Performing Department
College of Veterinary Medicine
Non Technical Summary
Aquaculture is the fastest growing food-production sector in the world, providing a significant supplement to, and substitute for, wild aquatic organisms. However, disease is a primary constraint to the growth of many aquaculture species and is now responsible for severely impeding both economic and socio-economic development in many countries of the world. Current methods of analyzing economically relevant diseases caused by infectious hematopoietic necrosis virus, Vibrio tubiashii, white spot syndrome virus, salmonid herpesvirus 1, infectious salmon anemia virus and mycobacteria spp. in diagnostic labs require sophisticated and expensive equipment, complex experimental procedures and highly trained personnel. We will develop a new field applicable rapid diagnostic method for the above-mentioned pathogens detectable within 30 mins using recombinase polymerase amplification (RPA) and Lateral flow technology. An isothermal DNA or RNA detection technology, RPA offers several advantages over other testing systems, including speed, sensitivity, specificity, and portability and is suitable for field-usable nucleic acid tests as well as high throughput lab testing. The validation of RPA and lateral flow assays will help veterinary diagnostic laboratory to serve our aquaculture industry clients and veterinarians to identify economically important infectious disease pathogens at Point-of-Care so that treatment or prevention can be initiated immediately. These field applicable tests are critical for the eradication of the above-mentioned economically significant aquaculture animal diseases in the United States.
Animal Health Component
100%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
Our major goals in this proposal is to develop a portable, sensitive, rapid on-field diagnostic method based on a novel Probe-based recombinase polymerase amplification (RPA) technology with lateral flow to detect genomes of infectious hematopoietic necrosis virus, Vibrio tubiashii, white spot syndrome virus, salmonid herpesvirus 1, infectious salmon anemia virus, mycobacteria spp., in aquatic animal samples at Point-of-Care within 30 minutes at room temperature without any sophisticated equipment.
Project Methods
Objective 1A: Primer and probe design. Screening for selecting efficient primers and probes using pathogen nucleic acid. Unlike real-time PCR primers and probes, the design and requirements of both RPA primers and probes are different. RPA Primers must be 30-35 bases and probe should be >40 bases. RPA works best at constant temperature (37-39°C) and Amplicons of 80-400bp are preferred. In contrast to PCR, the melting temperature of an oligonucleotide is not the critical factor for its performance as a primer. Melting of the whole DNA template is not required, instead, RPA employs recombinase-primer complexes to scan double-stranded DNA and facilitate strand exchange at cognate sites. The design of the probe is quite different. The probe contains a tetrahydrofuran abasic-site mimic (THF) [8] flanked in close proximity by nucleotides modified with a fluorophore and a quencher. Fluorescent signal generation by separation of a fluorophore and a quencher depends on cutting of the probe by double-strand specific Nfo exo enzyme. Primer and probe sequences will be selected from an alignment of nucleotide sequences of the genomes of infectious hematopoietic necrosis virus, Vibrio tubiashii, white spot syndrome virus, salmonid herpesvirus 1, infectious salmon anemia virus, mycobacteria spp., from GenBank. The alignment will be performed to select a highly conserved region for each pathogen. The RPA primers and probes will be optimized with Primer Designer version 3.0, to perform with an annealing temperature of 38°C. Additionally, the program checks for interactions between primers and probes. All primers and probes will be synthesized by Eurogentec, San Diego, California.Objective 1B: Recombinase Polymerase Amplification (RPA) technology for Lateral flow Device (LFD) ("dip stick") detection: Forward primer is labeled with FAM (green fluorescence) and reverse primer is labeled with Biotin (Fig 1). The first step is to conduct RPA reaction as described previously (Fig 1). The second step is to detect the RPA product by LFD as shown in Fig 2 below.Figure 1 5'Lateral Flow Device (forsite diagnostics, UK) (Fig 2 and 3): The dipstick device is designed for the in vitro qualitative detection of DNA or RNA amplification products labeled with FAM (fluorescein dye) and biotin. The single-use detector requires no specialist equipment for use, and will confirm the presence of suitably labeled-pathogen genome within 5 minutes.Figure 2The device will specifically capture double-stranded amplified DNA labeled with FAM and biotin formed during an amplification reaction in which 5' labeled primers are included. Once loaded onto the test strip, biotin in the amplified DNA binds to anti-biotin antibody linked to a latex bead, and as these complexes flow along the nitrocellulose membrane they are captured at the T-line composed of anti-FAM antibody (Fig 3). Only amplified DNA containing both labels will be captured to form the visible T-line. The strip contains a control line (C-line) so that negative results are indicated by the presence of a single blue line on the test strip. Positive results are recognized by the presence of 2 blue lines (Fig 3).Figure 3 T and C-line are positive: Test Positive T-line is negative and C-line is positive: Test negativeDip strip detection method is much more suitable for point-of-care applications because it virtually eliminates the need for instrumentation and it would provide qualitative results.Fluorescent detection: Any small electronic device with built-in UV or built-in LED light can then detect the glowing dye to determine whether the pathogen is present in the sample (Fig 2).OBJECTIVE 2: Development and Validation of real-time RPA technology and lateral flow assay in the detection of infectious hematopoietic necrosis virus, Vibrio tubiashii, white spot syndrome virus, salmonid herpesvirus 1, infectious salmon anemia virus, mycobacteria spp., genomes in aquaculture raised aquatic animal samples at Point-of-Care.Before proceeding to RPA assay and lateral flow methods on aquaculture aquatic samples, we need to standardize and validate RPA reactions for each pathogen and then validate RPA in spiked aquatic animal samples. Viruses and bacteria obtained from ATCC and/or collaborators will be used to spike the samples for validation of RPA assay and lateral flow. The pathogen DNA or RNA will be extracted using commercially available Isolation Kit (Life Technologies) or by alkaline lysis method and the copy numbers will be established using real-time PCR. For real-time RPA, the lyophilized pellets of RPA reagents (TwistDx, Cambridge, UK) will be resuspended in 50µl volume containing different copy number of pathogen templates, primers and probe and incubated at 38C for 20 mins. The real-time fluorescence reactions will be detected using hand-held fluorometer or Twista tube scanner (TwistDx, UK). The real-time reactions will be scored as positive when fluorescence exceeded 50 units. RPA can be readily applied to any DNA or RNA target. Lateral Flow device can be used to detect RPA reaction products and the results can be easily read as colored lines similar to 'dip stick'. RPA and Lateral flow is field applicable and requires no sophisticated equipments.