The SARS-CoV-2 pandemic has brought to light the need for expedient diagnostic testing. Cost and availability of large-scale testing capacity has led to a lag in turnaround time and hindered contact tracing efforts, resulting in a further spread of SARS-CoV-2. To increase the speed and frequency of testing, we developed a cost-effective single-tube approach for collection, denaturation, and analysis of clinical samples. The approach utilizes 1 µL microbiological inoculation loops to collect saliva, sodium dodecyl sulfate (SDS) to inactivate and release viral genomic RNA, and a diagnostic reaction mix containing polysorbate 80 (Tween 80). In the same tube, the SDS-denatured clinical samples are introduced to the mixtures containing all components for nucleic acids detection and Tween 80 micelles to absorb the SDS and allow enzymatic reactions to proceed, obviating the need for further handling of the samples. The samples can be collected by the tested individuals, further decreasing the need for trained personnel to administer the test. We validated this single-tube sample-to-assay method with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) and discovered little-to-no difference between Tween- and SDS-containing reaction mixtures, compared to control reactions. This approach reduces the logistical burden of traditional large-scale testing and provides a method of deployable point-of-care diagnostics to increase testing frequency.
The success of fundamental and applied nucleic acid (NA) research depends on NA purity, but obtaining pure NAs from raw, unprocessed samples is challenging. Purification using solid-phase NA extractions utilizes sequential additions of lysis and wash buffers followed by elution. The resulting eluent contains NAs and carryover of extraction buffers. Typically, these inhibitory buffers are heavily diluted by the reaction mix (e.g., 10x dilution is 1 µL eluent in 9 µL reaction mix), but in applications requiring high sensitivity (e.g., single-cell sequencing, pathogen diagnostics) it is desirable to use low dilutions (e.g., 2x) to maximize NA concentration. Here, we demonstrate pervasive carryover of inhibitory buffers into eluent when several commercial sample-preparation kits are used following manufacturer protocols. At low eluent dilution (2–2.5x) we observed significant reaction inhibition of polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and reverse transcription (RT). We developed a two-phase wash (TPW) method by adding a wash buffer with low water solubility prior to the elution step. The TPW reduces carryover of extraction buffers, phase-separates from the eluent, and does not reduce NA yield (measured by digital PCR). We validated the TPW for silica columns and magnetic beads by demonstrating significant improvements in performance and reproducibility of qPCR, LAMP, and RT reactions.
more » « less- NSF-PAR ID:
- 10153890
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Scientific Reports
- Volume:
- 10
- Issue:
- 1
- ISSN:
- 2045-2322
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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