CZ201361A3 - L1 reaction mixture for molecular detection of Potato Leaf Roll Virus (PLRV) using quantitative RT-PCR - Google Patents

Abstract

The present invention relates to a L1 reaction mixture for the molecular detection of potato coil virus (PLRV) by quantitative RT-PCR, which is characterized in that it contains 1 .mu.l of a sample of RNA tested, a reagent comprising a mixture of reverse transcriptase and DNA polymerase enzymes, a mixture of nucleotides, buffer containing magnesium chloride, MgCl.sub.2, sterile distilled water supplementing the reaction mixture to a final volume of 25 .mu.l primers with a specific nucleotide sequence of L-499F: AAG AAG TAT CCC TCA AAG CCT, L-675R: TTA GCG CGC CCT TGT AAA TCA AGC, 176 bp fragment, and L-567S probe: TG GAC GCT TGC GAG GTT GAT GAC TTT, the probe is labeled with fluorescent dye 6-carboxyfluorescein (FAM) and tetra is used as the quencher -methylcarboxyrhodamine (TAMRA).

Description

L1 reaction mixture for molecular detection of potato coil virus (PLRV) by quantitative RT-PCR

Field of technology

The solution relates to the L1 reaction mixture for the diagnosis of potato rot virus (PLRV) by quantitative RT-PCR, which is a serious quarantine of potato viroid disease, by molecular detection of viroid RNA by molecular reverse transcription - real-time polymerase chain reaction (QRT-PCR)

Prior art

The viral coil of potatoes is one of the serious viral diseases of potatoes, causing a reduction in yield of up to 80%. The coil virus is transmitted only by sucking insects and seedlings (Rasocha et al., 2007). The coil virus (PLRV) causes conical twisting of the leaves, first in the lower floor, later in the upper floors of the plants. The leaves are leathery, rustling on touch and cracking when pressed. Plants are lighter, chlorosis, usually of lower stature with shortened internodes. Tubers are usually smaller. The presence of potato leaf virus is mainly detected by laboratory methods, the longest and most often by immunoenzymatic ELISA (Clark and Adams, 1977).

The second procedure is the use of specific molecular probes. This test is based on the hybridization of an artificially prepared DNA probe (using genetic engineering methods) complementary to RNA viroid. PLRV binds to a solid support (nitrocellulose, nylon) and a DNA-RNA hybrid is detected. Because the PLRV cDNA is cloned, it can be obtained in sufficient quantities without great expense. This method is very sensitive, there is no need to inoculate intermediate hosts and lengthy RNA purification during sample preparation. The use of only partially purified juice from potato plants simplifies, speeds up and reduces the cost of sample preparation. Diagnostic DNA probes can be radioactively labeled (32P) _n ebo radioactively (biotin, digoxigenin). Hybridization detection techniques are now used in many variations not only for the detection of PLRV but also for other viruses.

The third option is procedures using RT-PCR and real-time RT-PCR techniques. The quantitative PCR method is based on determining the fluorescence of the PCR product in individual samples after each PCR cycle using fluorescent probes and determining the number of cycles required to reach the threshold fluorescence. The determination is performed on the basis of the measured changes in the fluorescence of the fluorescent probe. On the one hand, non-specific probes using SybrGreen I or fluorochrome-labeled probes specific for the amplified stretch of a given nucleic acid are used. There are several types of specific probes, but the most commonly used probes for quantification are linear hydrolysis probes, so-called TaqMan probes (Ptáček and Dědič, 2009).

Methods and procedures based on the detection of viral nucleic acid using specific primers for reverse transcription - polymerase chain reaction (RT-PCR) are much more sensitive than ELISA or hybridization methods and are currently more widely used. The quantitative RT-PCR method is one of the most sensitive diagnostic methods used and finds application in the detection of a wide range of potato pathogens. A method for the specific detection of PLRV using quantitative RT-PCR has not been proposed in the Czech Republic to date.

The essence of the invention

The above-mentioned shortcomings are eliminated by the reaction mixture L1 for molecular detection of potato coil virus (PLRV) by means of reverse transcription - real-time polymerase chain reaction (QRT-PCR), according to the invention. containing a mixture of reverse transcriptase and DNA polymerase enzymes, a mixture of nucleotides, a buffer containing magnesium chloride MgCl ₂ , sterile distilled water replenishing the reaction mixture to a final volume of 25 μΙ and primers with a specific nucleotide sequence

L-499F: AAG AAG TAT CCC TCA AAG CCT

L-675R: TTA GCG CGC CCT TGT AAA TCA AGC, amplifying 176 bp fragment, and probe

L-567S: TG GAC GCT TGC GAG GTT GAT GAC TTT, the probe is labeled with fluorescent dye 6-carboxyfluorescein (FAM) and tetramethylcarboxyrhodamine (TAMRA) is used as a quencher.

Primers and probes for PLRV detection by QRT-PCR (Ptáček and Dědič, 2009) were designed based on the sequence of the PLRV isolate (Plchová et al, 2009), numbers in the title indicate the location of primers and probes in the PLRV genome).

The L1 reaction mixture of the invention is prepared in a microtube and contains 1 μΙ of test RNA sample, any commercial reagent containing a mixture of reverse transcriptase and DNA polymerase enzymes, a mixture of oligonucleotides, MgCl ₂ magnesium chloride buffer, eg Brilliant II QRT-PCR 1-Step Master Mix, then sterile distilled water replenishing the reaction mixture to a final volume of 25 μΙ.

The actual QRT-PCR takes place in a real-time thermocycler in 0.2 ml PCR tubes (strips, plates) for 30 minutes at 48 ° C, followed by ten minutes of denaturation at 95 ° C and 40 cycles, containing 2 steps: 15 seconds at 95 ° C and 1 minute at 60 ° C.

The L1 reaction mixture according to the invention enables the exact determination of the potato borer virus (PLRV), which is of great importance in the production of seed potatoes in the Czech Republic.

The following examples merely illustrate the reaction mixture L1 according to the invention without limiting it in any way.

Exemplary embodiments

Example 1

The possibilities of its detection in 55 collection isolates of this virus were verified in RT-PCR and QRT-PCR procedures. Both total nucleic acid (NA) isolation methods and immunoassay (IC) techniques were used for self-testing. For RT-PCR, optimal results were obtained using primers according to Soliman, which detected all tested isolates. For the detection of PLRV by QRT-PCR, primer sets and TaqMan probes were designed based on sequence analysis of the Czech isolate (Plchová et al, 2009). Both non-specific labeling with SYBR Green and the use of a specific TaqMan probe were used to quantify viral NA amplificates in two-step QRT-PCR procedures. Detection of PLRV by QRT-PCR was possible and reliable in both DNA labeling procedures. The obtained results were compared with the ELISA method in terms of sensitivity and reliability.

Isolates of individual viruses from the collection of microorganisms (http://www.vurv.cz/collections/) maintained at VÚB Havlíčkův Brod, CZ, in vitro were used for our own experiments. A total of 59 samples were tested, of which 4 were negative controls.

Many different methods can be used to isolate total RNA, in terms of time consuming, quality of RNA obtained and reproducibility of the isolation process, it is optimal to use one of a wide range of commercial kits (Invitek, Roche, etc.). We recommend an RNA extraction procedure using the RNeasy Plant Total RNA Kit (QIAGEN).

QRT-PCR was performed in 0.2 ml PCR tubes (plates, strips), first 30 min at 48 ° C, then 10 min initial denaturation 95 ° C, 40 cycles (15 s 95 ° C, 60 s 60 ° C in real -time thermocycler, Brilliant II QRT-PCR Master Mix Kit, 1-Step, 600809 Stratagene, reaction volume 25 μΙ, 12.5 μΙ 2x master mix, 1.0 μΙ RT / RNase block enzyme (mixture of reverse transcriptase and RNasin), 0.1 μΙ primer L-499F 100 μΜ, 0.1 μΙ primer L-675R 100 μΜ, 1 μΙ probe 30 μΜ, 9.3 μΙ water, 1 μΙ RNA.

The QRT-PCR result was analyzed by measuring the dR fluorescence, as shown in FIG. 1, where QRT-PCR - example of amplification curves - PLRV detection.

Industrial applicability

The L1 reaction mixture for the diagnosis of potato blight virus (PLRV) by quantitative RT-PCR can be supplied to plant pathogen detection laboratories as a detection kit for simple immediate use, where the user only needs to add his test RNA and perform a QRT-PCR reaction according to precisely defined conditions.

List of authors' publications

PTÁČEK, J. - DĚDIČ, P. Detection and identification of potato coil virus (PLRV) isolates using molecular RT-PCR and QRT-PCR techniques, Scientific works - Potato Research Institute Havlíčkův Brod, 2009, 17, 25-35.

References

PEIMAN, M. - XIE, C. (2006): Sensitive detection of potato viruses, PVX, PLRV, and PVS in potato leaf and tuber. Australasian Plant Diseases Notes 1 (1): 41-46

PLCHOVÁ, H. - ČEŘOVSKÁ, N. - MORAVEC, T. - DĚDIČ P. (2009): Molecular analysis of Potato leafroll virus isolates from the Czech Republic. VIRUS GENES 39, 1, 153-155.

RASOCHA, V. - HAUSVATER, E. - DOLEŽAL, P. Mšice - significant carriers of viral diseases of potatoes, their occurrence, harmfulness and possibilities of protection, Potato Research Institute Havlíčkův Brod, s.r.o. 2007

Claims (1)

PATENT CLAIMS 1. L1 reaction mixture for molecular detection of potato coil virus (PLRV) by quantitative RT-PCR (QRT-PCR), characterized in that it contains 1 μΙ of a sample of the tested RNA, further a reagent comprising a mixture of reverse transcriptase enzymes and DNA polymerase, nucleotide mixture, buffer containing magnesium chloride MgCl ₂ , sterile distilled water replenishing the reaction mixture to a final volume of 25 μΙ and primers with a specific nucleotide sequence L-499F: AAG AAG TAT CCC TCA AAG CCT L-675R: TTA GCG CGC OCT TGT AAA TCA AGC, amplifying 176 bp fragment, and probe L-567S: TG GAC GCT TGC GAG GTT GAT GAC TTT, the probe is labeled with fluorescent dye 6-carboxyfluorescein (FAM) and tetramethylcarboxyrhodamine (TAMRA) is used as a quencher.