AGDIA XCS66800/0048 Tomato Brown Rugose Fruit Virus (ToBRFV) Isothermal Amplification Detection Kit

Overview

The AGDIA XCS66800/0048 Tomato Brown Rugose Fruit Virus (ToBRFV) Isothermal Amplification Detection Kit is a CE-IVD marked, ready-to-use molecular diagnostic solution designed for the rapid, sensitive, and specific detection of ToBRFV in tomato and pepper plant tissues—including leaves, stems, fruits, and seed coats. Unlike conventional PCR-based assays requiring thermal cycling, this kit leverages loop-mediated isothermal amplification (LAMP) technology, enabling robust nucleic acid amplification at a constant temperature (60–65 °C) within 30–45 minutes. Engineered for field-deployable and laboratory-based plant health monitoring, it delivers high analytical sensitivity (detection limit ≤10 copies/µL RNA equivalent) with minimal sample preparation—no RNA extraction or purification is required. The assay targets a highly conserved region of the ToBRFV RNA-dependent RNA polymerase (RdRp) gene, ensuring cross-strain reactivity across globally reported isolates, including those from North America, Europe, the Middle East, and Asia.

Key Features

• Single-tube, lyophilized LAMP master mix—stable at room temperature (2–30 °C) for ≥12 months; no cold-chain logistics required
• Integrated visual readout via colorimetric pH indicator (phenol red): positive result = yellow-to-pink transition; negative = orange-red
• Optional real-time fluorescence detection using SYTO 9 or calcein-Mn²⁺—compatible with standard qPCR instruments and portable fluorimeters
• Validated for use with crude plant extracts prepared by simple grinding in supplied extraction buffer (≤60 sec per sample)
• No requirement for RNase inhibitors, reverse transcriptase, or separate RT step—the reaction mix includes thermostable reverse transcriptase and DNA polymerase
• Includes internal control primers to monitor extraction efficiency and detect PCR inhibition

Sample Compatibility & Compliance

This assay is validated for fresh, frozen, or lyophilized symptomatic and asymptomatic leaf, petiole, fruit, and seed tissue from Solanum lycopersicum and Capsicum annuum. It demonstrates no cross-reactivity with closely related tobamoviruses—including Tobacco mosaic virus (TMV), Tomato mosaic virus (ToMV), and Pepper mild mottle virus (PMMoV)—as confirmed per ISO 17025-accredited validation reports. The kit complies with EPPO PM 7/133 (2023) diagnostic protocol requirements for ToBRFV and meets EU Regulation (EU) 2016/2031 phytosanitary import criteria. All reagents are manufactured under ISO 13485-certified quality management systems, and batch-specific Certificates of Analysis (CoA) and Certificates of Conformance (CoC) are provided with each shipment.

Software & Data Management

While the core assay operates without instrumentation, quantitative interpretation of fluorescence kinetics is supported via AGDIA’s free desktop application, LAMPQuant v2.1, which enables Ct-value calculation, amplification curve normalization, and export of CSV/Excel-compatible datasets. For regulated environments, the software supports user access controls, electronic signature workflows, and audit-trail generation compliant with FDA 21 CFR Part 11 and EU Annex 11 requirements. Raw fluorescence data files (.lqd) retain full metadata (operator ID, date/time stamp, instrument serial number, calibration log), facilitating GLP/GMP traceability during phytosanitary certification or seed lot release testing.

Applications

• Routine screening of greenhouse-grown tomato transplants prior to distribution
• Border inspection and quarantine verification at ports of entry (e.g., USDA APHIS, EFSA-designated laboratories)
• Seed health testing for certified seed producers (aligned with ISTA 2023 ToBRFV testing guidelines)
• Surveillance in commercial fields during early symptom emergence or pre-harvest risk assessment
• Research applications including host resistance evaluation, virus titer quantification, and co-infection profiling with other Tobamovirus species

FAQ

Is RNA extraction necessary before running the assay?
No. The kit includes an optimized lysis-extraction buffer enabling direct amplification from homogenized plant tissue—eliminating RNA isolation steps and reducing hands-on time to under 5 minutes per sample.
Can this kit be used on dried seed samples?
Yes. Validation data confirm reliable detection in surface-rinsed or scarified tomato and pepper seeds following the included seed-specific protocol (EPPO PM 7/133 Annex B-compliant).
What instruments are required for fluorescence readout?
Any real-time thermocycler or portable fluorimeter capable of excitation at 490 nm and emission detection at 520 nm (e.g., Bio-Rad CFX96 Touch, Qiagen Rotor-Gene Q, or Biomeme M1 Sample Prep System).
Does the kit include positive and negative controls?
Yes—each kit contains lyophilized synthetic ToBRFV RNA positive control (1×10⁴ copies/µL) and nuclease-free water negative control, both pre-aliquoted and calibrated against NIST-traceable reference material.
How should results be interpreted if the internal control fails?
Failure of the internal control signal indicates insufficient template recovery or presence of inhibitors; such samples must be re-extracted using diluted homogenate or subjected to charcoal-based cleanup prior to retesting.