Nucleic acid lateral flow assay with recombinase polymerase amplification: Solutions for highly sensitive detection of RNA virus

Journal Pre-proof Nucleic acid lateral flow assay with recombinase polymerase amplification: Solutions for highly sensitive detection of RNA virus Aleksandr V. Ivanov, Irina V. Safenkova, Anatoly V. Zherdev, Boris B. Dzantiev PII:

S0039-9140(19)31249-4

DOI:

https://doi.org/10.1016/j.talanta.2019.120616

Reference:

TAL 120616

To appear in:

Talanta

Received Date: 5 September 2019 Revised Date:

1 December 2019

Accepted Date: 2 December 2019

Please cite this article as: A.V. Ivanov, I.V. Safenkova, A.V. Zherdev, B.B. Dzantiev, Nucleic acid lateral flow assay with recombinase polymerase amplification: Solutions for highly sensitive detection of RNA virus, Talanta (2020), doi: https://doi.org/10.1016/j.talanta.2019.120616. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Reverse transcription Recombinase polymerase amplification (20 min, 37°C)

Adjusted parameters:

Primers and Conditions for amplification

Lateral flow assay (10 min, room temperature or 37°C)

Composition of detected complexes

Reached result: 260-hold growth of assay sensitivity

Nucleic acid lateral flow assay with recombinase polymerase amplification: Solutions for highly sensitive detection of RNA virus Aleksandr V. Ivanov, Irina V. Safenkova, Anatoly V. Zherdev, Boris B. Dzantiev* A.N. Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy of Sciences, 119071 Moscow, Russia *Corresponding author at: A.N. Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy of Sciences, Leninsky prospect 33, 119071 Moscow, Russia. Tel.: +7-495-954-3142. E-mail address: [email protected]

Abstract We propose nucleic acid lateral flow assay (LFA) coupled with reverse transcription recombinase polymerase amplification (RT-RPA) resulting from step-by-step multiparametric adjustments to both RT-RPA reactions and LFA interactions. The assay was realized for RNA virus detection using the example of potato virus X (PVX), a dangerous phytopathogen. The assay stages were adjusted for sensitive detection. (1) DNA target was designed and verified. A fragment (146 bp) of coat protein gene (gp5) and biotin-/fluorescein-labeled forward/reverse primers were chosen to produce target amplicons. (2) In a test strip, the construction advantage of the realization of the highest-affinity interaction (biotin–streptavidin in our research) through gold nanoparticle conjugate (streptavidin immobilized on the GNP surface) was demonstrated. (3) RPA with reverse transcription was adjusted including primer concentration, order of components’ mixing, and reaction temperature. Due to the adjustments, the assay was able to detect 0.14 ng PVX per g potato leaves at 30 min. The PVX assay was 260 times more sensitive than conventional lateral flow assay based on antibodies and demonstrated the same sensibility

1

as PCR detection. The proposed adjustments are applicable for ultrasensitive and rapid detection of various RNA viruses. Keywords: assay enhancement, isothermal amplification, nucleic acid lateral flow assay, recombinase polymerase amplification, potato virus X

2

1. Introduction RNA viruses are the most diverse pathogens, causing dangerous diseases in humans, animals, and plants [1]. In particular, the Zika virus, the Ebola virus, the influenza virus, and the human immunodeficiency virus, which cause high mortality and risks of future epidemics, are viruses containing RNA. Moreover, positive-strand (+) RNA viruses are especially dangerous because their genomic RNA can be translated into protein immediately upon entering the cell. It is therefore crucially important to develop simple, rapid, and highly sensitive detection methods of virus detection to prevent the spread of viral infections. The most reliable of these methods are traditional nucleic acid-based detection approaches based on reverse transcription PCR (RTPCR). However, rapid and convenient diagnoses are often limited due to the long reaction time, complicated protocols, and expensive equipment required. Competitive alternatives to PCR and PCR-derived techniques are isothermal amplification methods such as loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), strand displacement amplification, and helicase-dependent amplification. All listed isothermal approaches have been applied to the development of nonlaboratory assays for RNA virus detection [2]. Of them, lateral flow assay (LFA) is particularly useful due to its great convenience for users, the low cost of the components, and its rapid performance [3-5]. Beneficial results gained through a combination of nucleic acid LFA and LAMP have been shown from 2013, for detection of yellow head virus [6], to the present, for Zika virus [7] and senecavirus A [8]. The second perspective technique, a combination of nucleic acid LFA with RPA for detection of little cherry virus 2 [9] and plum pox virus [10] in plants, emerged in 2014. Thereafter, the list of RNA viruses expanded to include rice blackstreaked dwarf virus with sensitivity and specificity similar to RT-PCR [11] or H9N2 avian influenza virus RNA with 0.15 pg detection limit, which is 10 times more sensitive than that of conventional RT-PCR [12]. 3

Considering that all described assays are appropriate for RNA virus detection and further development, we propose to focus on an RPA approach that characterizes two main advantages relative to LAMP [13, 14]. The first is an assay temperature that is lower than that for LAMP (65 °C) and corresponds to a range of 30–42 °C. The second is that only two primers are needed for amplification, leading to a more predictable result in contrast to LAMP, for which the composition of the six primers remains ambiguous and influences LFA sensitivity [15]. Moreover, a large number of inhomogeneous products generated by LAMP can act negatively on lateral flow recognition. Undoubtedly, these advantages provide better conditions for the onestep detection of RPA products and homogeneous double-stranded DNA (dsDNA). All reported RPA-LFA developments for RNA virus detection were geared around optimization of specific primers and their application to commercial ready-to-use lateral flow tests for dsDNA recognition, followed by evaluation of sensitivity, specificity, and application to real samples. As far as we know, there are no systematic evaluations and grounded choice of solutions for development of a new RNA virus. The described reports focused on first of three main stages that should be adjusted for sensitive detection: 1) design of DNA target and primers; 2) construction of test strip including gold nanoparticle conjugate and coating of the test zone; and 3) adjustments of RPA with reverse transcription including primer concentration, temperature of reaction, and so on. We realized step-by-step adjustments for RNA virus detection using the example of potato virus X (PVX), a dangerous phytopathogen that causes serious potato damage (necrotic streaks, mild or severe mosaic, crinkling and rugosity of leaves with dwarfing of the plant, extensive top necrosis) especially in mixed infection with potato viruses Y or S, and followed by economic losses [16]. PVX contains (+) sense genomic RNA with length 6.4 kb and about 1300 pieces of coat protein forming an extended filament structure [17]. We aim to establish solutions for highly sensitive detection of RNA viruses with RPA-LFA to develop an on-site PVX nucleic acid detection method. The proposed solutions could easily be adapted for any positive-sense 4

RNA viruses. Moreover, there is no RPA-LFA, as well as other RPA-derived methods, for PVX detection, unlike other plant viruses described in the review of Babu et al. [18]. The development of a sensitive, rapid, and simple method of PVX detection is a critical task for agricultural applications to facilitate timely detection of infection and take action to prevent yield losses [16]. 2. Materials and Methods 2.1. Materials PVX, potato virus Y (PVY), and potato virus S (PVS) were provided by Dr. Yu. A. Varitsev (A.G. Lorch All-Russian Potato Research Institute, Korenevo, Russia). Monoclonal antibodies specific to fluorescein (anti-FAM antibodies) came from Bialexa (Russia). Streptavidin

and

anti-mouse

antibodies

were

obtained

from

Imtech

(Russia).

Tris(hydroxymethyl)aminomethane, ethanolamine hydrochloride, tetrachloroauric (III) acid trihydrate, sucrose, bovine serum albumin (BSA), 1-hydroxy-2,5-pyrrolidinedione (Nhydroxysuccinimide [NHS]), and biotin-amidohexanoyl-6-aminohexanoic acid NHS ester were purchased

from

Sigma-Aldrich

(USA).

3-(Ethyl-iminomethylideneamino)-N,N´-

dimethylpropan-1-amine hydro-chloride (EDC) was purchased from ThermoFisher (USA). Biotin- and FAM-labeled primers, RT-qPCR mix for detection of PVX, and RNA extraction kit were produced by Syntol (Russia). Taq polymerase, deoxynucleotides, MMLV reverse transcriptase, RNase inhibitor (RNasin) and unlabeled primers, and PCR mix for SYBR Green detection were produced by Evrogen (Russia). RPA TwistDx basic kits were purchased from TwistDx (UK). The salts and compounds used in buffer solutions were of analytical or chemical grade. Phenol and chloroform were molecular biology grade. Solutions were prepared using water deionized by a Milli-Q system produced by Millipore (USA). The LFA test strips were assembled using membranes manufactured by Advanced Microdevices (India): working nitrocellulose membranes CNPC-12, a glass fiber membrane for applying a conjugate PT-R5, a membrane for applying the sample GFB-R4, and the final adsorbing membrane AP045. 5

2.2. Extraction of genomic RNA of PVX Genomic RNA of PVX was extracted according to the phenol‒chloroform method with LiCl2 precipitation [19]. Briefly, 100 µg of PVX in 50% glycerol (100 µL) was mixed with 100 µL phenol‒chloroform solution pH 5.5 and incubated for 5 min at 20 °C with shaking. The sample was centrifuged for 10 min at 12000 g. Then, 80 µL of 3 M LiCl2 was added to 100 µL in the water phase and incubated for 30 min at 4 °C to precipitate genomic RNA. The precipitate was centrifuged at 12000 g for 5 min then dissolved in 50 µL mQ. The RNA solution was applied at Mini Spin desalting column (ThermoFisher, USA). RNA was checked by electrophoresis in 1% Tris-borate-EDTA-agarose gel. 2.3. Primers design Primers for reverse transcription and RT-PCR were designed using Primer Blast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/), (http://biotools.nubic.northwestern.edu/OligoCalc.html),

OligoCalc and

Multiple

Primer

Analyzer

ThermoFisher online software. Three primer sets for RPA were designed based on the conserved region of the coat protein gene (gp5) of PVX with the sequence available from the NCBI database according to the instruction manual provided by TwistDx (UK), the manufacturer of the RPA reaction. Details of the final primers are given in Supporting Information, Section 1. 2.4. Reverse transcription RNA of PVX To produce cDNA of PVX reverse transcription, 70 ng (approx. 3 fmol) of PVX RNA was incubated with 20 pmol PVXgp5 reverse primer (see Supporting Information, Section 1) for 1 min at 70 °C. Then, commercial buffer (Evrogen), dNTP mix 1 mM each, 2 mM DTT, 10u RNasin, and 100 u MMLV reverse transcriptase was added and incubated for 60 min at 37 °C, before heating at 70 °C was used to stop the reaction. Produced cDNA was purified using the conventional phenol‒chloroform method followed by ethanol precipitation [19]. 2.5. Obtaining target DNA fragments

6

To produce gp5 gene for the following application, we performed PCR with 200 nM primers and gp5 DNA with 714 bp as a template. To obtain amplicons flanked by biotin and fluorescein (FAM) on the 5´-ends with length of 146 bp, we used a 5' labeled forward primer (gp5 RPA fw 1) and 5' biotin-labeled reverse primer (gp5 RPA rev 1) (see Supporting Information, Section 1). Annealing temperature was 55 °C, and PCR continued for 40 cycles. PCR was performed using the BioRad T100 Thermal Cycler (USA). The PCR products were analyzed and purified by electrophoresis in 1% Tris-acetate-EDTA-agarose gel using SYBR Green staining. The target DNA fragments were extracted from the gel using a DNA extraction kit (Evrogen, Russia), followed by DNA precipitation with 85 mM sodium acetate, pH 5.2, and 70% ethanol at -70 °C for 1 h. The pellet was centrifuged, dried, and dissolved in 10 mM Tris-HCl, pH 8.0, with 0.1 mM EDTA. 2.6. qPCR and RT-qPCR for PVX detection RT-qPCR and qPCR were performed using the Roche Light Cycler® 96 (Roche, Switzerland) with an estimation of cycle threshold (Ct). We used a commercial RT-qPCR kit for detection of PVX produced by Syntol (Russia) and RT-qPCR performed with designed primers in this study. The commercial RT-qPCR kit was used according to the manufacturer’s protocol. Briefly, an obtained DNA gp5 gene fragment (714 bp) was serial-diluted from 106 to one copy. Then, 5 µL of diluted DNA was added to 20 µL mix PCR mix and 0.5 µL Taq polymerase mixed with reverse transcriptase supplied by the manufacturer. Protocol included 45 °C for 900 sec, 95 °C for 300 sec followed by 45 cycles with 15 sec denaturation at 95 °C, and 40 sec annealing and elongation at 60 °C. The signal was detected via FAM channel. Samples with Ct more than 37 were considered as negative as per the manufacturer. Serial dilutions of viral RNA from 108 to 100 copies were detected in the same way.

7

For qPCR with designed primers (3 RPA sets), we used PCR reaction mix with SYBR Green (Evrogen, Russia) and 200 nM primers; 5 µL of DNA solution at various concentrations was added. Protocol included 95 °C for 600 sec followed by 45 cycles with 10 sec denaturation at 95 °C, 15 sec annealing at 55 °C, and 30 sec elongation at 72 °C. For RT-qPCR, 50 u MMVL, 2 mM DTT and 4 u RNasin, and 5 µL of RNA solutions at various concentrations were added into the reaction mix as described above. The following protocol was used: 37 °C for 15 min followed by the method described above. Melting curves analysis was performed to discriminate amplification products. 2.7. RPA and RT-RPA for PVX detection RPA was performed according to manufacturer protocol with modifications. Primers (gp5 RPA fw1 labeled biotin and gp5 RPA rev1 labeled FAM) were added at various concentrations from 150‒480 nM (volume varied from 1‒2.4 µL). SYBR Green (Evrogen, Russia) was added to 1‒50 µL RPA mix containing rehydration buffer, 14 mM magnesium acetate. DNA gp5 gene fragment (714 bp) solution with different amount of copies (from 100 to 105) was added (10 µL) after other components. Reaction was performed at 37 °C for 20 min. Fluorescent signal was detected by Light Cycler 96 (Roche, Switzerland). Threshold time was measured as time threshold (Tt). Melting curves analysis was performed to discriminate amplification products. RT-RPA for genomic PVX RNA was carried out as described above with the addition of 300 u MMLV, 10 u RNasin, and 12 mM DTT. Reaction condition, fluorescent detection during amplification, and melting analysis was executed using the same protocol as DNA amplification. RT-RPA reactions were performed at 37 °C for 20 min using BioRad T100 Thermal Cycler (USA). 2.8. Conjugation of GNP-antibody and GNP-streptavidin conjugates for LFA Anti-FAM antibodies (and streptavidin) at a chosen concentration by flocculation curve [20] (10 µg/mL for streptavidine and 20 µg/mL for anti-FAM antibodies) were added to the GNP solution synthesized according to the Frens method [21] (see Supporting Information, Section 2) 8

and mixed for 1 h. Free GNP surface was blocked with BSA (final concentration of 0.25%). The conjugates were separated through centrifugation at 10,000 g for 30 min at 4 °C. The synthesized conjugates were suspended in 10 mM Tris buffer, pH 7.4, containing 0.25% BSA, 0.25% Tween 20 detergent, and 1% sucrose. The conjugates were stored at 4 °C. The conjugates were characterized using a Libra S80 spectrophotometer (Biochrom, Cambridge, United Kingdom) (see Supporting Information, Section 2). 2.9. Preparation of lateral flow test strips Test strips were prepared using plastic supports with the nitrocellulose membrane, glass fiber membrane with conjugate, adsorbed pad, and sample pad. We used two schemes: 1) GNP– streptavidin conjugate, anti-FAM antibodies (1 mg/mL) in the test zone and biotinylated antimouse antibodies (1 mg/mL) in the control zone; 2) GNP–anti-FAM antibodies conjugate, streptavidin (1 mg/mL) in the test zone and biotinylated anti-mouse antibodies (1 mg/mL) in the control zone. Biotinilation of anti-mouse antibodies was performed as described [22]. All reagents were dispersed by an IsoFlow dispenser (Imagene Technology, USA) at 0.15 µL per 1 mm membrane width in 0.05 M potassium phosphate buffer, pH 7.4, containing 0.1 M NaCl, 10% glycerol, and 0.03% sodium azide. The GNP conjugates were deposited onto glassfiber membranes from a solution with OD520 of 4; the conjugate load was 3.2 µL per 1 mm of strip width. Preparation and assembly of test strips were carried out according to the previously described protocol [20]. With one exception, for part of the tests we used liquid GNP conjugate (in the concentration equivalent to the dried conjugate) to determine the effect of premixing GNP conjugate and DNA. 2.10. RT-RPA-LFA for PVX detection RT-RPA stage was performed as described in the section “RPA and RT-RPA for PVX detection” without SYBR Green. The order in which components (dried components, magnesium acetate, and RNA) were added varied. The LFA was performed at either room temperature or 37 °C. We tested the following samples: 1) DNA (tenfold serial dilutions of DNA 9

amplicons with 146 bp ranging from 0.04‒205 nM); 2) DNA with primers (primer concentrations from 15‒620 nM); 3) DNA with primers and all compounds for RPA; and 4) RNA (isolated from potato leaves extract containing PVX) with primers and all compounds for RPA. For RT-RPA-LFA, 5 µL of the RT-RPA mix solution after 20 min of reaction was added to 65 µL of 0.05 M potassium phosphate buffer, pH 7.4, containing 0.1 M NaCl and 0.05% Triton X-100 (PBST) and applied at strip. The test strip was vertically submerged into the tested sample (or sample with liquid GNP conjugate) for 2 min, before being taken out and placed on the horizontal surface. The qualitative results were estimated visually after 10 min, and quantification of results was carried out as described previously [23]. For the calibration curve, each measurement was conducted in duplicate. 2.11. Analysis of potato samples All infected and healthy potato leaves (35 samples from Moscow region, Russia) were kindly provided by Dr. Yu. A. Varitsev (A.G. Lorch All-Russian Potato Research Institute, Russia). Extraction of total RNA from plant samples was performed using commercial kit of Syntol (Russia) or RNeasy Plant Mini Kit (Qiagen, USA) following the procedure described by the manufacturer. In both cases, the procedures included homogenization and incubation in extracting solutions, separation of a solid fraction by centrifugation and the following specific isolation of RNA molecules by magnetic carriers (Syntol, Russia) or functionalized membranes (Qiagen, USA). Detailed protocols are given in Supplementary information, Section 3. The final volume of total RNA solution was 100 µL. Rough homogenate (1 g potato leaves: 20 mL commercial extraction buffer by Syntol [Russia] or Qiagen [USA]) from healthy plant was used for dilution-infected extracts and serial dilution with purified PVX. To check cross-reactivity, purified PVS and PVY were added to the extract of the healthy leaves at 1 µg/mL (0.02 µg/g) for experiments with specificity of the test. All samples were tested using proposed RT-RPA-LFA. Control of PVX in the samples was carried out via commercial RT10

qPCR test (Syntol, Russia) as described above or LFA without amplification based on antibodies specific to PVX coat protein and described by Panferov et al. [24]. 3. Results and Discussion 3.1. Obtaining specific nucleic acid fragments of PVX and primer selection Coat protein gene (gp5) of PVX was chosen for specific detection due to being the most abundant in the infected plant cell [17]. Coat protein was coded not only in genomic full-size RNA but in subgenomic mRNA [17]. Moreover, gp5gene is located at 3'-end of PVX RNA and thus is the most effective for reverse transcription. The genomic RNA was extracted from purified PVX using phenol‒chloroform and evaluated as approx. 4‒6 kb (see Supporting Information, Section 3, Fig. S2A), which corresponds to the RNA genome of PVX [25]. Based on the extracted PVX RNA genome, cDNA was obtained by reverse transcription followed by PCR. The size of amplified DNA (714 bp) was visualized by electrophoresis in agarose gel at a level of approx. 700 bp (see Supporting Information, Section 4, Fig. S2B). This DNA was used for optimization of qPCR and to obtain target DNA amplicons for LFA. For RPA, primes were designed based on the desired length of target amplicons (100–300 bp), coating different annealing spots at annealing temperature corresponding to 55 °C. After rejecting primers prone to the formation of dimers, three sets of primers providing amplicons with 146 bp (set 1), 260 bp (set 2), and 200 bp (set 3) were selected (see Supporting Information, Section 1). To evaluate the sets for the following procedures, we performed qPCR. Serial-diluted gp5 cDNA from 106 to one copy per reaction volume was used as a template, and a control detection was qPCR with a commercial kit. We found that qPCR with designed primers detected 10 copies of DNA for set 1 (Fig. 1A, curve 1), which was comparable with commercial kit detection (Fig. 1A, curve 4). The sensitivity for primers set 2 and 3 was about 100 copies (Fig. 1A, curves 2, 3). Summarizing the collected data, we concluded that set 1 is most appropriate for PVX detection.

11

We then checked sensitivity of detecting the PVX RNA with the same primers (set 1) in RT-qPCR. In this experiment with SYBR detection, the detection limit was 1000 copies of PVX RNA (Fig. 1B, curve 1); commercial RT-qPCR kit detected the similar minimal RNA amount equal to 500 copies (Fig. 1B, curve 2). Reduction of reverse transcription efficiency, even for commercial kits, is a well-known problem that can be caused by various reasons as summarized by Schwaber et al. [26]. Therefore, PCR detection has shown the proposed primers are just as sensitive as the commercial kit in detecting PVX DNA and RNA. However, reverse transcription decreased the sensitivity of the detection. 3.2. RT-RPA for PVX RNA The chosen primers’ set 1 was used for RPA. To optimize the primers’ concentration, we performed RPA with the primers from 150‒480 nM (following the range established by the manufacturer) and 10,000 copies of gp5 DNA. We found that the primers in the concentration above 300 nM have a similar time threshold (Tt) (Section 5, Fig. S3A) and do not yield an unspecific product (Fig. S4B). We then performed RPA with 300 nM of primers to estimate its sensitivity. Dependencies of Tt on DNA copies are shown in Figure 2A (raw fluorescence plots are given in Supporting Information, Section 5, Fig. S5). We found that sensitivity of RPA was 500 copies of DNA (see Fig. 2A). However, analyzing melting curves, we discovered the presence of a specific product even in the sample with 100 copies of the initial DNA template (Fig. 2B). Note that SYBR Green shows any dimerization, but in LFA, only dimerization between forward and reverse primers is significant. In this case, the emerging duplex will have biotin and FAM on different ends and will form a complex as the target amplicon with streptavidin and anti-FAM antibodies. However, probability of the duplex formation between forward and reverse primers of set 1 is very low, because the maximum number of overlapping nucleotides contains 3xGC and 6xAT bp. For RT-RPA, the RNA at the amount from 100 to 105 copies was added to the RT-RPA mix wherein nonspecific signal of nontarget duplex was still visible, so melting curves were 12

analyzed to discriminate the specific signal (Fig. 2C). As a result, it was found that RT-RPA detected 1000 copies of RNA, which corresponded to the sensitivity of RT-PCR. 3.3. Optimization of LFA for detection of RPA product The product of RT-RPA for detection in LFA is dsDNA-flanked biotin at one end and FAM at the opposite end. To optimize LFA conditions, we investigated different modes of LFA with differing constructions of test strip, in particular gold nanoparticle conjugate and coating of the test zone. DNA fragment (146 bp in length) based on the primers’ set 1 (FAM-labeled forward primer, biotin-labeled reverse primer) was synthetized and purified as the standard of target DNA for LFA optimization. The size of target DNA was verified by electrophoresis in agarose gel (see Supporting Information, Section 4, Fig. S2C). For binding biotin, we used streptavidin with high affinity to biotin [27]. For binding FAM, monoclonal anti-FAM antibodies were chosen with equilibrium constant determined by the surface plasmon resonance (SPR) method and equaled (1.2 ± 0.3) x 10-9 M (for more details, see Supporting Information, Section 6). To detect formation of the ternary (streptavidin–dsDNA with biotin and FAM–anti-FAM antibodies) on the lateral flow membrane, GNPs were chosen as a most common colored label to ensure sufficient sensitivity in detection [28]. GNPs with spherical shape and 17.3 ± 1.5 nm in diameter were confirmed by transmission electron microscopy; absence of aggregates was confirmed by dynamic light scattering and spectrum (for more details, see Supporting Information, Section 2). The formation of the ternary (anti-FAM antibodies–dsDNA with biotin and FAM–streptavidin or conjugate GNP–streptavidin) complex was confirmed by SPR method and is described in detail in Supporting Information, Section 6. We performed LFA in different modes to detect dsDNA: (1) streptavidin immobilized in the test zone, GNP‒anti-FAM antibodies conjugate located in the conjugate pad; (2) streptavidin immobilized in the test zone, GNP‒anti-FAM antibodies conjugate incubated with target dsDNA preliminary; 13

(3) anti-FAM antibodies immobilized in the test zone, GNP‒streptavidin conjugate located in the pad; (4) anti-FAM antibodies immobilized in the test zone, GNP‒streptavidin conjugate incubated with dsDNA preliminary. Results showed that in the test zone lateral flow strips with anti-FAM antibodies were more sensitive than test strips with streptavidin (Fig. 3A, B). Preliminary incubation of dsDNA with the GNP conjugates yielded a sensitivity similar to the corresponding mode with GNP conjugates dried on the pad (Fig. 3A, B). Comparison of stained zones for a larger range of DNA concentrations, given in Supplement Information, Section 7, indicates that the both binding proteins (anti-FAM antibodies and streptavidin), when applied to the membrane, were distributed over the same area for both LFA schemes. The limit of detection (LOD) for 146 bp dsDNA was equal to 160 pM (5•109 copies per reaction) for both (1) and (2) LFA modes, 22 pM (7•108 copies) for (3) mode, and 19 pM (6•108 copies) for (4) mode. Thus, our results showed that the most sensitive assay was obtained if the pair with the greatest affinity (biotin–streptavidin in our study) was involved in the interaction through GNP conjugate (streptavidin immobilized on the GNP surface). Since the concentration of proteins deposited on the test zone of the working membrane was much higher than the concentration of the GNP conjugate, that is logical to use a more affine interaction for interactions with GNP conjugate. 3.4. Effect of RPA components on the detection of dsDNA in LFA The second task was to determine the conditions in which components of RPA mix do not influence lateral flow rate. Excess RPA components in the sample can reduce flow rate, which is negative for LFA. Moreover, these excess components can cause competition in LFA of the biotin-/FAM-labeled primers with target dsDNA-flanked biotin/FAM for binding sites in the test zone and on the GNP conjugate. Therefore, we conducted experiments for all LFA modes to determine the effect of primers on the signal in the test zone. Primers were added in PBST at various concentrations from 15‒620 nM simultaneously with dsDNA (10 nM). Test zones of 14

strips and their color intensities after this assay are shown in Fig. 3C, D. A specific signal in the test zone was decreased, starting from the final concentration of each primer equal to 75 nM for modes (1), (2), and (4). In LFA mode (3), there was no signal reduction (see Fig. 3C, D). In the case of dsDNA absence, the primers did not give a signal, which confirms the absence of forward/reverse primers’ duplex. Previously (see section “RT-RPA for PVX RNA”), we found that the concentration of primers in the RPA mixture for PVX amplification should be at least 300 nM. Accordingly, to ensure a noncompetitive concentration of primers (75 nM) in the LFA, the dilution of the RPA mixture should be at least 4 times. However, this dilution led to a significantly decrease in lateral flow rate. We found that 14-fold dilution (after combining 5 µL of RPA mixture and 65 µL of PBST) was optimal for lateral flow and complex formation. Thus, the sample for lateral flow assay included no more than 22 nM of primers. Unfortunately, aggregation and change of color from red to purple in the test and control zones appeared in LFA mode containing GNP–streptavidin conjugate with addition of dsDNA and primers to the RPA mix. This process led to a false positive signal. Accordingly, components (DTT, KAc, MgAc, ATP, PEG) of the RPA mix were checked separately in LFA. We found that among the listed above components in RPA buffer, DTT caused the aggregation effect for GNP–streptavidin conjugate (Supporting Information, Section 8). The following reasons can be suggested for this effect. Streptavidin from Streptomyces avidinii, unlike antibodies, contains no cysteine residues which provide free thiols to form the strong covalent Au-S bonds. Thus, streptavidin can be displaced from the GNP surface by DTT that is smaller and much more reactive toward gold surface [29]. After displacement, GNPs become prone to aggregation under conditions of high ionic strength and multicomponent RPA solution. Moreover, DTT may itself be the cause of aggregation of bare GNPs [30]. Therefore, in choosing the LFA scheme, we were forced to prefer a less sensitive (8 times) LFA mode with streptavidin immobilized in test zone due to aggregation. Note that 15

commercial test strips use this LFA mode. Therefore, we see good potential for increasing the sensitivity of RPA-LFA in obtaining GNP–streptavidin conjugate stable to DTT. 3.5. Optimization of RT-RPA with LFA for PVX The following optimizations (order of component addition, temperature) were performed in the assay at the addition of purified PVX to the sample. PVX was added to potato leaf rude extract (1 g potato leaves: 20 mL extraction buffer), forming a concentration range from 1.4 µg PVX per gram of leaf tissue (72 ng/mL, see concentration conversion in Supporting Information, Section 9) to 0.014 fg/g. After total RNA extraction as described in the section “Analysis of potato samples,” presence of PVX RNA was detected by RT-RPA-LFA using three variants of addition order of RPA components: (a) liquid components (primers, DTT, MMLV, etc.) then 2.5 µL magnesium acetate (280 mM) was added, then 10 µL RNA (after isolation from potato leaves containing PVX), then lyophilized RPA mix; (b) lyophilized RPA mix was dissolved in liquid components with RNA, then magnesium acetate; (с) lyophilized RPA mix was dissolved in liquid components, then magnesium was added, then RNA. The impact of addition order was found to be highly significant. The most effective variant was (a) with LOD equal to 0.14 ng/g (Supporting Information, Section 10). For (b) option, LOD was higher than 200 ng/g, and for (c) option, LOD was higher than 1000 ng/g (see details in Supporting Information, Section 9). A significant influence of the order of reagents’ addition was previously described by Lillis et al. [31]; however, the effect was less. Interestingly, the order of the reagent addition recommended by the manufacturer corresponds to (b) option. Perhaps this is not a universal rule for all RT-RPA-LFA systems. To eliminate the duplex formation between forward and reverse primers during LFA at room temperature, we also performed an assay at 37 °C and found a permanent absence of 16

nonspecific color in the test zone. Calibration curve obtained at 37 °C is shown in Fig. 4. The LOD of RT-RPA LFA was 0.14 ng/g (2•106 RNA copies per gram or 5•103 RNA copies per reaction) without visible background (Fig. 4), which is appropriate to detect disease without symptoms caused by PVX [32]. Total assay time was 30 min (from obtaining of leaves to result). The produced test strips showed good reproducibility after 7 months of storage at room temperature. After storage, neither the signal intensity nor the LOD had worsened (The calibration dependence obtained after the storage is presented in Supporting Information, Section 11). These results confirm the high stability of the LFA components used for the assay. The reactivity of RPA components was also stored that accords to data of their manufacturer, TwistDX. To confirm an absence of cross-reactivity, we performed the test with a sample of serialdiluted PVX with presence of 1 µg/mL (20 µg/g) of other potato pathogens—PVS and PVY. These viruses also have single-strand (+) RNA. No cross-reactivity with either virus was shown. Moreover, we plotted dependence of color intensity in the test zone on PVX concentration at constant high concentration (20 µg/g) of PVS and PVY. Calculated LOD was 0.014 ng/g (see details in Supporting Information, Section 12). Moreover, potato leaf extracts spiked by purified PVX were tested by RT-qPCR commercial kit (Syntol, Russia) with obtained LOD of 0.14 ng/g, RT-qPCR with SYBR Green detection, and our primers with LOD of 1.4 ng/g (see details in Supporting Information, Section 13). Therefore, sensitivity of the developed RT-RPA-LFA was the same as that of PCR detection. However, more sensitive RT-qPCR are known: Mortimer-Jones et al. detected about 80 PVX RNA transcript [33], and Agindotan et al. detected 200 particles of PVX in a PCR mix sample [34]. RT-qRPA detected PVX with LOD of 1.4 ng/g after melting curve analysis as described in the section “RT-RPA for PVX RNA” (see details in Supporting Information, Section 13). Thus for PVX detection, the appearance of amplified targets was more successful by GNP-based LFA rather than by fluorescence detection with SYBR Green. 17

3.6. RT-RPA-LFA of real infected plant samples Total RNA was extracted from leaves of infected plants. The same leaves were also used for LFA assay based on antibodies specific to coat protein of PVX. Healthy plants were used as a negative control. Infected plants by PVX (24 plants) and healthy plants (11 plants) were confirmed by RT-qPCR (Supporting Information, Section 14). All samples were tested using the proposed RT-RPA-LFA, the general scheme of which is shown in the Fig. 5. The results obtained by the RT-RPA-LFA fully coincided with the results of RT-qPCR (Supporting Information, Section 14). We evaluated serial-diluted extracts for two positive samples using RT-qPCR: LFA without amplification based on antibodies specific to PVX and developed RT-RPA-LFA. Results are shown in Fig. 6 and demonstrate minimal detected dilution equal to 1 x 106 for RT-qPCR (Fig. 6A), 0.26 x 106 for RT-RPA-LFA (Fig. 6B), and 1 x 103 for LFA (Fig. 6C). Therefore, the introduction of RPA significantly (250 times) increased the sensitivity of LFA. The developed RT-RPA-LFA method is as highly sensitive as RT-qPCR, but it does not require thermal melting and performed at low temperature (37 °C), so there is no need for a thermocycler. RT-RPA-LFA reduces the cost and time of diagnosis and so can be used at resource-limited conditions. Note that the result of convenient LFA is consistent with LFA reported previously with LOD of PVX, which was 2 ng/mL (20 ng of PVX per g of potato leaves) [35, 36]. Previously, we reported immune LFA with nanoparticle aggregation amplification resulting in LOD of 0.25 ng/mL (25 ng/g) [37], with alkaline phosphatase amplification resulting in LOD of 0.3 ng/mL (3 ng/g) [24], and with magnetic concentration resulting in 0.5 ng/mL (15 ng/g) [38]. Additionally, even LFA with 240-fold enhancement due to gold enlargement of GNP in test zone provided 0.017 ng/mL (0.34 ng/g) [39]. Thus, the RT-RPA-LFA test exceeds the most sensitive LFA tests based on antibodies to PVX. 4. Conclusion

18

In this study, we first developed a test for RT-RPA-LFA detection of PVX providing LOD similar to PCR (0.14 ng PVX per gram of plant tissue). This assay reduces the cost and time of diagnosis, can be used at resource-limited conditions, and includes reverse transcription and RNA amplification without an additional stage. The developed assay was successfully applied to detect PVX in the infected samples, even 260,000-fold diluted. We found significant differences for the primers’ set designed based on coat protein gene gp5. The optimal results were obtained for the set producing DNA amplicon with 146 bp. Among following adjustments unassociated with target and primer design, five of the most important issues should be noted. (1) The most sensitive assay could be obtained if the pair with the greatest affinity (biotin–streptavidin in our study) is involved in the interaction through GNP conjugate (streptavidin immobilized on the GNP surface). (2) DTT in RPA buffer caused aggregation for GNP–streptavidin conjugate. (3) Another important issue was associated with primers’ concentrations. Concentration of upper 75 nM in the LFA sample could decrease color intensity due to competition between biotin-/FAM-labeled primers and target dsDNA-flanked biotin/FAM for binding sites in the test zone and on the GNP conjugate. (4) We found an appropriate sequence of components’ addition—addition of sample to liquid compotes, then dissolution of lyophilized RPA mix followed by addition of magnesium acetate. (5) We showed that only one order of RPA sample mixing was appropriate. These adjustments will be useful to consider and apply when developing any RT-RPA-LFA for RNA virus detection.

Acknowledgements The study was financially supported by the Russian Science Foundation (Grant 16-16-04108).

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Figure captions Fig. 1. Comparison of primer sets for detection of (A) DNA by qPCR with SYBR detection using primer set 1 (curve 1), set 2 (curve 2), set 3 (curve 3), and commercial kit with FAM detection (curve 4); (B) RNA by RT-qPCR using primers designed in this study (set 1) and SYBR detection (curve 1) and commercial kit with FAM detection (curve 2). Dash line shows upper significant Ct for commercial kit. Fig. 2. Detection of DNA and RNA using RPA based on primers’ set 1. (A) Dependence of threshold time on DNA copies; (B) Melting curves of RPA products with different initial quantities of gp5 DNA; (C) Melting curves of RT-RPA products with different initial quantities of PVX RNA. Fig. 3. Effect of LFA mode on the detection of purified 146 bp dsDNA. For all figures numerals 1‒4 correspond to modes: 1 – streptavidin is immobilized in the test zone, GNP‒anti-FAM antibodies conjugate is located in the conjugate pad; 2 – streptavidin is immobilized in the test zone, GNP‒anti-FAM antibodies conjugate is incubated with target dsDNA preliminary; 3 – anti-FAM antibodies are immobilized in the test zone, GNP‒streptavidin conjugate is located in the pad; 4 – anti-FAM antibodies are immobilized in the test zone, GNP‒streptavidin conjugate is incubated with dsDNA preliminary. (A) Test zone of strips after LFA of 146 bp dsDNA; (B) Color intensities depending on DNA concentrations. Concentration (nM) of gp5 RPA fragment DNA shown above the strips; (C) Test zone of strips after LFA of 146 bp dsDNA (10 nM) with competition of FAM-/biotin-labeled primers; (D) Histogram of color intensity of test zone after detection of 10 nM ds DNA at different primer concentrations. Concentration (nM) of primers shown above the strips. Fig. 4. LFA of RPA plant samples extracts with serial-diluted purified PVX. Fig. 5. The scheme of RT-RPA-LFA proposed for PVX detection

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Fig. 6. Analysis of extracts of infected leaves. (A) Calibration plots of RT-qPCR for two diluted infected plant extracts. Dash line indicates limit cycle for reliable detection of PVX; (B) LFA after RPA of serial-diluted infected plant extracts; (C) Conventional LFA based on antibodies specific to PVX for serial-diluted infected plant extracts. Numbers 1 and 2 correspond to infected plants 1 and 2. Ctrl – control strips after LFA of RNA extraction from noninfected plant.

26

A

15 14

Threshold time, min

13 12 11 10 9 8 7 6 0

102

103

Target DNA, copies

104

105

-dF/dT

B 1.6x10

Number of initial of gp5 DNA, copies

-3

1.4x10

-3

1.2x10

-3

1.0x10

-3

8.0x10

-4

6.0x10

-4

4.0x10

-4

2.0x10

-4

DNA product

0 100 500 1000 10000 100000

0,0 60

65

70

75

80

85

Temperature, C

90

95

100

С 1.4x10

-3

1.2x10 -3 1.0x10 -3

-dF/dT

8.0x10 -4

non-target Number of initial of PVX RNA, copies duplex 0 100 1000 10000 100000

DNA product

6.0x10 -4 4.0x10 -4 2.0x10 -4 0.0 65

70

75

80

85

Temperature, C

90

95

100

Color intensity, arb.units

B

50 1

40 30

4 2 3

20 10 0 0

0,1

1

Target DNA, nM

10

00 14

0 14

14

0. 01 4 0. 14 1. 4

0 0. 00

14

Initial PVX concentration, ng/g

Lateral flow assay (10 min, room temperature or 37°C)

Y

YY Y

Y

Y

Y YY

Y

YY Y

Y

YY Y

Y YY

reverse transcription

Y YY

Reverse transcription Extraction of total RNA from plant samples recombinase polymerase amplification (RT-RPA) (conditions dependce on using kit) (20 min, 37°C)

Y

Y

gp5 gene amlification

Y YY

Test strip Y

Y YY

Y

Y

YY Y

Y YY

YY Y

146 bp FAM/biotin labeled target DNA

YY Y

FAM labeled primer (35 nt) Biotin labeled primer (35 nt) MMLV reverse transcriptase BsuI polymerase UvrY recombinase single strand binding protein

Sample with target DNA

Y

One-step RT-RPA

Y

Isolated RNA

Y

Infected or helthy leaf

Y

GNP – antiFAM-antibody conjugate ternary complex in the test zone complex in the control zone

6 3

ct rl

26 0 3 0* 10 10 3 6

*1

10 65

4*

25

16

1

Dilution:

ct rl

10

25

64

16

1

3

6

Dilution:

Highlights Recombinase polymerase amplification – lateral flow assay for PVX was developed Detection limit was 0.14 ng potato virus X (PVX) per g potato leaves Sensitivity was 260-fold increased as compared with antibody-based lateral flow assay Multiparametric adjustments for detection of RNA virus were proposed

Declaration of interests ☒ 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. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: