Duplex Lateral Flow Assay for the Simultaneous Detection of Yersinia

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Duplex lateral flow assay for the simultaneous detection of Yersinia pestis and Francisella tularensis Miriam Jauset-Rubio, Herbert Tomaso, Mohammad Soror El-Shahawi, Abdulaziz Saleh Omar Bashammakh, Abdulrahman Obaid Al-Youbi, and Ciara K. O' Sullivan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03105 • Publication Date (Web): 08 Oct 2018 Downloaded from http://pubs.acs.org on October 8, 2018

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Analytical Chemistry

Duplex lateral flow assay for the simultaneous detection of Yersinia pestis and Francisella tularensis Miriam Jauset-Rubio a*, Herbert Tomaso b, Mohammad S. El-Shahawi c, Abdulaziz S. Bashammakh Abdulrahman O. Al-Youbi c and Ciara K. O´Sullivan a,d*

c, a

INTERFIBIO Consolidated Research Group, Department of Chemical Engineering, Universitat

Rovira I Virgili, 43007 Tarragona, Spain b

Friedrich-Loeffler-Institut, Institute of Bacterial Infections and Zoonoses, Naumburger Strasse 96a,

07743 Jena, Germany c

Department of Chemistry, Faculty of Science, King Abdulaziz University, P. O. Box 80203, Jeddah

21589, Kingdom of Saudi Arabia. c

Institució Catalana de Recerca I Estudis Avancats, Passeig Lluís Companys 23, 08010 Barcelona,

Spain * To whom correspondence [email protected]

should

be

addressed,

email:

[email protected];

Abstract High-risk pathogens such as Francisella tularensis and Yersinia pestis are categorised as highly hazardous organisms that can be used as biological weapons. Given the extreme infectivity of these potential biowarfare agents, a rapid, sensitive, cost-effective and specific method for their detection is required. Here, we report the multiplexed amplification detection of genomic DNA from Francisella tularensis and Yersinia pestis. Amplification was achieved using isothermal recombinase polymerase amplification, exploiting tailed primers, followed by detection using a nucleic-acid lateral flow assay. Excess primers were removed using a novel fishing strategy, avoiding the use of post-amplification purification that requires centrifugation and infers additional assay cost. The entire assay is completed in less than 1 hour, achieving limits of detection of 243 fg (1.21 x 102 genome equivalent) and 4 fg (0.85 genome equivalent) for Francisella tularensis and Yersinia pestis, respectively. Introduction According to the Centre for Disease Control and Prevention (CDC), bioterrorism refers to biological agents (microbes or toxins) that can be used as weapons to further personal or political agendas. Since these biological agents are relatively easy and inexpensive to obtain, can be easily disseminated, can cause widespread fear and panic, and are difficult to distinguish from naturally occurring infectious disease outbreaks, bioterrorism has become an attractive weapon1. These biological agents have been classified in three main groups (category A, B and C), depending on their potential risk. Category A refers to high priority agents, which can pose a risk to national security, resulting in high mortality such as is the case for both Francisella tularensis (F. tularensis) and 1 ACS Paragon Plus Environment

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Yersinia pestis (Y. pestis)2, with these microorganisms causing serious zoonotic diseases in humans called tularaemia and plague, respectively. Under natural conditions, infection may occur through direct contact with infected animals, animals’ tissues, bodily fluids, ingestion of meat from infected animals, drinking contaminated water, bites from arthropods or inhalation of infectious droplets3–5. In order to reduce the impact of these pathogens by enabling rapid appropriate treatment of infected people, whilst also avoiding the rapid spread of the disease in the population, accurate, rapid inexpensive and easy-to-use diagnostic tools are essential for their detection. To date, different methodologies have been developed and reported for the detection of these microorganisms. Culture-based methods for pathogen identification are the gold standard and are highly sensitive. However, these methods are not adequate for rapid detection, as they are timeconsuming, involve biosafety measures, and can be difficult to cultivate2. In contrast, molecular methods can be equally or more sensitive and can provide higher speed and specificity. The most commonly used molecular method is the polymerase chain reaction (PCR) and its variants, real-time PCR or multiplex PCR6–10. Alternative methods, include immunochemistry for pathogen detection such as the enzyme linked immunosorbent assay (ELISA)11–15. Nanoconstructs, which exploit the combination of capture antibodies immobilised on magnetic beads with genetically engineered apoferritin nanoparticles conjugated with multiple quantum dots and detection antibody have also been reported16,17. Although these techniques provide high sensitivity, they involve complex sample preparation protocols that may not facilitate a rapid enough detection of pathogens. Immunochromatographic assays have been widely used18–20, and whilst rapid, these assays suffer from low sensitivity. Various commercial kits are available based on these methodologies including the SMART II Yersinia pestis Anti-F1 detection kit or Francisella tularensis detection kit (New Horizons Diagnostics Corporation)21,22, Plague Biothreat Alert or Tularemia Biothreat Alert (Tetracore)23, BADD Plague (ADVNT)24,25 and ABICAP classic test kit – Y. pestis or F. tularensis (Senova GmbH)26. The ABICAP, based on immunofiltration, is the most sensitive commercial kit, with a limit of detection (LOD) of 104 CFU / ml. Alternative DNA amplification methods based on isothermal amplification techniques have been developed, overcoming the limitations of PCR as most of them do not require thermal cycling, operate at constant temperature, and have reduced assay times. One example is reported by Feng et al., for the detection of Y. pestis using loop-mediated isothermal amplification (LAMP) combined with magnetic bead capture DNA, achieving a LOD of 2.3 x 103 CFU in simulated spleen and lung samples27. Although the authors demonstrated a higher sensitivity as compared to PCR, LAMP requires the design of a set of four to six primers, which can make multiplexed amplification extremely complicated. In addition, a higher temperature (60-65ºC) and longer assay time is required with LAMP, in contrast to an alternative method of isothermal amplification, namely recombinase polymerase amplification (RPA), which operates at 37 oC and can be completed in less than 10 minutes28. In 2012, Euler et al., exploited real time-RPA for the detection of F.tularensis29, and subsequently developed a panel for the detection of biowarfare agents following the same methodology30. Our group recently reported on an approach for solid-phase RPA based on the immobilisation of the forward primer on a microtiter plate well / gold electrode surface, with solution based reverse primer, resulting in surface tethered amplicons, which could be quantitatively detected31–33. In a different approach, again exploiting RPA, we described the use of tailed primers, resulting an amplicon with a duplex flanked by two single stranded DNA tails, which 2 ACS Paragon Plus Environment

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was then detected on a nucleic acid lateral flow assay34. This tailed amplicon facilitates direct detection via hybridization to a surface immobilised capture probe and a reporter probe labelled to gold nanoparticles, combining the characteristics of lateral flow assays such as inexpensiveness, rapidity, portability and ease-of-use, with the benefits of isothermal amplification, achieving detection limits of 1 × 10-11 M (190 amol). Expanding on this preliminary work, we developed a duplex nucleic acid lateral flow assay for the simultaneous detection of F. tularensis and Y. pestis based on the combination of RPA and tailed primers. Assay parameters were optimised to obtain similar amplification of both targets with synthetic DNA and then applied to the detection of genomic DNA isolated from real samples.

Materials and methods Materials Phosphate buffered saline (PBS, 10 mM phosphate, 138 mM NaCl, 2.7 mM KCl, pH 7.4), Phosphate buffered saline Tween (PBS-Tween, 10 mM phosphate, 138 mM NaCl, 2.7 mM KCl, 0.05% v/v Tween 20, pH 7.4), Proteinase K, Skimmed milk powder, sodium citrate, sulphuric acid and 3,3’,5,5’Tetramethylbenzidine (TMB), gold (III) chloride trihydrate (HAuCl4) were purchased from Sigma (Spain). Agarose powder and Low range DNA ladder were obtained from Fischer Scientific (Spain). Gel red was provided by VWR International Eurolab S.L (Spain). Primers and synthetic sequences for the detection of Y. pestis target gene pla and F. tularensis target gene tul4 were designed using BLAST tools. All DNA oligonucleotides were purchased from Biomers (Germany). The genomic DNA was obtained from the Institut für Bakterielle Infektionen und Zoonosen, Germany. Primer and probe sequences can be found in Table S-1. Recombinase polymerase amplification (RPA) reaction. RPA was performed according to the manufacturer’s instructions (TwistDX, Cambridge, UK). Briefly, for the singleplex amplification, master mix was prepared in a tube with 480 nM of each primer, 1x rehydration buffer, 14 mM magnesium acetate and the desired concentration of the single stranded DNA, and adjusted to a final volume of 50 µl with Milli Q water. For the duplex amplification, some minor modifications were introduced: 100 nM of each specific primer for Y. pestis, whilst 1000 nM of each specific primer for F. tularensis was used, as well as 1.5x rehydration buffer instead of 1x. The reactions proceeded at 37ºC for 30 minutes in the case of synthetic DNA whilst for genomic DNA the reaction was extended to 45 minutes. All the reactions were stopped with 0.1 mg/ml of Proteinase k for 10 minutes at 22ºC. Amplicons were analysed using a 2.6% w/v in 1x Tris-BorateEDTA buffer (1x TBE) agarose gel.

Fishing strategy to remove excess primers Thirty microliters of 10 mg/ml streptavidin magnetic beads (SiGMAG, Spain) were incubated with 100 µl of 20 µM biotinylated fishing probe in PBS buffer for 10 minutes at 22ºC under tilt rotation. Subsequently, the magnetic beads were washed thoroughly with PBS and the RPA product was added to the beads for a 10 minutes incubation. Finally, the magnetic beads were placed close to 3 ACS Paragon Plus Environment


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the magnet for 2 minutes and the supernatant, which was free of excess primers, was collected. To test the assay, the samples before and after the assay were characterised in 2.6% w/v agarose gel in 1x Tris-Borate-EDTA buffer (1x TBE) and using the ELONA assay as described below. Enzyme linked oligonucleotide assay (ELONA). One hundred microlitres of 100 nM biotinylated capture probe in PBS were pipetted into each well of a neutravidin coated microtitre plate. The plates were subsequently blocked with 200 µl per well of 2% w/v skimmed milk powder in PBS-Tween to avoid non-specific binding to the wells. The resulting RPA product was added to the functionalised neutravidin plates (50 µl per well), followed by the addition of 50 µl per well of 5 nM reporter probe labelled with horseradish peroxidase (HRP). Finally, 50 µl per well of TMB substrate was added to react with HRP and 5 minutes later the reaction was stopped with 50 µl per well of 1 M H2SO4. The absorbance was read at 450 nm (SpectraMax 340PC384, bioNova Scientifics S.L.). Each step was incubated for 20 minutes at 22ºC and between steps 200 µl per well of PBS-Tween was used as washing buffer. To explore the sensitivity of the assay, amplification was carried out with different starting concentrations of ssDNA (10-9 M, 10-11 M, 10-13 M, 10-15 M, 10-17 M, 0 M). The limit of detection (LOD) was calculated using GraphPad Prism Software, which calculates the LOD of the assay based on the interpolation of the value resulting from the formula zero concentration (blank) value + 3× standard deviation of this value using the sigmoidal 4PL model. Duplicate measurements were performed for each concentration. Lateral flow assay Gold nanoparticles (AuNPs) with an approximate average diameter of 13 nm were prepared and conjugated to DNA reporter probe previously described34,35. The membrane used was HF180 nitrocellulose (Millipore, Germany), and the absorbent pad was composed of cotton litter fibbers grade 320 (Ahlstrom, Sweden). The test and control lines were prepared by drawing a line with a tip containing 20 µl of a pre-incubated (15 minutes at 22ºC) mixture of 1.7 mg/ml of streptavidin and 67 µM of the respective biotinylated capture probe in PBS buffer in a membrane of 3 cm wide by 30 cm long. Subsequently, the membrane was allowed to dry at 22ºC for 1 hour, followed by blocking with 1% w/v skimmed milk powder and 0.1% v/v empigen detergent for 15 minutes, under shaking conditions. The membrane was left to dry, again at 22ºC for approximately 2 hours and then stored at 4ºC until use. The test strips were cut in strips of 4 mm width (M70, Advanced Sensor Systems P.Ltd.). Ten microliters of reporter probe conjugated with AuNPs were mixed with 2 µl of RPA product, 2 µl of 10x PBS buffer and 6 µl Milli Q water. The mixture was incubated for the optimised time of 3 minutes at 22ºC before being wicked onto the test strip (Figure S-5). In order to explore the sensitivity of the assay, RPA was carried out with different starting concentrations of ssDNA (10-9 M, 10-11 M, 10-13 M, 10-15 M, 10-17 M, 0 M) and with different starting concentrations of genomic DNA (50 pg, 5 pg, 0.5 pg, 0.05 pg, 0.005 pg, 0.0005 pg, 0 pg). A Smartphone camera was used to take an image of the strip and the intensity of the bands were analysed using Image J software. These values were plotted in GraphPad Prism software, which calculates the LOD of the assay as zero 4 ACS Paragon Plus Environment

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concentration (blank) value + 3× standard deviation of this value using the sigmoidal 4PL model. Both duplicate strips and measurements were performed for each concentration. Results and discussion Post-amplification purification using Proteinase K In most reports of RPA to date, the use of a purification kit following amplification and prior to product detection is required. For example, in the case of visualisation of RPA amplicons using gel electrophoresis, the single stranded DNA binding proteins (SSBs) result in other bands, complicating analysis. This purification step incurs additional costs, assay time and requires inherently laboratory based equipment such as centrifuges. To simplify this purification step, the use of Proteinase K was explored. Proteinase K is a broad-spectrum serine protease, discovered in extracts of the fungus Engyodontium album (formely Tritirachium album)36, and used in a wide range of molecular biology applications to digest unwanted proteins, such as nucleases from DNA or RNA preparations from microorganisms, cultured cells, and plants 37–40. A variety of assays were performed to check the effectiveness of the Proteinase K and to elucidate optimal conditions. In an initial experiment, an unpurified RPA amplicon, an amplicon purified using a commercial kit (DNA clean and concentrator, Zymoresearch) as well as an amplicon treated with 1 mg/ml of Proteinase K for 30 minutes at 55ºC were visualised on an agarose gel. In the case of the unpurified amplicon an intense smear rather than a clear band was observed, whilst when the purification kit or Proteinase K was used a clear band was observed. A smear was also observed for the unpurified non-template control, presumably due to the primers, whilst in the purified nontemplate control, no smears or bands were observed as the primers had been removed/digested (Figure S-1a). To further optimise the Proteinase K purification, different temperatures (Figure S1b), assay times (Figure S-1c) and concentrations of Proteinase K (Figure S-1d) were evaluated, with the best results observed being 0.1 mg/ml of Proteinase K, 22ºC and 10 minutes incubation. These conditions were thus used for all further experiments. Singleplex detection Primarily, the design of the tailed primers in terms of specificity and cross-reactivity was evaluated by amplifying each target with its specific primers and then with each other´s primers, as well as amplifying each target in the presence of both sets of primers and no cross-reactivity was observed (Figure S-2). As a proof-of-concept, neutravidin coated microtiter plates were used to immobilise biotinylated capture probes specific for each Y. pestis and F. tularensis, mimicking the lateral flow assay format to be developed. The RPA amplicons, duplexes flanked by single stranded tails, were added to the wells of the microtiter plate and allowed to hybridise to the immobilised capture probe, and subsequently to the reporter probe conjugated with horseradish peroxidase (HRP) (Figure 1a). The LODs for each of F. tularensis and Y. pestis were calculated to be 2.3 x 10-15 M (Figure 1b) and 1.3 x 10-14 M (Figure 1c), respectively.

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Figure 1: Singleplex ELONA assay: a) Schematic representation; b) Francisella tularensis detection; c) Yersinia pestis detection.

Optimisation of multiplexed amplification Multiplexed amplification results in reduced assay costs and time, whilst garnering additional information from a single sample41, and to this end, the simultaneous amplification of Y. pestis and F. tularensis was optimised to achieve similar amplification levels of both targets. In initial experiments, an equal amount of primers from both targets (480 nM) with different buffer concentrations was evaluated, based on the observation reported by Henegarium et al., who highlighted that when one amplicon is less efficiently amplified in PCR as compared to another, varying the buffer concentration can solve this problem42. A slight improvement in the amplification of F. tularensis was observed using 1.5x rehydration buffer. However, in all cases, Y. pestis amplifies faster, thus biasing detection rates (Figure 2a). Indeed, in our laboratory, we have observed with a range of diverse targets that there are some sequences that are amplified using RPA much more rapidly and efficiently than others, but we have not been able to find an explanation for this as there appears to be no correlation between amplification rates and GC content of melting temperature of target / primers. To address this issue, we explored the possibility of balancing primer ratios, decreasing the amount of the more sensitive primers and increasing the amount of less sensitive 6 ACS Paragon Plus Environment

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primers43. Primers for the Y. pestis target were thus decreased to 100 nM, and a range of concentrations of the primers for the F. tularensis target (100-1000 nM) were evaluated, and optimum results in terms of equivalent amplification were obtained using a 1:10 ratio (Figure 2b). Finally, 30 minutes was observed to be optimal to obtain an equal amplified amount of both targets (Figure 2c).

Figure 2: Duplex RPA optimisation: a) Different buffer concentrations; b) Different primers concentrations; c) Amplification time (5, 10, 20, 30 and 40 minutes). NT corresponds to nontemplate control.

Fishing sequences to remove excess primer Whilst in the case of the F. tularensis and Y. pestis targeted in this work it is critical to detect as low levels as possible, information regarding their exact concentration is not of immediate importance. However, in general, exact quantitative measurement of targets detected using lateral flow is needed and to this end, it is important to remove any excess primers that may compete with the tailed amplicons for binding to the probe immobilised on the test lines, resulting in falsely lower signal intensity. One solution to avoid this issue is the optimisation of the primers to use the minimum possible during the amplification step, thus exhausting the primers. However, not only does this require very careful optimisation for each target, but also in the case of multiplexing, it becomes increasingly difficult to optimise, particularly given the ratios of primers required to obtain equivalent amplification levels of each target. As described above, in this work, 1 µM of F. tularensis primers are required, resulting in a high amount of unconsumed primers following amplification. To efficiently remove these excess primer prior to lateral flow detection, we included a specific sequence in the primers, which would allow any primers not incorporated in a duplex amplicon, to be captured, or “fished” via hybridisation to a complementary sequence immobilised on the surface of, for example, magnetic beads (Figure 3a). This sequence is also present in the RPA amplicons, but as it is part of the double stranded duplex, it is not available for hybridisation. Following amplification, the RPA product was incubated with functionalised magnetic beads, and the tube was 7 ACS Paragon Plus Environment


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then placed close to a magnet and the excess primer-free amplicon was directly wicked on to the lateral flow. In order to determine the time required to remove all excess primers, a range of incubation times was studied and 10 minutes observed to be optimal (Figure S-3). In addition, an agarose gel electrophoresis evaluation was performed to confirm that all excess primers were removed during the duplex amplification (Figure S-4). Future work will focus on incorporating this excess primer fishing step onto the lateral flow An enzyme linked oligonucleotide assay (ELONA) assay was performed to demonstrate that the combination of Proteinase K and this “fishing” procedure results in the same clean amplicon product as achieved using commercial purification kits, but decreasing the cost around 3 times for each sample (from 1.5€/sample to 0.45€/sample) and avoiding the use of inherently laboratory based equipment such as a centrifuge (Figure 3b). Unpurified RPA products, as well as Proteinase K purified RPA products were also tested. When no treatment was performed, higher signals were observed both for the positive and negative controls, indicative of non-specific binding, which could be attributed to the single stranded DNA binding proteins (SSBs), binding to DNA primers/probes. Whilst Proteinase K eliminated this background, decreasing the signal of both controls, in the case of the positive controls a lower signal is observed than that obtained when incorporating the “fishing” step due to the presence of excess primers, which compete for binding to the immobilised probes.


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Figure 3: Incorporation of fishing sequences: a) Schematic representation of fishing assay; b) ELONA assay comparing the amplicon without treatment, treated with Proteinase K, treated with Proteinase K + fishing sequences and purified with a commercial kit (DNA clean & Concentrator). YP, FT and NC correspond to Y. pestis, F. tularensis and Negative control (non-template) respectively.

Lateral flow assay Once duplex amplification using tailed primers and fishing sequences had been demonstrated using a microtiter plate format, the developed assay was transferred to a lateral flow assay format. The lateral flow was based on the immobilisation of three biotinylated capture probes, for the test line 1 (Y. pestis), test line 2 (F. tularensis) and control line. On the test lines, the immobilised probe is complementary to the tail in the 5′ region of the amplified DNA (Y. pestis and F. tularensis respectively), meanwhile on the control line, the probe is complementary to the reporter probe conjugated with AuNPs. The reporter probe is complementary to the tail on the 3′ end of the amplified DNA, and the same tail on the 3’ end is used for both Y. pestis and F. tularensis. Upon formation of the sandwich at the test lines and hybridisation at the control line, the red colour of the gold nanoparticles can be observed (Figure 4a).

Figure 4: Lateral flow assay: a) Schematic representation of duplex lateral flow assay; b) Proof-ofconcept on lateral flow strips.

In order to demonstrate the specificity of the assay, singleplex and duplex amplifications were detected on the strip (Figure 4b). To establish the sensitivity achievable with singleplex and duplex detection with lateral flow detection, a range of concentrations of synthetic DNA were amplified 9 ACS Paragon Plus Environment


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using RPA. The test line was visible to the naked eye at concentrations as low as 0.1 pM (140 fg) and 10 pM (19.48 pg) for F. tularensis and Y. pestis, respectively, when individually amplified (Figure 5), whilst duplex amplification resulted in a higher sensitivity of 1 fM (1.42 fg) in the case of F. tularensis, and the same of 10 pM (19.48 pg) for Y. pestis (Figure 6a). Using a Smartphone camera and Image J software, the LOD was improved by ca. two orders of magnitude. The differences in the LOD between the two species could be due to hybridisation efficiencies between the complementary tail sequences or the length of the amplicon, where one amplicon could hybridise more rapidly than the other.

Figure 5: Lateral flow assay: a) Visual limit of detection of Francisella tularensis and Yersinia pestis; b) Limit of detection of Francisella tularensis and Yersinia pestis calculated by Image J software.

To validate the assay using real samples, genomic DNA extracted from F. tularensis and Y. pestis was amplified by RPA for 30 minutes followed by a 10 minute incubation with functionalised magnetic beads to remove excess primers and subsequent lateral flow assay, achieving detection limits of 243 fg (1.21 x 102 genome equivalent) and 4 fg (0.85 genome equivalent)44, respectively, using Image J software (Figure 6b). In this case, Y. pestis had a better LOD, which can be explained by the fact that the gene pla is located on the pPCP1 plasmid, being repeated with a high copy number in the genome45. These detection limits are comparable to those reported in the literature for the singleplex detection of these pathogens9,46.


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Figure 6: Lateral flow assay: a) Limit of detection of duplex lateral flow assay using synthetic ssDNA; b) Limit of detection of duplex lateral flow assay using genomic DNA.

Conclusions We have reported the first example of a duplex recombinase polymerase amplification-nucleic acid lateral flow, exploiting tailed primers and fishing sequences combined with the treatment of Proteinase K to facilitate rapid, facile and cost-effective purification compatible with use at the point-of-need. To demonstrate the functionality of the developed assay, genomic DNA from F. tularensis and Y. pestis was simultaneously isothermally amplified using RPA and detected in lateral flow assay, achieving LODs of 243 fg and 4 fg, respectively. Future work is focusing on the integration of amplification, purification and detection on a single lateral flow strip.

Acknowledgements The authors are grateful to King Abdulaziz University, under the financing of the collaborative project “Selection and application of aptamers against anabolic steroids” for funding.



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Associated Content: [Details about the optimisation process of Proteinase K, singleplex amplification, fishing assay and the oligonucleotides used in this work].

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Duplex Lateral Flow Assay for the Simultaneous Detection of Yersinia

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