二步中，我们并行对所有单个 DNA 分子进行 LAMP 扩增。使用这种新方案，我们通过以下方式将 dRT-LAMP 的绝对效率（从实际计数计算的浓度与预期浓度之比）提高了 10 倍，从约 2%提高到约 23%：（i）使用更有效的逆转录酶，（ii）引入 RNase H 以破坏 DNA:RNA 杂交，以及（iii）仅在 RT 步骤中添加 BIP 引物。我们还使用这种两步法来定量从四个患者样本中纯化的 HIV RNA，并发现在某些情况下，定量结果对患者 HIV RNA 的序列高度敏感。从这项工作中我们学到了以下三点：（i）数字扩增技术，包括 dLAMP 和 dPCR，可能会提供足够的稀释曲线，但效率却很低，从而提供了低估真实浓度的定量值。在考虑该方法提供绝对定量之前，必须进行仔细的验证；（ii）dLAMP 对靶核酸序列的敏感性需要用携带全谱突变的患者样本进行额外验证；（iii）对于多步数字扩增化学，例如逆转录与扩增的组合，微流控装置可用于将这些步骤彼此分离，并在不同的、单独优化的条件下进行这些步骤，以提高效率。
