RU2663719C1 - Method for determining genotoxicity of xenobiotics based on analysis of damage to mitochondrial dna of earth bumblebee (bombus terrestris) - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to biotechnology. Method for determining the genotoxicity of xenobiotics based on an analysis of damage to mitochondrial DNA of earth bumblebee (Bombus terrestris). Method comprises feeding ground bumblebees () with the tested xenobiotics throughout the day, sampling of biological material and isolation of DNA from pectoral muscles of the bumblebee, determination of the quantitative level of damage to mitochondrial DNA by real-time PCR at an elongation temperature of 66.1±0.5 °C for 5 minutes using Encyclo-polymerase corresponding to Encyclo-buffer and other standard reaction components with primer pairs having specific sequences, comparison of the quantitative level of damage to the mitochondrial DNA of the experimental and control groups. Higher level of damage in the experimental group is an indicator of the genotoxicity of xenobiotics. Sequences of primers for determining the amount of damage in mitochondrial DNAand the number of its copies:Present invention can be used in testing various xenobiotics, including pesticides, for genotoxicity.EFFECT: technical result consists in development of highly sensitive, highly reproducible and relatively inexpensive way to determine the genotoxicity of xenobiotics.1 cl, 2 dwg, 1 ex

Description

The present invention relates to microbiology and relates to measurement methods using microorganisms, namely nucleic acids. This invention can be used in testing various xenobiotics, including pesticides, for genotoxicity.

Chemical protection of agricultural plants involves the widespread use of pesticides to control weeds, pests, fungal diseases and other diseases of agricultural plants. Pesticides belong to the class of xenobiotics, compounds alien to the body, which cannot be included in the metabolism and cause serious harm to the body. The relevance of the problem of xenobiotic toxicity has risen sharply in recent years due to the crisis in the decrease in the number of pollinating insects. The problem of reducing pollinators is a food safety issue as they pollinate 75% of all crops eaten by humans (Potts, SG, Biesmeijer, J.C., Kremen, C., Neumann, P., Schweiger, O., & Kunin , WE (2010). Global pollinator declines: trends, impacts and drivers. Trends in ecology & evolution, 25 (6), 345-353).

Damage to the mitochondria of pollinating insects is one of the effects of pesticide toxicity. Even partial inhibition of mitochondrial respiration by pesticides, especially in the flight muscles, can lead to a decrease in the flying activity of pollinators, an increase in the rate of production of reactive oxygen species (ROS), which will lead to DNA damage, subsequent mutations and death of insects.

Mitochondrial DNA is a convenient marker of oxidative stress, which is why it is often used to identify the early stages of various diseases. The prototype is patent No. RF 2243558 (IPC G01N 33/48, C12Q 1/68, publ. 05/20/2000), which relates to a method for assessing an atherosclerotic state in a subject by mitochondrial DNA damage, comprising the following steps: (a) taking a blood sample from the specified subject; (b) determining the quantitative level of mitochondrial DNA damage in said blood sample by a quantitative PCR method, wherein said DNA is treated with FAPY glycosylase prior to said PCR amplification to determine 8-oxo-G damage, or quantifying the level of nuclear DNA damage by quantitative PCR for amplification of a product of 16.2 kbp .; (c) comparing the quantitative level of mitochondrial DNA damage in a blood sample of said subject with the quantitative level of mitochondrial DNA damage in a blood sample of a control subject not suffering from atherosclerosis, where a higher quantitative level of mitochondrial DNA damage in said subject with an increased risk than that of said control subject is an indicator of atherosclerosis in the specified subject.

However, this method is not applicable for the analysis of insect mtDNA damage, since in them, the average AT composition of mtDNA ranges from 69.5% to 84.9%, while in vertebrates it varies between 53% and 66% (Arunkumar, K.P., & Nagaraju, J. (2006). Unusually long palindromes are abundant in mitochondrial control regions of insects and nematodes. PLoS One, 1 (1), e110). The mitochondrial genome of B. terrestris is 17,400 bp in length. and AT composition of 85%.

The present invention is to develop a method for testing and identifying hazardous pesticides that can adversely affect pollinating insects, causing damage to mitochondrial DNA, which can adversely affect insect pollinating abilities or cause the death of a significant part of the population.

The technical result consists in the development of a highly sensitive, well reproducible and relatively inexpensive method for determining the genotoxicity of xenobiotics.

The technical result is achieved by the fact that in the method for determining the genotoxicity of xenobiotics based on the analysis of damage to mitochondrial DNA of bumblebee (Bombus terrestris), including the collection of biological material, DNA extraction, determination of the quantitative level of mitochondrial DNA damage by real-time PCR, comparison of the quantitative level of mitochondrial DNA damage experimental and control groups, where a higher level of damage in the experimental group is an indicator of gene flow xenobiotics, according to the invention, ground bumblebees (Bombus terrestris) are preliminarily fed with the tested xenobiotics throughout the day, DNA is isolated from the bumblebee’s pectoral muscles, PCR is carried out at an elongation temperature of 66.1 ± 0.5 ° C for 5 minutes using Encyclo polymerase corresponding Encyclo buffer and other standard components of the reaction with pairs of primers having specific sequences.

The primer sequences for determining the amount of damage in B. Terrestris mitochondrial DNA and the number of copies thereof are given in the Sequence Listing.

In FIG. 1 shows an electrophoregram of fragment amplification at an elongation temperature gradient, where M is a marker of fragment lengths of 1 Kb; 1 - 72 ° C; 2 - 71.1 ° C; 3 - 69.3 ° C; 4 - 66.1 ° C; 5 - 62.3 ° C; 6 - 59.2 ° C; 7 - 57.1 ° C; 8 - 56 ° C. In FIG. Figure 2 shows a diagram of the number of lesions in 1, 2, 3 fragments of B. terrestris mitochondrial DNA after exposure to rotenone (n = 6).

The PCR method of long fragments is based on the assumption that the presence of damage in DNA, for example, single-stranded breaks, modified bases or their adducts, will interfere with DNA polymerase and slow down the accumulation of PCR product. Thus, the accumulation rate of the PCR product will be inversely proportional to the number of damaged DNA molecules. The method is based on the selection of primers for the genome region of interest and its subsequent amplification in order to detect the number of DNA damage. However, due to the high AT composition of B. terrestris mitochondrial DNA, the possibilities for selecting a large number of primers were significantly limited. As a result, the developed kit includes three pairs of primers for amplification of fragments of mitochondril DNA about 2000 bp in length. directly to identify the amount of damage in the given amplicons (see the List of sequences). One pair of primers was also selected to amplify the reference short fragment necessary to determine the number of copies of mitochondrial DNA.

Due to the high AT composition of the bumblebee mtDNA amplified regions, the optimum elongation temperature is about 66 ° C. Higher elongation temperatures lead to an increase in the number of non-specific reaction products, presumably as a result of breaking the DNA chain. At lower temperatures, amplification of products of a given length does not occur (Fig. 1).

In addition, it is not recommended to use DNA isolation methods that use aggressive reagents (phenol, chloroform, etc.) that can further damage mitochondrial DNA.

Example.

The method for determining the amount of damage in mitochondrial DNA is as follows.

To initiate damage, a group of 6 bumblebees was fed with rotenone, which was added to sugar syrup (55-65%). It is slightly toxic to humans and mammals, but extremely toxic to insects. Used rotenone at the rate of 0.49 μg / bumblebee, which corresponds to half the dose of LD50, which does not cause death in bumblebees, but theoretically should cause damage to mitochondrial DNA. In parallel, a control group of bumblebees of the same age was kept under the same conditions with nutritious syrup without the addition of a xenobiotic.

Bumblebees were killed 24 hours after the start of the experiment.

Isolation of total DNA can be carried out by any commercially available method based on sorption columns.

When setting up long-chain real-time PCR, the following reaction mixture was used: 10X Encyclo buffer 2 μl; 50X Encyclo polymerase mixture 0.4 μl; a mixture of dNTP (10 mM each) 0.4 μl; 20X SYBR-green 1 μl; DNA template (3-10 ng / μl) 1 μl; primers (forward + reverse mixture) 1 μl; MilliQ water 14.2 μl. Reaction protocol: preliminary denaturation of 95 ° С for 3 min, denaturation at the beginning of the cycle - 95 ° С, 20 sec, annealing of primers - 54 ° С - 30 sec, elongation - 66.1 ° С - 5 min.

The calculation of the amount of mitochondrial DNA damage was performed according to the following formula:

Damage to

where Δcor = Cq of a short target fragment - Cq of a short control fragment, Δlength = Cq of a long target fragment - Cq of a long control fragment.

Mortality in the group of bumblebees treated with rotenone was absent. It was shown (Fig. 2) that rotenone in 24 hours causes significant damage to the mitochondrial DNA of B. terrestris. In 1 fragment, 1.06 ± 0.25 lesions / 10 kb were observed. In 2 fragment, 2.4 ± 0.26 lesions / 10 kb were detected. The greatest number of lesions was observed in 3 fragment, which corresponds to the D-loop (2.85 ± 0.09 lesions / 10 kb). Thus, this method can be used to detect mitochondrial DNA damage in xenobiotic concentrations that do not cause insect death, but can reduce their motor activity and, as a result, adversely affect pollination properties.

The proposed method for determining the amount of mitochondrial DNA damage of B. terrestris is highly sensitive, well reproducible and relatively inexpensive. The analysis can be carried out in laboratories that have a thermal cycler with a fluorescence detection system for real-time PNR, a centrifuge, an electrophoresis chamber and related reagents and consumables.

Sequence listing

Primers

Claims (2)

A method for determining the genotoxicity of xenobiotics based on the analysis of damage to mitochondrial DNA of bumblebee (Bombus terrestris), including the collection of biological material, DNA extraction, real-time PCR determination of the level of mitochondrial DNA damage by real-time PCR, comparison of the quantitative level of mitochondrial DNA damage of the experimental and control groups, where more a high level of damage in the experimental group is an indicator of xenobiotic genotoxicity, characterized in that feeding groundbum bumblebees (Bombus terrestris) is preliminarily carried out with a tested xenobiotic throughout the day, DNA is isolated from the bumblebee’s pectoral muscles, PCR is carried out at an elongation temperature of 66.1 ± 0.5 ° C for 5 minutes using Encyclo polymerase and its corresponding Encyclo- buffers with pairs of primers having the following sequences:

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