CZ25101U1 - L1 reaction mixture for molecular detection of potato leaf roll virus (PLRV) using quantitative RT-PCR - Google Patents

Description

Technical field

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

Background Art

Potato virus coil is a serious potato viral disease causing a yield reduction of up to 80%. The coil virus is transmitted only by absorbent insects and seedlings (Rasocha et al., 2007). The coil virus (PLRV) causes filamentous twisting of the leaves, first in the lower palate, later in the higher palate. The leaves are stiff, leathery when they touch, crack when crushed. Plants are lighter, chlorous, usually of lower stature with shorter habitats. Tubers are usually smaller. The presence of the potato coil virus is predominantly determined by laboratory methods 15, the longest and most often by the ELISA A method (Clark and Adams, 1977).

The second approach is to use specific molecular probes. This assay is based on hybridization of engineered DNA probe (using genetic engineering methods) complementary to viroid RNA. PLRV binds to a solid support (nitrocellulose, nylon) and a hybrid is detected

DNA-RNA. Since the PLRV cDNA is cloned, it can be obtained in large quantities without much expense. This method is very sensitive, there is no need to inoculate the intermediate host and to purify the RNA at length in the preparation of samples. The use of only partially purified potato juice simplifies, accelerates and reduces sample preparation. DNA diagnostic probes can be radiolabelled ( ³² P) or non-radioactive (biotin, digoxigenin). Hybridization detection techniques are now used in many variations not only for PLRV detection, but also for other viruses.

A third possibility is the use of RT-PCR and real-time RT-PCR techniques. The quantitative PCR method is based on the determination of the fluorescence of the PCR product in each sample after each PCR cycle using fluorescent probes and the number of cycles required to reach threshold fluorescence. The determination is made on the basis of measured fluoro s30 fluence probe changes. Both a non-specific probe using SybrGreen I or a fluorochrome labeled probe specific for the amplified region of a given nucleic acid is used. There are several types of specific probes, but the most commonly used probes for quantification are linear probes of the so-called TaqMan probe (Ptáček and Dědič, 2009).

Methods and procedures based on the detection of viral nucleic acid by specific reverse transcription-polymerase chain reaction (RT-PCR) primers are more sensitive than ELIS A or hybridization procedures and are more widely used today. 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. The method for the specific detection of PLRV by quantitative RT-PCR has not been proposed to date in the Czech Republic.

The essence of the technical solution

The above drawbacks are remedied by the L1 reaction mixture for the molecular detection of potato coil virus (PLRV) by reverse transcription of real-time polymerase chain reaction (QRT-PCR), according to a technique that contains a mixture of 1 µl of RNA sample to be tested, Reagent containing a mixture of reverse transcriptase and DNA polymerase enzymes, a mixture of nucleotides, magnesium chloride MgCl2 buffer, sterile distilled water supplementing the reaction mixture to a final volume of 25 μΐ, and primey of a specific nucleotide sequence

- 1 CZ 25101 Ul

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, wherein the probe is labeled with fluorescent dye 6-carboxyfluorescein (FAM) and tetramethylcarboxyrhodamine (TAMRA) is used as quencher.

Proteins and probes for the detection of PLRV by QRT-PCR (Ptáček and Dědič, 2009) were designed based on the PLRV isolate sequence (Plchová et al, 2009), numbers in the name indicate the location of primers 10 and the probe in the PLRV genome).

The LI reaction mixture according to the invention is prepared in a microtube and contains 1 µl of a sample of RNA tested, any commercial reagent containing a mixture of reverse transcriptase and DNA polymerase enzymes, a mixture of oligonucleotides, a magnesium chloride MgCl ₂ buffer, eg Brilliant II QRT-PCR 1-Step Master Mix followed by sterile distilled water supplementing the reaction mixture to a final volume of 25 μΐ.

Own QRT-PCR takes place in real-time thermocycler in 0.2 ml PCR tubes (strips, plates) for 30 min at 48 ° C, followed by 10 min denaturation at 95 ° C and 40 cycles containing 2 steps: 15 seconds at 95 ° C and 1 minute at 60 ° C.

The reaction mixture LI according to the technical solution enables exact determination of the potato coil virus (PLRV), which is of great importance in the production of seed potatoes in the Czech Republic.

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

Exemplary embodiments

Example 1

55 virus isolates of this virus were tested for their detection in RT-PCR and QRT-PCR procedures. Both self-isolation (NA) and immunobinding (IC) techniques were used for self-testing. L-RT-PCR results were obtained using Soliman primers to detect all isolates tested. To detect PLRV by QRT-PCR, primer sets and TaqMan probes were plotted on the basis of sequence analysis of the Czech isolate (Plchova et al, 2009). To visualize viral NA amplifications in two-step QRT-PCR procedures, both non-specific staining with SYBR Green was used, as well as using its own specific TaqMan probe. Detection of PLRV by QRT-PCR was possible and reliable in both DNA labeling procedures. The results were compared with ELISA in terms of sensitivity and reliability.

The isolates of individual viruses from the collection of microorganisms (http://www.vurv.cz/collections/) maintained in V LIB Havlíčkův Brod, CZ were used for in-house experiments. A total of 59 samples were tested, including 4 negative controls.

Many different techniques can be used to isolate total RNA, it is optimal to use one of a wide variety of commercial kits (Invitek, Roche, etc.) in terms of time consuming, RNA quality and reproducibility of the isolation process. We recommend the RNA extraction process using 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 of 95 ° C, 40 cycles (15 sec 95 ° C, 60 sec 60 ° C in real time thermocycler, Brilliant II QRT-PCR Master Mix Kit, 1-Step, 600809 Stratagene, reaction volume 25 μΐ, 12.5 μΙ

CZ 25101 Ul

2x master mix, 1.0 μΐ RT / RNase block enzyme (mixture of reverse transcriptase and RNasin), 0.1 μΐ adequate L-499 F 100 μΜ, 0.1 μΐ primer L-675R ΙΟΟμΜ, l μΐ probe 30μΜ, 9, 3 μΐ water, 1 μΐ RNA.

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

Industrial usability

The Ll Reaction Mixture for Potato Whirlwind Virus Detection (PLRV) by Quantitative RT-PCR can be supplied to plant pathogen detection laboratories as a ready-to-use detection kit, where the user is only required to add a sample of the tested RNA and perform a QRT-PCR reaction according to conditions.

io List of authors' publications

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

References

PEIMAN, Μ. - 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 cho20 robots, their occurrence, harmfulness and possibilities of protection, Potato Research Institute

Havlickuv Brod, s.r.o. 2007.

Claims (1)

PROTECTION REQUIREMENTS 1. A reaction mixture L1 for the molecular detection of Potato Scrub Virus (PLRV) by quantitative RT-PCR (QRT-PCR), characterized in that it contains a 1 μΐ sample of tested RNA, a reagent containing a mixture of reverse transcriptase enzymes and DNA polymerase; mixture of nucleotides, buffer containing magnesium chloride MgCl ₂ , sterile distilled water supplementing the reaction mixture to a final volume of 25 μΐ 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 30 amplifying a fragment of 176 bp, and a probe L-567S: TG GAC GCT TGC GAG GTT GAT GAC TTT, wherein the probe is labeled with fluorescent dye 6-carboxyfluorescein (FAM) and tetramethylcarboxyrhodamine (TAMRA) is used as the quencher.