A Fast and Accessible Method for the Isolation of RNA, DNA, and Protein To Facilitate the Detection of SARS-CoV-2

Abstract

Management of the coronavirus disease 2019 (COVID-19) pandemic requires widespread testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A main limitation for widespread SARS-CoV-2 testing is the global shortage of essential supplies, among them RNA extraction kits. The need for commercial RNA extraction kits places a bottleneck on tests that detect SARS-CoV-2 genetic material, including PCR-based reference tests. Here, we propose an alternative method we call PEARL (precipitation-enhanced analyte retrieval) that addresses this limitation. PEARL uses a lysis solution that disrupts cell membranes and viral envelopes while simultaneously providing conditions suitable for alcohol-based precipitation of RNA, DNA, and proteins. PEARL is a fast, low-cost, and simple method that uses common laboratory reagents and offers performance comparable to that of commercial RNA extraction kits. PEARL offers an alternative method to isolate host and pathogen nucleic acids and proteins to streamline the detection of DNA and RNA viruses, including SARS-CoV-2.

Keywords: DNA and protein; SARS-CoV-2; field deployable; rapid isolation of RNA; virus detection.

FIG 1

(A) PEARL workflow. (B) Comparative RT-qPCR analysis of the levels of SARS-CoV-2 nucleocapsid (N1) and RNase P RNA sequences in deidentified SARS-CoV-2-positive and -negative clinical specimen samples after RNA extraction using PEARL or an RNA extraction kit (QIAamp Mini Elute virus spin kit) in nasopharyngeal swab samples. Dotted lines indicate a limit of detection of 36 cycles. We obtained C_q values for N1 below our limit of detection in 9 negative samples out of 67. No C_q values were obtained for N1 in the remaining 58 samples. Data points correspond to the reciprocal of the C_q value (1/C_q), which is directly proportional to input RNA. P values and Spearman’s correlation coefficient (ρ) are shown.

FIG 2

(A) RT-qPCR analysis of the levels of KSHV (LANA and GFP) and host β-actin (ACTB) mRNAs and (B) their corresponding genomic sequences. (C) Western blot analysis of the expression of KSHV (LANA and GFP) and host (HSP70) proteins. Inf., infected; Uninf., uninfected.

FIG 3

(A) RT-qPCR analysis of the levels of ZIKV nonstructural proteins NS1 and NS5 and host β-actin (ACTB) mRNAs. (B) qPCR analysis of the expression host ACTB genomic DNA sequences in ZIKV-infected samples. (C) Western blot analysis of expression of ZIKV (NS2B) and host (HSP70) proteins. *, nonspecific band.