Preliminary Validation of Direct Detection of Foot-And-Mouth Disease Virus within Clinical Samples Using Reverse Transcription Loop-Mediated Isothermal Amplification Coupled with a Simple Lateral Flow Device for Detection

<div><p>Rapid, field-based diagnostic assays are desirable tools for the control of foot-and-mouth disease (FMD). Current approaches involve either; 1) Detection of FMD virus (FMDV) with immuochromatographic antigen lateral flow devices (LFD), which have relatively low analytical sensitivity, or 2) portable RT-qPCR that has high analytical sensitivity but is expensive. Loop-mediated isothermal amplification (LAMP) may provide a platform upon which to develop field based assays without these drawbacks. The objective of this study was to modify an FMDV-specific reverse transcription–LAMP (RT-LAMP) assay to enable detection of dual-labelled LAMP products with an LFD, and to evaluate simple sample processing protocols without nucleic acid extraction. The limit of detection of this assay was demonstrated to be equivalent to that of a laboratory based real-time RT-qPCR assay and to have a 10,000 fold higher analytical sensitivity than the FMDV-specific antigen LFD currently used in the field. Importantly, this study demonstrated that FMDV RNA could be detected from epithelial suspensions without the need for prior RNA extraction, utilising a rudimentary heat source for amplification. Once optimised, this RT-LAMP-LFD protocol was able to detect multiple serotypes from field epithelial samples, in addition to detecting FMDV in the air surrounding infected cattle, pigs and sheep, including pre-clinical detection. This study describes the development and evaluation of an assay format, which may be used as a future basis for rapid and low cost detection of FMDV. In addition it provides providing “proof of concept” for the future use of LAMP assays to tackle other challenging diagnostic scenarios encompassing veterinary and human health.</p></div

Titration of epithelial suspensions and subsequent analysis with the RT-LAMP-LFD assay.

<p>A 10% epithelial homogenate (w/v) containing 10⁶ TCID₅₀/mL FMDV was diluted in nuclease free water as described below. These dilutions were each assayed in duplicate with the RT-LAMP-LFD protocol, and analysed with AGE and on an LFD. (a) Application of 5 uL of the “neat” 10% epithelial homogenate added directly to the RT-LAMP-LFD reaction, (b) 1 in 3 dilution, and (c) 1 in 5 dilution, (d) 5 ul of nuclease free water added to the reaction mixture.</p

Comparison between the RT-qPCR, RT-LAMP and RT-LAMP-LFD assays from field epithelial suspensions.

<p>Comparison between the RT-qPCR, RT-LAMP and RT-LAMP-LFD assays from field epithelial suspensions.</p

The effect of isothermal temperature on the end point limit of detection of both the RT-LAMP (un-labelled internal primers) and RT-LAMP-LFD (labelled internal primers) reaction.

<p>The effect of isothermal temperature on the end point limit of detection of both the RT-LAMP (un-labelled internal primers) and RT-LAMP-LFD (labelled internal primers) reaction.</p

Limit of detection between the RT-qPCR (Ct value), RT-LAMP (Tp value) and RT-LAMP LFD assays.

<p>(a) RT-qPCR amplification Ct values corresponding to each of the 10-fold dilutions of RNA standards; (b) RT-LAMP amplification result with Tp values given for each RNA copy number; (c) AGE analysis of RT-LAMP-LFD reaction, spanning the same RNA standards as in (a) and (b); (d) The RT-LAMP-LFD reactions from (c) applied to the LFD device. +/− indicates that out of 4 identical replicates of a given RNA concentration applied to a specific assay (a–d), there were a mixture of positive and negative results.</p

Comparative limit of detection between Svanova LFD, RT-qPCR, RT-LAMP-LFD and the RT-LAMP assays.

<p>10 fold dilutions of FMDV containing epithelial suspensions were analysed using the Svanova LFD device (a) by applying the suspensions directly to the device. RNA was extracted from each suspension and subsequently analysed using either the RT-qPCR (giving a Ct value) (b) the RT-LAMP assay read using PicoGreen fluorescence(giving a Tp value) (c) or by mixing each 10 fold dilution 1∶5 with nuclease free water, and directly adding this to the RT-LAMP-LFD assay for subsequent detection with the LFD (d).</p

RT-LAMP-LFD reactions utilising a simple desk top water bath as the isothermal heat source.

<p>(a) RT-LAMP-LFD products analysed using AGE; (b) The RT-LAMP-LFD products from (a) applied to the LFD.</p

Ct values generated from FMDV 3D rRT-PCR performed on sections of 14 separate LFDs and corresponding values generated for MagNA pure extracted RNA from the equivalent original epithelial suspensions in parallel.

<p>LP: Loading Pad; WS: Wicking strip; NB: Nitrocellulose below Ab Band (NB); Nitrocellulose Ab Band (AbB); Nitrocellulose above Ab band (NA). LFDs spanned four serotypes O (red dots) (LFDs TUR 8/1969, BAR 2/2008, KUW 2/2008, SAU 3/2008, ZAM 5/2008, HKN 10/2005, IRN 53/2006, UKG 7B/2007, A (blue dots) (LFDs BAR 4/2009, IRN 1/2008, KEN 8/2008, TUR 20/2006, IRN 36/2007), Asia 1 (grey dots) (IRN 15/2001) and SAT 1 (yellow dots) (ZAM 5/2008).</p

Ct values generated from FMDV 3D rRT-PCR performed on sections of LFDs in dilution series from serotype O isolate (BAR 2/2008) compared against the visual detection on the Ag LFD.

<p>LP: Loading Pad; WS: Wicking strip; Nitrocellulose Ab Band (AbB)(+)  =  genome detected; (-)  =  No Ct.</p

Box plot of Ct values generated from FMDV 3D rRT-PCR performed on sections of LFDs from serotype SAT 1 isolate (ZAM 5/2008) eluted at one month after LFD testing.

<p>LFDs had either been stored at room temperature (RoomT) (green bar) or 37°C (red bar). LP: Loading Pad; WS: Wicking strip; Nitrocellulose Ab Band (AbB). Dotted blue line represents baseline data (Ct values from LFDs washed on day one).</p