SwabExpress: An End-to-End Protocol for Extraction-Free COVID-19 Testing

Abstract

Background: The urgent need for massively scaled clinical testing for SARS-CoV-2, along with global shortages of critical reagents and supplies, has necessitated development of streamlined laboratory testing protocols. Conventional nucleic acid testing for SARS-CoV-2 involves collection of a clinical specimen with a nasopharyngeal swab in transport medium, nucleic acid extraction, and quantitative reverse-transcription PCR (RT-qPCR). As testing has scaled across the world, the global supply chain has buckled, rendering testing reagents and materials scarce. To address shortages, we developed SwabExpress, an end-to-end protocol developed to employ mass produced anterior nares swabs and bypass the requirement for transport media and nucleic acid extraction.

Methods: We evaluated anterior nares swabs, transported dry and eluted in low-TE buffer as a direct-to-RT-qPCR alternative to extraction-dependent viral transport media. We validated our protocol of using heat treatment for viral inactivation and added a proteinase K digestion step to reduce amplification interference. We tested this protocol across archived and prospectively collected swab specimens to fine-tune test performance.

Results: After optimization, SwabExpress has a low limit of detection at 2-4 molecules/µL, 100% sensitivity, and 99.4% specificity when compared side by side with a traditional RT-qPCR protocol employing extraction. On real-world specimens, SwabExpress outperforms an automated extraction system while simultaneously reducing cost and hands-on time.

Conclusion: SwabExpress is a simplified workflow that facilitates scaled testing for COVID-19 without sacrificing test performance. It may serve as a template for the simplification of PCR-based clinical laboratory tests, particularly in times of critical shortages during pandemics.

Fig. 1.

Polyester anterior nares swabs are both comfortable and easy to use. (A, B), Study participants’ (n = 35) self-reported (A) discomfort and (B) confidence during self-administration of an anterior nares swab at home. (C), Boxplot depicting the RT–qPCR C_t values for RNaseP from self-administered anterior nares swabs (ANS) and mid-turbinate (MT) swabs.

Fig. 2.

Extraction-free RT–qPCR set-up and test performance. (A), Assay layout of the EF-RT–qPCR test. One sample is assayed in 4 wells on a 384-well plate. Each sample is tested for 2 probes, in duplicate. RNase P is assayed in each well. (B), Mean C_t values for 67 specimens processed by EF-RT–qPCR and extraction-based RT–qPCR. Reactions with no amplification by one preparation protocol are demarcated with red or blue points as indicated.

Fig. 3.

Addition of Proteinase K improves test performance. (A), Observed percentage of test failures with and without the addition of proteinase K. (B), Archived samples with C_t > 28 reprocessed with either KingFisher Flex Extraction (left), Extraction-Free RT–PCR (middle), or SwabExpress (Extraction-Free RT–PCR + ProK) (right). Colors signify the number of samples and their classifications. (C), Box and whisker plots depicting the average delta C_t between replicate wells for SARS-Cov2 positive specimens. Red points indicate outliers. ΔC_t values were more consistent on addition of proteinase K. (D), Mean C_t values of matched specimens run through the automated KingFisher extraction system (left) or using SwabExpress (SE). Specimens that were detected in only 1 of the 2 protocols are displayed as green points.

Fig. 4.

SwabExpress workflow. (1) Anterior nares swabs are collected and (2) transported dry to the lab. On receipt, (3) each swab is then hydrated with low-TE buffer, aliquoted into a 96-well plate, and (4) proteinase K is added to every well. (5) The eluted specimens are digested and heat-inactivated in a laboratory oven before (6) they are loaded as the template in a RT–qPCR reaction. The cost listed includes reagents and consumables.