TCDVd Detection Kits

PDD-360

TCDVd Detection Kits

TCDVd End-Point RT-PCR Kit is designed for the detection of Tomato Chlorotic Dwarf Viroid (TCDVd) specific viroid RNA.

TCDVd End-Point RT-PCR Kit is designed for the detection of TCDVd-specific RNA based on the use of end-point RT-PCR technology. This kit is designed for research use only and not for
use in diagnostic procedures. The kit includes Master Mix and primers for the specific amplification of a 294 nucleotide region of the TCDVd genome, as well as a positive control and
negative control to confirm the integrity of the kit reagents. In addition, the kit contains a loading dye and a DNA ladder to facilitate the analysis of the results. The detection of TCDVd-specific RNA is based on end-point RT-PCR technology, utilizing Reverse transcription polymerase chain reaction (RT-PCR) for the amplification of specific TCDVd RNA sequences. For analysis of the RT-PCR data, the RT-PCR reaction is loaded on an agarose RNA gel along with the provided DNA ladder for qualitative analysis.

1. Before use, suitable amounts of all components should be completely thawed at room temperature, mixed by gentle vortexing or by pipetting, and centrifuged briefly.
2. Work quickly on ice.
3. The recommended minimum number of DNA samples tested per PCR run is 6.

Viroid

1. Follow universal precautions. All specimens should be considered as potentially infectious and handled accordingly.
2. Ensure that a suitable lab coat, disposable gloves, and protective goggles are worn when handling specimens and kit reagents.
3. Use sterile pipette tips with filters. Use proper pipetting techniques and maintain the same pipetting pattern throughout the procedure to ensure optimal and reproducible values.
4. Ensure that appropriate specimen collection, transport, storage, and processing techniques are followed for optimal performance of this test.

1. MDx 2X RT-PCR Master Mix 350 µL
2. TCDVd Primer Mix 70 µL
3. TCDVd Positive Control 50 µL
4. Nuclease-Free Water 1.25 mL
5. Loading Dye 100 µL
6. DNA Ladder 100 µL
7. Product Insert 1

1. Appropriate Real-Time PCR Instrument with FAM and HEX filter channel
2. DNA Purification Kit
3. Disposable powder-free gloves
4. Benchtop microcentrifuge
5. Micropipettors
6. Sterile pipette tips with filters
7. PCR tubes
8. Vortex mixer
9. PCR reaction preparation station (Optional)

Tomato Chlorotic Dwarf Viroid (TCDVd) is a viroid which causes disease in both field and green-house tomatoes. The viroid is a single-stranded RNA molecule consisting of 300 nucleotides, and is closely related to the Potato Spindle Tuber Viroid. Infected tomato plants show different symptoms depending on the tomato variety, age of the plant, plant vigour and climatic conditions. Some symptoms of TCDVd infection include stunting, overall bunchiness, reduced leaf and fruit, leaf chlorosis, downward bending of leaves and even death of plants. TCDVd has been detected on tomato plants in Europe, Canada and the United States. The detection of TCDVd infection by symptoms is challenging since many other viroids and viruses can produce similar symptoms. Therefore, detection using PCR is the most effective method available.

PCR

The specificity of TCDVd End-Point RT-PCR Kit is first and foremost ensured by the selection of the TCDVd-specific primers, as well as the selection of stringent reaction conditions. The TCDVd primers were checked for possible homologies to all plant viroids in GenBank published sequences by sequence comparison analysis.

All kit components should be stored at -20°C upon arrival. Repeated thawing and freezing (> 2 x) of the Master Mix and Positive Control should be avoided, as this may affect the performance of the assay. If the reagents are to be used only intermittently, they should be frozen in aliquots. All reagents can be stored for 1 year at -20°C without showing any reduction in performance.