Yersinia pestis detection using biotinylated dNTPs for signal enhancement in lateral flow assays

Highlights

• Ultrasensitive molecular detection of Yersinia pestis using biotinylated dNTPs and tailed primer is demonstrated.

• Optimisation of ratio of biotinylated dNTPs: natural dNTPs is carried out using PCR and ELONA.

• Optimised parameters were exploited using RPA.

• Lateral flow assays for detection of single tailed amplicon and biotinylated dNTPs with SA-AuNPs was developed.

• Real samples were tested.

Abstract

Due to the extreme infectivity of Yersinia pestis it poses a serious threat as a potential biowarfare agent, which can be rapidly and facilely disseminated. A cost-effective and specific method for its rapid detection at extremely low levels is required, in order to facilitate a timely intervention for containment. Here, we report an ultrasensitive method exploiting a combination of isothermal nucleic acid amplification with a tailed forward primer and biotinylated dNTPs, which is performed in less than 30 min. The polymerase chain reaction (PCR) and enzyme linked oligonucleotide assay (ELONA) were used to optimise assay parameters for implementation on the LFA, and achieved detection limits of 45 pM and 940 fM using SA-HRP and SA-polyHRP, respectively. Replacing PCR with isothermal amplification, namely recombinase polymerase amplification, similar signals were obtained (314 fM), with just 15 min of amplification. The lateral flow detection of the isothermally amplified and labelled amplicon was then explored and detection limits of 7 fM and 0.63 fg achieved for synthetic and genomic DNA, respectively. The incorporation of biotinylated dNTPs and their exploitation for the ultrasensitive molecular detection of a nucleic acid target has been demonstrated and this generic platform can be exploited for a multitude of diverse real life applications.

Introduction

Yersinia pestis is a high-risk pathogen categorised as a highly hazardous organism that can be used as a biological weapon since it is relatively easy and inexpensive to obtain, and is difficult to distinguish from naturally occurring infectious disease outbreaks [1]. Y. pestis is responsible for plague, a zoonotic disease, with dissemination of the infection occurring through direct contact with infected animals, animal tissue, bodily fluids, ingestion of the meat from infected animals, drinking contaminated water, bites from arthropods or inhalation of infectious droplets [2].

Different methodologies have been reported for the detection of Y. pestis, offering a more rapid alternative to the gold standard technique of culture-based methods, whilst having the same or even higher sensitivity, including the use of molecular techniques [[3], [4], [5], [6], [7]], immunochemistry [[8], [9], [10]] or the use of nanoconstructs [11]. However, these techniques cannot be deployed for use in situ at the point of need and in order to reduce the impact of this potential biowarfare agent, an accurate, rapid, inexpensive and easy-to-use analytical tool is essential.

Lateral flow assays (LFAs) have been developed for rapid detection at the point of need. These tests are single-use and the vast majority of those reported to date are based on immunochromatographic assays generating detectable colored bands with a qualitative yes or no answer, with the best known assay being the pregnancy test [12], and LFAs have evolved to be semi-quantitative or quantitative [13].

Various commercial kits are available exploiting antibodies, including the SMART II Yersinia pestis Anti-F1 detection kit (New Horizons Diagnostics), Plague Biothreat Alert (Tetracore), BADD Plague (Advnt Biotechnologies) and the ABICAP classic test kit – Y. pestis (Senova). However, none of these immunotests have been demonstrated to have both high sensitivity and high specificity, and thus there is still a mature need for the development of reliable diagnostic kits for use at the point of need (Table S1) [14].

As an alternative to immunodetection, molecular detection can combine both high sensitivity and specificity. Nucleic acids can be easily amplified producing multiple copies of DNA and consequently, improving the limit of detection. Furthermore, a set of primers can be designed to amplify a unique region specific for the microorganism of interest and avoid cross-reactivity with other related species. Moreover, molecular detection could also be deployed as lateral flow assays [6].

There are a wide range of paper analytical devices that have been developed for the detection of amplified products, commonly referred to as nucleic acid amplification tests (NAAT). There are two main types of NAAT: nucleic acid lateral flow immunoassays (NALFIA) and nucleic acid lateral flow assays (NALF). NALFIA is the most commonly reported and uses primers modified with small molecules, including digoxigenin (Dig), carboxyfluorescein (FAM), fluorescein isothiocyanate (FITC) or biotin, which are detected using antibodies or streptavidin, either immobilised at the test and control lines, or linked to reporter molecules [[15], [16], [17], [18], [19]]. NALF, on the other hand, does not use hapten labelled amplicons and instead detects DNA using capture and reporter oligonucleotide probes. There are far less reports of NALF, which can be attributed to more complicated assays requiring dT or dA probes [20,21], asymmetric PCR [22,23], or alternative methods for the post-amplification generation of single stranded DNA to hybridise with the capture and reporter probes.

Specifically referring to the detection of Yersinia pestis, the combination of different isothermal DNA amplification techniques including loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA) and thermophilic helicase-dependent isothermal DNA amplification (tHDA) combined with lateral flow dipsticks for the detection of biothreat agents based on NALFIA assays was recently reported. The authors demonstrated that either LAMP or RPA gave 100% specificity against a wide range of related microorganisms tested without the need for a thermocycler, further approaching the realisation of diagnostics for deployment to the point-of-need [24]. However, the NALFIA assay they used requires hapten labelled primers for immunobased detection, which is not only more expensive than the primers, capture and reporter probes needed for NALF assays, but also introduces issues of protein stability [25]. Due to this reason, NALF assays were developed by our group based on isothermal RPA amplification combined with the use of tailed primers, which result in an amplicon comprising the target duplex flanked by two single stranded DNA tails designed to be complementary to capture and reporter probes, facilitating direct detection via hybridization [25,26]. This unique type of amplicon is obtained thanks to an internal modification on the oligonucleotides consisting in a propyl chain (C3), positioned between the primer binding site and the single stranded tail, acting as “stopper”. Most recently, this approach was applied for the simultaneous duplex detection of Yersinia pestis and Francisella tularensis in a multiplex lateral flow assay, achieving LODs of 4 fg and 243 fg, respectively [27].

In the work described here, we have extended our previous work to develop a more sensitive NALF assay, using the detection of Yersinia pestis as a model system. We combined the use of a forward tailed primer which hybridises to the immobilised capture probe with the incorporation of biotinylated dNTPs to enhance the signal of the assay. Assay parameters were optimised using synthetic DNA and then applied to the detection of genomic DNA. The developed platform is generic in nature and can be applied to the detection of all types of nucleic acids using optical or electrochemical transduction by just applying a specific set of primers for each target of interest.