TH19989Y - A primer kit for the identification of becomovirus in cucurbit plants, and a method for identifying becomovirus using this primer kit. - Google Patents

Abstract

This invention develops a primer kit for the identification of begonia viruses in cucurbit plants and a method for identifying begonia viruses using the primer kit. The primer kit can identify three types of begonia viruses: SLCCNV, SLCuYV, and ToLCNDV, which are the three main types of begonia viruses found in cucurbit plants in Thailand. The primer kit for identifying begonia viruses in cucurbit plants consists of a forward primer (Beg-Cu-F), a first reverse primer (SLC1314-R), a second reverse primer (SYN955-R), and a third reverse primer (TOLC1524-R). This primer kit can be used to detect SLCCNV, SLCuYV, or ToLCNDV, individually or in combination, using PCR or multiplex-PCR methods. It can identify all three types simultaneously without cross-reaction. This method is compatible with DNA extracts from healthy plant samples and has a sensitivity for detecting all three pathogens equivalent to standard PCR methods. Multiplex PCR can detect plants from cultivated fields infected with SLCCNV, SLCuYV, or ToLCNDV, both in cases of single-type infection and simultaneous infection with more than one pathogen in various scenarios.

Claims (8)

1. The primer kit for identifying Begomovirus in cucurbit plants consists of: a forward primer (Beg-Cu-F) with the following nucleotide sequence: 5'- GAA CTA TGK CGA AGC GWC CA -3'; a first reverse primer (SLC1314-R) with the following nucleotide sequence: 5'- GTC AAG GTG CAG GTA ATG TTC-3'; a second reverse primer (SYN955-R) with the following nucleotide sequence: 5'- CTT CCC TGC TTC CTG GTG AT-3'; and a third reverse primer (TOLC1524-R) with the following nucleotide sequence: 5'- AAG AGC ATT CGC AGG AGG CGA G-3'. 2. A primer kit for the detection and identification of begonia virus in cucurbit plants, pursuant to claim 1, whereby the forward primers are specific to SLCCNV, SLCuYV, and ToLCNDV. 3. A primer kit for the identification of begonia virus in cucurbit plants, pursuant to claim 1, whereby the first reverse primer line is specific to SLCCNV. 4. A primer kit for the identification of begonia virus in cucurbit plants, pursuant to claim 1, which includes a reverse primer, second line, specific to SLCuYV. 5. A primer kit for the identification of begonia virus in cucurbit plants, pursuant to claim 1, which includes a third reverse primer line specific to ToLCNDV. 6. A primer kit for the identification of begonia virus in cucurbit plants, pursuant to claim Patent 1, which states that the primer kit can be used to detect one or more begonia viruses such as SLCCNV, SLCuYV, or ToLCNDV using PCR or multiplex-PCR methods. 7. The method for classifying begonia species using primers according to Patent 1 has the following components, mixture, and conditions for the multiplex PCR reaction: a. The multiplex PCR reaction mixture consists of a DNA polymerase buffer at a concentration of 1X and a magnesium chloride solution. At a concentration of 1.5 millimolar, deoxyribonucleotide triphosphate (dNTPs) containing dATP, dCTP, and dGTP at concentrations of 0.3 millimolar each, forward primer Beg-Cu-F at a concentration of 0.3 micromolar, reverse primer SLC1314-R at a concentration of 0.2 micromolar, reverse primer TOLC1524-R at a concentration of 0.2 micromolar, reverse primer SYN955-R at a concentration of 0.1 micromolar, DNA polymerase at a volume of 0.625 units, DNA extract of the sample to be investigated, 2 to 5 microliters, distilled water free of DNAs and RNAs to adjust the total reaction volume to 50 microliters. b. The conditions used in the multiplex PCR reaction include the following time and temperature: Initial reaction. Denaturation is performed at 95°C for 30 seconds. The polymerase chain reaction (PCR) consists of incubation at 95°C for 30 seconds, at 60°C for 50 seconds, and at 68°C or 72°C for 1 minute and 10 seconds, for a total of 30 cycles. The final DNA strand splitting reaction is performed at 68°C or 72°C for 5 minutes. 8. A method for identifying the species of Begonia virus, as per claim 7, where the samples used for testing can be selected from cucurbit plant extracts or DNA extracts from whiteflies.