RU2581026C2 - Synthetic oligodeoxyribonucleotides for amplification and detection of endogenous internal control in research of biological material of cattle by polymerase chain reaction in real time - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed is a kit containing two oligodeoxyribonucleotide primers and fluorescent-marked DNA-probe.

EFFECT: invention provides the effective amplification and detection of endogenous internal control in research of biological material of cattle by PCR test on a real-time basis and reduces unreliable result rate.

1 cl, 2 dwg, 3 tbl, 1 ex

Description

The invention relates to the field of biotechnology, molecular genetic diagnostics, molecular biology, in particular to a set of oligodeoxyribonucleotides for amplification and detection of a species-specific fragment of cattle genomic DNA (hereinafter - Bovine Cattle) by real-time polymerase chain reaction (PCR) containing two oligodeoxyribonucleotide primer (forward and reverse) and one fluorescently-labeled DNA probe.

The method of polymerase chain reaction (PCR) is widespread in studies of biological material of animals (cells, tissues, blood and other biological fluids) and is used in laboratory practice for genotyping, diagnosis of genetic pathologies, establishing biological affinity, identifying the genetic material of infectious disease pathogens, etc. The essence of the method is the multiple copying (amplification) of a specific DNA fragment in the test sample of biological material using the term stable DNA polymerase and a pair of oligodeoxyribonucleotide primers to an amount sufficient for its detection by any of the known methods - electrophoretic, hybridization-enzyme or hybridization-fluorescence (Rebrikov D.V., Samatov G.A., Trofimov D.Yu. and others. PCR in "real time"; 2nd edition - M: BINOM. Laboratory of knowledge, 2009. - 223 p.). In order to detect the accumulation of amplification products in real time, in addition to a pair of primers, a fluorescently-labeled DNA probe is also required. The PCR method is highly sensitive and specific and allows you to detect the target DNA fragment, even if it is presented in a single copy. Other advantages of the method are the possibility of quantitative measurements and low time spent on analysis.

There are a number of problems in the study of the biological material of cattle by PCR, affecting the obtaining of reliable results, including those arising during the laboratory formulation of PCR, which consists in the following. A test mixture containing the main components such as a buffer solution, a mixture of deoxynucleotide triphosphates (dNTPs), oligodeoxyribonucleotide primers, fluorescent DNA probes, DNA polymerase is introduced into a sample of the studied biological material, and an amplification reaction is carried out (DNA-Technology. Fundamentals of polymerase chain reaction. Methodical manual. Moscow, 2012, pp. 10-12. Website: http://www.dna-technology.ru/files/images/metodichki/OsnoviPCR.pdf, 2015). If the desired DNA is present in the sample of the analyzed sample, then during the amplification reaction it is denatured (DNA is transferred from the double-stranded form to single-stranded when hydrogen bonds between complementary base pairs are broken under high temperatures), annealing (oligodeoxyribonucleotide primers are attached to single-stranded target DNA) and elongation (after annealing of the primers, DNA polymerase starts the synthesis of the second DNA chain from the 3′-end of the primer). Throughout the reaction, the results of PCR are recorded in real time using the hybridization-fluorescence method using modern technological means that simplified the measurement technique and made a more accurate approach to assessing PCR results.

However, in the sample of the studied sample of biological material in which the DNA must be a priori, the recorded PCR results may indicate the fact of its absence due to a number of "technical" reasons:

- DNA loss during sample preparation due to non-compliance with the instructions for DNA extraction or the use of low-quality reagents;

- DNA destruction due to non-compliance with the rules for storage and / or transportation of samples;

- contamination of the sample with impurities that inhibit PCR (for example, such strong inhibitors as hemoglobin, heparin).

Thus, in PCR technology, one of the important factors affecting the accuracy and reliability of the results is the quality of technical performance by laboratory personnel of all stages of the study, including the collection of biological material and its preparation for PCR (Medvedeva T.V., Skvortsova R.G. ., Kuzmenko VV, Ogarkov OB PCR analysis in the clinical laboratory. Textbook. Irkutsk. 2009. INTERNET website: https: //www.google.ru/url? Sa = t & rct = ^: j & q = & esrc = s & source = web & cd = 28 & ved = 0CEk QFjAHOBQ & url = http% 3A% 2F% 2Fasld.baikal.ru% 2Fasld% 2Fdocs% 2FObzor% 2F11% 2F1.doc & ei = xBwuVe7dHMrjywOBoIGYCA & usg = AFQjCNEY4AcJAOzCysgusR78hNEFnxg8Cw & bv m = bv.90790515, d.bGQ & cad = rjt, twenty 15 g.).

There is a generally accepted approach in which, in the course of laboratory PCR, positive and negative, as well as internal amplification controls (the so-called internal control samples) are used to reduce the number of unreliable results. At the same time, the internal control of amplification is a specific DNA sample that passes through all stages of the tests, as well as a sample of the studied biological material. Real-time PCR technology allows you to register signals from the desired DNA and signals from the internal amplification control in a single tube containing the biological material under study. Evaluation of the results of the study of biological material by PCR is carried out taking into account the results of the analysis of positive and negative controls, as well as taking into account the internal control of amplification (internal control sample).

It is also known that internal control samples are divided into two types: endogenous and exogenous (“Real-time PCR. 2nd edition, revised. Edited by Dr. D.V. Rebrikov. - M: Binom. Laboratory of Knowledge. 2009, pp. 160-161). An exogenous internal sample is added to the sample of the test biological material immediately before the start of the study. An endogenous internal sample, in contrast to an exogenous one, is initially present in the sample of the biological material under study, being part of the genomic DNA of the animal being studied. Hereinafter, the internal control sample of the endogenous type is referred to as “endogenous internal control”.

The endogenous internal control (hereinafter - EVK) during PCR is a specific fragment of the genomic DNA of the studied animal, amplified with oligodeoxyribonucleotides and detected with a fluorescently labeled DNA probe. EVC allows one to judge the effectiveness of PCR, namely, the accumulation of a DNA fragment of endogenous internal control during PCR will be evidence of a normal amplification reaction. The use of EVCs makes it possible to exclude the receipt of false negative research results due to the absence or insufficient amount of DNA for detection in the sample taken for analysis, improper preparation of the reaction mixture, the presence of reaction inhibitors, or inadequate quality of the reagents used.

The difficulty in choosing oligodeoxyribonucleotides used for the amplification and detection of endogenous internal control is that they must have strict species specificity. In the absence of species specificity in the primers and fluorescently-labeled DNA probe used to amplify and detect endogenous internal control, false negative results of the study are possible. If, however, the selected oligodeoxyribonucleotide primers and DNA probe have an affinity for non-target DNA sequences, then false-positive results of the study are possible. The correct choice of a combination of a pair of primers and a fluorescently labeled DNA probe allows for strictly species-specific amplification and detection of the target DNA fragment by real-time PCR.

Known endogenous internal control, which can be taken as a prototype, used to reduce the percentage of erroneous PCR results in the Leukemia test system for detecting cattle leukemia virus by polymerase chain reaction (http://www.interlabservice.ru/upload/ iblock / 195 / Leykoz.MANUAL.150413.pdf, 2015). As such control, a cattle α-actin gene fragment is used with the presence of a specific band on the electrophoregram at the level of 582 bp

The disadvantage of using a bovine α-actin gene fragment as an endogenous internal control is that it is not species-specific for cattle, as it is also found in other animal species and has a high level of structural similarity (more than 90% identity) in different vertebrate species. This reduces the reliability of its use for internal PCR control in the study of biological material for the detection of cattle leukemia virus, since it does not provide sufficient grounds to assert that it is a fragment of cattle DNA that is amplified in the test sample.

The objective of the invention is the creation of a set of synthetic oligodeoxyribonucleotides for amplification and detection of endogenous internal control, which could be used in any study of biological material of cattle by polymerase chain reaction in real time.

The technical result of the claimed invention is the expansion of the spectrum of oligodeoxyribonucleotides, allowing reliable amplification and detection of endogenous internal control in the study of biological material of cattle by polymerase chain reaction in real time, and reducing the frequency of false PCR results.

This result is achieved by the development of a set of synthetic oligodeoxyribonucleotides for amplification and detection of endogenous internal control in the study of biological material of cattle by polymerase chain reaction in real time, containing two oligodeoxyribonucleotide primers and a fluorescently-labeled DNA probe, which have the following structure:

Direct primer 5 ′ → 3 ′

Reverse Primer 5 ′ → 3 ′

Fluorescently-labeled DNA probe 5 ′ → 3 ′

Within the framework of a patented technical solution, the developed oligodeoxyribonucleotides are species-specific for cattle and can reliably identify cattle DNA, while a real-time polymerase chain reaction fragment is used as an endogenous internal control in cattle biological material by real-time polymerase chain reaction, which encodes a species-specific cattle retrotransposon L1-2_BT.

Testing of oligodeoxyribonucleotides was carried out using fifteen DNA samples isolated from whole blood of cows of the Kalmyk, Ayrshire breeds and black-and-white holsteinized cattle. It was experimentally shown that the selected oligodeoxyribonucleotides provide reliable synthesis and detection of a cattle DNA fragment with a length of 96 nucleotide pairs.

Characterization of a set of oligodeoxyribonucleotides and a site of amplified cattle DNA

 Oligodeoxyribonucleotide primers proposed for patenting flank a section of the cattle genome with a length of 96 pairs of nucleotides encoding the L1-2_BT retrotransposon sequence. The use of a fluorescently-labeled DNA probe in a kit with patented oligodeoxyribonucleotide primers allows the detection of accumulation of the amplification product (endogenous internal control) in real-time mode.

It is important to note that the optimization of PCR conditions was carried out using sets of domestic commercially available reagents and enzymes intended for mass use in laboratory practice, which allows you to quickly and reliably apply this invention in veterinary and research laboratories.

List of graphic materials

 Figure 1 presents the electrophoregram of the amplification products of genomic DNA (endogenous internal control) cattle of the Kalmyk breed (1-5), Ayrshire breed (6-10) and black-and-white holsteinized cattle (11-15) using patented oligodeoxyribonucleotides. “K-” is a negative control. M - marker of DNA lengths of 100-1000 bp (MWM-100RL, Dialat Ltd.).

In FIG. 2. presents graphs of the accumulation of PCR products (endogenous internal control) in real-time mode when analyzing 15 genomic DNA samples from Kalmyk, Ayrshire breeds and black-and-white holsteinized cattle using patented oligodeoxyribonucleotides. “K-” is a negative control. The abscissa shows the number of PCR cycles, the ordinate shows the fluorescence intensity.

The technique of constructing a set of oligodeoxyribonucleotides. The selection of synthetic oligodeoxyribonucleotides for amplification and detection of endogenous internal control in the study of biological material of cattle by polymerase chain reaction in the "real time" mode was carried out on the basis of the L1-2_BT retrotransposon sequence obtained by us by sequencing DNA fragments with a length of about 500 nucleotide pairs in cows black and motley holsteinized cattle (Glazko V.I., Kosovsky G.Yu., Kovalchuk S.N., Arkhipov A.V., Petrova I.O., Dedovich G.O., Glazko TT Inverted repeat of microsatellite (AGQ6G flanks DNA regions with regions of homology to retrotransposons in the cattle genome // Innovative technologies in medicine - 2014. - 2 (03). - P. 63-79). Selection of the fragment of retrotransposon L1- 2_BT as an endogenous internal control in studies of the biological material of cattle by real-time PCR due to its species-specificity for cattle (Jurka, J. L1 family from cow. Repbase Reports, 2009, V. 9 (2), P. 598-59; Adelson D.L., Raison J.M., Edgar R.C. Characterization and distribution of retrotransposons and simple sequence repeats in the bovine genome. Proc Natl Acad Sci USA, 2009, V. 106 (31), P. 2855-2860).

Using the BLAST server (http://blast.ncbi.nlm.nih.gov/Blast.cgi), genome regions were found in the international GenBank database (http://www.ncbi.nlm.nih.gov/genbank/) Cattle Bos taurus Hereford Btau_4.6.1 and Bos taurus UMD 3.1, highly homologous to the sequenced fragment of the retrotransposon L1-2_BT. Using the ClustalW2 server (http://www.ebi.ac.uk/Tools/msa/clustalw2/), the obtained retrotransposon L1-2_BT sequences were analyzed and their conserved regions were found, on the basis of which oligodeoxyribonucleotide sequences were selected.

The choice of oligodeoxyribonucleotides was based on the following criteria: length of 18-24 nucleotide pairs; the annealing temperature of oligodeoxyribonucleotide primers is similar and is in the range of 60-70 ° C; the temperature of the annealing of the DNA probe is higher than the annealing temperature of oligodeoxyribonucleotide primers; the absence in the sequence of oligodeoxyribonucleotide of secondary structures with a melting point above its annealing temperature; lack of self-and complementarity between the 3′-ends of the selected oligonucleotides (Rebrikov D. V., Samatov G. A., Trofimov D. Yu. et al. PCR in “real time”; 2nd edition - M .: BINOM. Laboratory of knowledge, 2009. - 27 p.).

As a result of studying the structure of the retrotransposon L1-2_BT of the cattle genome, oligodeoxyribonucleotide primers and a fluorescently-labeled DNA probe for real-time polymerase chain reaction were calculated and synthesized (Table 1).

Cattle DNA extraction. DNA was extracted from the whole blood of fifteen cows of the Kalmyk, Ayrshire breeds and black-and-white Holstein cattle using the Magna ™ DNA Prep 200 reagent kit (Izogen Laboratory LLC, Russia) according to the manufacturer's protocol.

Real-time polymerase chain reaction

The amplification reaction was carried out in 20 μl of a PCR mixture (Table 2) containing 1 × Taq amplification buffer, 3 mm MgCl ₂ , 0.2 mm each nucleoside triphosphate, 1 active units of HS Taq DNA polymerase, 0.2 μM forward and reverse oligodeoxyribonucleotide primer , 0.1 μM oligodeoxyribonucleotide fluorescent probe (all reagents manufactured by CJSC Evrogen, Russia).

Real-time PCR was performed according to the amplification program indicated in the table. 3. Detection of fluorescence intensity was carried out on the ROX channel on a LightCycler 98 instrument (Roche, Switzerland). The results were evaluated by the presence of a reaction product with a length of 96 nucleotide pairs (Fig. 1) and an exponential increase in fluorescence (Fig. 2).

Testing of patented oligodeoxyribonucleotides

 Patented oligodeoxyribonucleotides were tested on 15 DNA samples from Kalmyk and Ayrshire cows and black-and-white holsteinized cattle. It was experimentally shown that the selected oligodeoxyribonucleotides provide synthesis and detection of the target cattle DNA fragment with a length of 96 nucleotide pairs (Fig. 1, 2). In FIG. Figure 1 shows an electrophoregram of amplification products of cattle genomic DNA from Kalmyk breed (1-5), Ayrshire breed (6-10) and black-and-white holsteinized cattle (11-15) using patented oligodeoxyribonucleotides. As a result of PCR using patented oligodeoxyribonucleotides in all the studied samples, an amplification product of 96 bp was detected. In FIG. Figure 2 shows real-time PCR product storage graphs using patented oligodeoxyribonucleotides. The presence of a 96 bp amplification product and the exponential increase in fluorescence in all the samples studied serves as an endogenous internal control and is an indicator that: 1) the reaction mixture is correctly composed; 2) there are no PCR inhibitors in the reaction mixture; 3) cattle DNA is present in samples taken in PCR.

Thus, the claimed set of synthetic oligodeoxyribonucleotides provides amplification and detection of a fragment of genomic DNA with a length of 96 pairs of nucleotides, which can serve as an endogenous internal control when examining biological material of cattle by polymerase chain reaction in real time.

Claims (1)

A set of synthetic oligodeoxyribonucleotides for amplification and detection of endogenous internal control in the study of biological material of cattle by polymerase chain reaction in real time, containing two oligodeoxyribonucleotide primers and a fluorescently-labeled DNA probe, which have the following structure:
direct primer 5 ′ → 3 ′
F: 5′-ACTCCCAGCAGAGAACTAGC - 3 ′
reverse primer 5 ′ → 3 ′
R: 5'-AGTCCTTACCCTAAACAATGC-3 ′
5 ′ → 3 ′ fluorescently-labeled probe
Probe: 5'-ROX-CTCGCGTCCGCCACCAGCAAG-BHQ2.

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