A fully-enclosed prototype ‘pen’ for rapid detection of SARS-CoV-2 based on RT-RPA with dipstick assay at point-of-care testing

Abstract

A fully-enclosed prototype ‘pen’ for rapid detection of SARS-CoV-2 based on reverse transcriptase isothermal recombinase polymerase amplification (RT-RPA) with dipstick assay was developed. The integrated handheld device, consisting of amplification, detection and sealing modules, was developed to perform rapid nucleic acid amplification and detection under a fully enclosed condition. After RT-RPA amplification with a metal bath or a normal PCR instrument, the amplicons were mixed with dilution buffer prior to being detected on a lateral flow strip. To avoid aerosol contamination causing false-positive, from amplification to final detection, the detection ‘pen’ had been enclosed to isolate from the environment. With colloidal gold strip-based detection, the detection results could be directly observed by eyes. By cooperating with other inexpensive and rapid methods for POC nucleic acid extraction, the developed ‘pen’ could detect COVID-19 or other infectious diseases in a convenient, simple and reliable way.

It is well known that, PCR is an important tool for molecular diagnosis due to its sensitivity and specificity [1], [2]. Because of its complexity with elaborate thermal cycling, it is challenging to apply PCR into low-cost, easy-to-operate POC diagnosis [3], [4]. As a complementary technology to PCR, isothermal amplification exhibits its superiority in POC diagnosis due to its simple heating strategy with short amplification time [5], [6]. Different microfluidic systems based on isothermal amplification were developed for POC diagnosis [7], [8].

For PCR or isothermal amplification, with fluorescence probes, real-time or end-point detection can be performed [9], [10]. Alternatively, as an easy-to-use method, dipstick assay can be adopted to detect the amplicons with a lateral flow strip [11], [12]. Especially, when colloidal gold lateral flow strip is applied, the detection result can be directly observed by eyes. Different diagnosis systems based on both isothermal amplification and dipstick assay were developed for POC diagnosis in resource poor settings [13], [14], [15].

Based on both RT-RPA and dipstick assay, a prototype detection ‘pen’ was developed to perform rapid nucleic acid diagnosis at POC testing. Especially, different from other similar devices running in an open-air environment [16], [17], [18] which were easier to cause false-positive, the developed method could effectively reduce the risk of aerosol contamination since the ‘pen’ was fully enclosed with good airtightness. Assisted by a regular heating device, rapid nucleic acid diagnosis could be achieved with the ‘pen’ within 25 min

The disposable ‘pen’ was designed by integrating different functional modules into a fully enclosed device. As shown in Fig. 1(I) and (II), the ‘pen’ includes three major modules, e.g., a bottom amplification module, a middle detection module, and a top sealing module. Dry RT-RPA reagents were lyophilized and pre-stored inside the reaction tube. The bottom head of the reaction tube has similar dimensions to those of a regular 200 μl PCR tube, allowing the ‘pen’ to be conveniently heated with a regular heating device. To allow 50 μl RPA reagent to be conveniently mixed with 500 μl dilution buffer, the top head of the reaction tube was enlarged, and connected to the detection module to form a large mixing chamber (volume > 550 μl) comprising the reaction tube and the bottom part of the detection module. As shown in Fig. 1(III), the detection module consists of three components, a colloidal gold lateral flow strip, a buret for dilution buffer pre-storage and mixing, and a casing module to hold different components. The capture reagents, Anti-FITC antibody and biotin were immobilized, respectively, on the test (T) and control (C) lines of the strip. In dipstick assay, the FITC-labeled amplicons are captured by the Anti-FITC-labeled test line of the strip, and streptavidin-labeled colloidal gold particles are captured by the biotin-labeled control line of the strip. To hold the strip before or during dipstick assay, two positioning stops, e.g., the top and bottom stops, were fabricated on the casing module. As shown in Fig. 1, initially, the strip is held by the top stop, and just before dipstick assay, it is pushed down by fingers until it reaches the bottom stop when the elastic top stop is pushed away by the strip itself. The position of the bottom stop is determined by allowing part of the sample pad of the strip to stay below the top surface of the diluted amplicons, which allow lateral flow not to be affected by the top air bubbles. Similarly, to temporarily hold the buret before amplicon dilution, another positioning stop was fabricated on the top of the casing module. As shown in Fig. 1, before dilution, the buret is manually pushed down due to the gentle shrinkage of its elastic surface, and after dilution, the buret is manually pulled up to avoid unexpected reagent sucking. The amplification module is connected to the detection module with tight-fit design to avoid aerosol contamination. As shown in Fig. 1(I) and (II), the sealing module is made from a regular air balloon or other similar elastic material. Due to the elasticity of the air balloon, the strip or the buret can be manually pushed down or pulled up through its thin surface from outside. Moreover, when the ‘pen’ is heated, the air balloon is helpful to hold the inside expanded air to reduce the risk of air leakage. To avoid aerosol contamination, the top head of the detection module is tightly sealed by the air balloon. With tight-fit between each two modules, the satisfied airtightness of the ‘pen’ is achieved. The detailed information about the assembly of the device and the procedure of the detection can be found in the Supplementary Material.

Fig. 1.

Prototype detection ‘pen’ for nucleic acid diagnosis. (I) Picture of the ‘pen’; (II) and (III) Two exploded views respectively for the ‘pen’ and the detection module.

The viscosity of the RT-RPA reagent was measured by a viscometer (Lovis 2000 M/ME, Anton-Paar, Austria), and is 10 times larger than that of water at room temperature. Therefore, it is difficult for the RT-RPA reagent to be smoothly absorbed by the lateral flow strip or flow along the strip based on capillary force due to its high viscosity. To resolve this issue, the RT-RPA reagent needs to be diluted to reduce its viscosity. For rehydration and RPA amplification, the reaction temperature is usually sufficient to achieve efficient mixing. Therefore, active mixing was introduced only at the stage of lateral flow test after amplification. To reduce the technical risk of manual mixing, an easy-to-operate buret was integrated into the ‘pen’ for both dilution buffer storage and mixing actuation. After amplification, once the dilution buffer was discharged into the reaction tube, active mixing could be performed by fingers. As shown in Fig. 2, to clearly show the difference of viscosity between the RT-RPA reagent and the dilution buffer, and meanwhile to compare the mixing efficiency among three different mixing modes, 50 μl peach dye-labeled RT-RPA reagent and 500 μl green dye-labeled dilution buffer are loaded into a regular 1.5 ml tube in experiments. Test results showed that deionized water could be used as the dilution buffer to achieve satisfied detection results. For mode I, with diffusion-based mixing, two reagents cannot be efficiently mixed even after 10-min incubation. For model II or III, efficient mixing can be achieved, respectively, with buret- or vortex-based mixing since two different color dyes are thoroughly mixed. Therefore, with buret-based manual mixing, after a couple of cycles, the RT-RPA reagent can be efficiently diluted. The detailed information about evaluation of airtightness of the ‘pen’ can be found in the Supplementary Material.

Fig. 2.

Comparison between with and without active mixing.

Performance of the prototype ‘pen’ was further evaluated by detecting synthetic RNA of SARS-CoV-2. The synthetic standard RNA templates (National Institute of Metrology, China) with different concentrations were used as the test sample, and the ORF1ab gene of SARS-CoV-2 was amplified and detected. The RT-RPA reaction mix, comprising two types of primers, different sets of enzymes, and other required reagents, was lyophilized and pre-stored inside the reaction tube by the provider (Amplification Future Co., Ltd., Changzhou, China). Before amplification, 42.5 μl R buffer, 2.5 μl B buffer (Amplification Future Co., Ltd., Changzhou, China) and 5 μl synthetic RNA as the templates, which achieved a total reaction volume of 50 μl, was added into the reaction tube to hydrate the lyophilized reagent. More detailed information about the RNA template, the reagent, and the protocol can be found in the Supplementary Material. The information about gel electrophoresis of RT-RPA-amplified SARS-CoV-2 of the prototype device can be found in the Supplementary Material. As shown in Fig. 3(A), after the ‘pen’ is fully assembled, it is partly inserted into a portable device to heat the reaction tube at 39 ℃ for RT-RPA amplification. After that, the amplicon is diluted with manual mixing before dipstick assay. As shown in Fig. 3(B), to provide clear detection result, instead of showing the whole ‘pen’, its middle part with part of the lateral flow strip is shown. Each experiment with different concentration has been repeated for three times. As shown in Fig. 3(B-I), for each test implemented on the ‘pen’ with active mixing, the positive sample (100 or 10 copies/test) can be successfully or consistently detected by the lateral flow strip. In contrast, as shown in Fig. 3(B-II), without active mixing, when the amplicon is not well mixed, the positive sample with the concentration of 10 copies/test cannot be successfully or consistently detected due to the high viscosity of the RT-RPA reagent. Without active mixing, for three low positive samples (10 copies/test), two of them don’t show an observable test line, and the rest one shows a quite weak test line. Similarly, without active mixing, for three medium positive samples (100 copies/test), the signal intensity of their test lines is significantly lower than that with active mixing. Negative test is confirmed as shown in Fig. 3(B-III). The detailed information about control experiments on benchtop devices can be found in the Supplementary Material. Since the ‘pen’ is at the stage of prototype, more performance evaluation on sensitivity and specificity especially with clinical samples will be done when mass-production is almost ready after further optimization in the future.

Fig. 3.

Detection to synthetic RNA of SARS-CoV-2. (A) Heating of the detection ‘pen’; (B-I) With active mixing; (B-II) Without active mixing; (B-III) Negative control.

The requirement for dilution of amplicons before dipstick assay could be a potential limitation of our work. To simplify the system, in the future, more efforts can be made to reduce the viscosity of the reagent or optimize the properties of lateral flow strip to enable the smooth flow of the reagents without dilution. Comparing to the traditional method which operates in an open environment with multiple standard lab devices, the fully-sealed prototype detection ‘pen’ is helpful to reduce not only the risk of aerosol or environment contamination, but also the reliance on outside tools.

CRediT authorship contribution statement

Qiang Fu: Device fabrication, Experimentation, Writing – original draft. Yunping Tu: Device fabrication, Data analysis, Writing – original draft. Lun Cheng: Device fabrication, Experimentation. Lulu Zhang: Methodology and Experiment design. Xianbo Qiu: Conceptualization, Methodology, Project management, Manuscript review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary material

Supplementary material

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Data availability

Data will be made available on request.