Kit and method for detecting copy number of mtDNA in drosophila melanogaster

Abstract

The invention discloses a kit and method for detecting the copy number of mtDNA in drosophila melanogaster, the kit comprises a drosophila melanogaster genome DNA extraction reagent and a drosophila melanogaster mtDNA amplification primer, high-purity genome DNA can be efficiently extracted from trace drosophila melanogaster tissue, the ratio of A260 / A280 to A260 / A230 is close to 2.0, and the downstream molecular experiment requirement is met. Meanwhile, a pair of qPCR primers for specifically amplifying a drosophila melanogaster mtDNA ND5 gene region is designed and screened out, and experiments prove that the amplification efficiency of the primer pair is as high as 98.4%, the linear relation is excellent (R is equal to 0.9995), the sensitivity is high, no primer dimer and non-specific amplification exist, and the performance is remarkably superior to that of other contrast primers. Therefore, the kit can be used for detecting the copy number of the mtDNA in the drosophila melanogaster, and an efficient, sensitive and reliable special tool is provided for researching the mitochondrial function of the drosophila melanogaster and related diseases.

Description

Technical Field

[0001] This invention relates to the field of detection, specifically to a kit for detecting mtDNA copy number in Drosophila melanogaster, and also to a detection method using the kit. Background Technology

[0002] Mitochondrial DNA (mtDNA) is a small, circular, double-stranded DNA molecule found in the mitochondria of eukaryotic cells, possessing unique genetic characteristics. The mtDNA of the fruit fly *Drosophila melanogaster* is approximately 19.5 kb in length, containing 37 genes encoding 13 key proteins involved in oxidative phosphorylation, 22 tRNAs, and 2 rRNAs.

[0003] Compared with the nuclear genome, mtDNA has the following characteristics: (1) a strict maternal inheritance pattern, which facilitates pedigree tracing; (2) a lack of introns and effective repair mechanisms, resulting in a significantly higher mutation rate than nuclear DNA; and (3) multiple copy characteristics, with each cell containing hundreds to thousands of copies. These characteristics make mtDNA an important molecular marker for studying energy metabolism, aging, disease, and evolution.

[0004] The "mitochondrial aging hypothesis" posits that the accumulation of mtDNA mutations is a key driver of aging. Fruit flies, with their short lifespan, are ideal models for studying aging. Detecting mtDNA mutations and copy number changes in fruit flies allows for direct assessment of the aging process and the effectiveness of interventions such as drugs and diet. Parkinson's disease and Alzheimer's disease have been found to be closely related to mitochondrial dysfunction; therefore, fruit fly models can be constructed and their pathogenic mechanisms studied by detecting specific mtDNA mutations or deletions. Furthermore, mtDNA abnormalities also affect energy metabolism and are associated with metabolic syndromes such as obesity and diabetes; therefore, detecting mtDNA copy number can also be used to study metabolic diseases.

[0005] Drosophila and humans share a high degree of conservation in genes and cell signaling pathways; approximately 75% of human disease-related genes have homologous genes in Drosophila. This makes Drosophila useful for disease research, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. Drosophila can simulate pathological processes such as protein aggregation and neuronal death. Therefore, there is an urgent need for a kit that can rapidly extract genomic DNA from Drosophila and determine the mtDNA copy number. Rapid and accurate quantitative analysis of mtDNA in Drosophila is of great significance for mitochondrial research, and the Drosophila model can serve as a "preclinical bridge," accelerating the translation from basic research to clinical applications. Summary of the Invention

[0006] In view of this, one objective of the present invention is to provide a kit for detecting the mtDNA copy number in Drosophila melanogaster; a second objective of the present invention is to provide a method for detecting the mtDNA copy number in Drosophila melanogaster for non-diagnostic purposes; and a third objective of the present invention is to provide the use of the kit in preparing products for detecting the relative copy number of mitochondrial DNA in Drosophila melanogaster.

[0007] To achieve the above objectives, the present invention provides the following technical solution: 1. A kit for detecting mtDNA copy number in Drosophila melanogaster, the kit comprising Drosophila genomic DNA extraction reagent and Drosophila mtDNA amplification primers; The Drosophila genomic DNA extraction reagent includes Buffer 1, Buffer 2, and Washing Buffer; each 25 ml of Buffer 1 contains 25 mL of 1 mol / L, pH 8.0 Tris·HCl, 250 μL of 0.5 mol / L, pH 8.0 EDTA, 250 μL of 5 mol / L NaCl, 500 μL of 10% SDS, 250 μL of β-mercaptoethanol, 25 ml of 20 mg / mL proteinase K, and the remainder is water; Each 25ml of Buffer 1 contains 12.5ml of anhydrous ethanol, 250μL of 5mol / L NaCl, 1mol / L pH8.0 Tris.HCl, and the remainder is water; The Washing Buffer consists of 150 ml of anhydrous ethanol, 2 ml of 1 mol / L pH 8.0 Tris-HCl, and 2 ml of 5 mol / L NaCl per 200 ml. The Drosophila mtDNA amplification primers are shown in SEQ ID NO.7 and SEQ ID NO.8.

[0008] 2. A method for detecting the mtDNA copy number of Drosophila melanogaster for non-diagnostic purposes, comprising the following steps: a) Extract genomic DNA from Drosophila melanogaster using the Drosophila genomic DNA extraction reagent of claim 1; b) Using the DNA extracted in step a as a template, amplify the Drosophila mtDNA using the described primers via real-time quantitative PCR; c) Calculate the relative copy number of mtDNA based on the Ct value obtained in step b.

[0009] In the preferred embodiment of the present invention, the qPCR reaction system in step b is as follows: 5 μL of 2×SYBR Green Pro Taq HS Premix, 0.25 μL of 40×Dilution Buffer, 0.5 μL of 10 μM forward primer, 0.5 μL of 10 μM reverse primer, 1 μL of template DNA, and dd H2O to a final volume of 10 μL.

[0010] In a preferred embodiment of the present invention, the qPCR reaction program in step b is as follows: 95°C for 3 minutes; then 40 cycles of 95°C for 10 seconds and 60°C for 15 seconds; finally, melting curve analysis is performed.

[0011] 3. The use of the kit in the preparation of a product for detecting the relative copy number of mitochondrial DNA in Drosophila melanogaster.

[0012] The beneficial effects of the present invention are as follows: The present invention provides a kit for detecting Drosophila mtDNA, including a kit for rapid extraction of Drosophila genomic DNA and primers for detecting mtDNA in Drosophila melanogaster.

[0013] The genomic DNA extraction kit of this invention is more suitable for extraction from Drosophila than conventional genomic DNA extraction kits. Drosophila are covered with a chitinous exoskeleton, and larvae / pupae also contain chitin. Conventional genomic DNA extraction kits are difficult to use for complete lysis. Therefore, this kit increases the concentration of SDS and adds β-mercaptoethanol to Buffer 1 specifically for Drosophila tissue. SDS functions to dissolve cell and nuclear membranes, while β-mercaptoethanol can disrupt protein disulfide bonds and inhibit certain oxidases. Therefore, increasing the concentration of SDS and adding β-mercaptoethanol allows for more complete lysis of Drosophila tissue, resulting in higher extraction efficiency.

[0014] The specific qPCR-mtDNA amplification primers ND5-F and ND5-R designed in this invention have a standard curve of y=3.36x+13.74, an amplification efficiency of 98.4%, and an R² of 0.9995, indicating that the primers have excellent amplification efficiency and perfect linearity. Compared with other qPCR-mtDNA primers, they have higher specificity and sensitivity, and can more accurately detect the relative copy number of mtDNA. Attached Figure Description

[0015] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration: Figure 1 Agarose gel electrophoresis images of PCR amplification products (A: TransTaq High Fidelity; B: TransStartFastPfu DNA Polymerase). Figure 2 Agarose gel electrophoresis of amplified samples at different template concentrations; Figure 3 Standard curves for each primer group (A: Standard curve for primer ND5; B: Standard curve for primer mt1; C: Standard curve for primer mt2; Standard curve for primer mt4). Figure 4 This kit was used to detect Polg1RNAi and mtDNA in a Drosophila colon cancer model. Detailed Implementation

[0016] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0017] The reagents and raw materials used in this invention are as follows: 1 mol / L Tris.HCl (pH 8.0) (Sangon Biotech, China), 0.5 mol / L EDTA (pH 8.0) (Sangon Biotech, China), 5 mol / L NaCl (Sangon Biotech, China), 10% SDS (Sangon Biotech, China), 20 mg / ml Proteinase K (Tiangen Biotech, China), Anhydrous Ethanol (Kolon Biotech, China), SpinColumn (Tiangen Biotech, China), SYBR Green Pro Taq HS premixed qPCR kit (including tracer dye) (Aikerui Biotechnology, China), high-purity low-electroosmotic agarose (Qingke Biotechnology, China), nucleic acid dye (Lingda Biotechnology, China), 2× TransTaq High Fidelity (HiFi) PCR SuperMix I (China Tiangen Biotech). TransStart FastPfu DNA Polymerase (China Tiangen Biotech). Trans 2K Plus DNA Marker (China TransGen Biotech), β-mercaptoethanol (China Solarbio).

[0018] Example 1: Extraction of genomic DNA from Drosophila melanogaster (1) Preparation of Drosophila genomic DNA extraction reagent Preparation of Buffer 1: 2.5 mL of 1 mol / L Tris·HCl (pH 8.0); 250 μL of 0.5 mol / L EDTA (pH 8.0); 250 μL of 5 mol / L NaCl; 500 μL of 10% SDS; 250 μL of β-mercaptoethanol; 25 mL of 20 mg / mL proteinase K; and bring the volume to 25 mL with dd H2O.

[0019] Preparation of Buffer 2: 12.5 mL anhydrous ethanol; 250 μL 5 mol / L NaCl; 500 μL 1 mol / L Tris·HCl (pH 8.0); bring the volume to 25 mL with dd H2O.

[0020] Preparation of Washing Buffer: 150 mL anhydrous ethanol; 2 mL 1 mol / L Tris·HCl (pH 8.0); 2 mL 5 mol / L NaCl; and dd H2O to a final volume of 200 mL.

[0021] Construct a Drosophila genomic DNA extraction kit using the configured Buffer1, Buffer2, and Washing Buffer.

[0022] Compared to other kits, the composition of Buffer 1 in this kit is more conducive to the extraction of Drosophila tissue. In genomic DNA extraction, SDS (sodium dodecyl sulfate) is a strong anionic detergent that can disrupt the lipid bilayer of cell membranes, nuclear membranes, and organelle membranes, releasing cellular contents. Conventional kits use SDS concentrations of 0.5%-1%. Because Drosophila tissue, compared to other common tissues such as blood and cells, is covered by a chitinous exoskeleton, and larvae / pupae also contain chitin, this kit increases the SDS concentration to 2%. Simultaneously, β-mercaptoethanol is added to Buffer 1. β-mercaptoethanol is a reducing agent that can break the disulfide bonds between proteins in chitin, softening the chitin-protein complex and making it easier to physically break down. Therefore, increasing the SDS concentration and adding β-mercaptoethanol allows for more complete lysis of Drosophila tissue, resulting in higher extraction efficiency.

[0023] (2) Genomic DNA was extracted from fruit flies using a fruit fly genomic DNA extraction kit. The specific steps are as follows: ① Take 8 males MHC-GAL4 × W 1118 Fruit flies and 8 males MHC-GAL4 × UAS-Polg1RNAiAdd the thoracic segments of Drosophila to 200 μL Buffer1, grind the Drosophila tissue thoroughly with a grinding rod, vortex until completely suspended, and place in a 65℃ metal bath for 30 min; ② Add 200 μL Buffer2 and mix by inverting the container 6-8 times; ③ Centrifuge at 12000 rpm for 5 min, add the supernatant obtained from centrifugation to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and put the adsorption column back into the collection tube; ④ Add 600 μL Washing Buffer, centrifuge at 12000 rpm for 1 min, and discard the waste liquid; ⑤ Repeat ④; ⑥ Place the adsorption column back into the collection tube, centrifuge at 12000 rpm for 2 min, and discard the waste liquid; ⑦ Transfer the adsorption column to a new EP tube, open the cap and let it air dry for 2-5 minutes; ⑧ Add 50-100 μl of dd H2O to the center of the adsorption membrane, let stand at room temperature for 2-5 min, and centrifuge at 12000 rpm for 2 min; ⑨ To improve the yield of genomic DNA extraction, the solution obtained from centrifugation in step ⑧ can be added back to the center of the adsorption membrane and centrifuged at 12,000 rpm for 2 min to obtain Drosophila genomic DNA.

[0024] The concentration and purity of DNA are shown in Table 1.

[0025] Table 1. Concentration and purity of Drosophila genomic DNA extracted. As can be seen from Table 1, the A260 / A280 and A260 / A230 of each group of genomic DNA are all around 2.0, indicating that the DNA extracted by the genomic DNA extraction kit of this invention has high purity and meets the requirements for PCR amplification.

[0026] Example 2: Primer Sequence Design and Detection (1) Primer design Download the Drosophila melanogaster mtDNA map from the NCBI website. Based on the qPCR primer design principles: ① target amplicon length controlled between 80-200 bp; ② primer length 18-25 bp; ③ Tm value between 58-62°C; ④ guanine-cytosine (GC) content maintained between 40-60%, four pairs of qPCR-mtDNA primers were designed and synthesized by Qingke Biotechnology Co., Ltd. The specific sequences are shown in Table 2.

[0027] Table 2. qPCR-mtDNA primer sequences (2) Detection of primers PCR amplification and agarose gel electrophoresis: The agarose gel extracted in Example 1 was then subjected to PCR amplification and agarose gel electrophoresis. MHC-GAL4 ×W 1118 -1 Genomic DNA was used as a template for the PCR reaction, using two DNA polymerases: TransStartFastPfu DNA Polymerase TransTaq HighFidelity and four sets of qPCR-mtDNA primers: mt1-F / mt1-R, mt2-F / mt2-F, mt3-F / mt3-R, and ND5-F / ND5-R were used for PCR amplification. TransTaq The high Fidelity reaction system is as follows: Template 1 μL; Forward Primer (10 μM) 1 μL, Reverse Primer (10 μM) 1 μL, 10× TransTaq HiFi Buffer Ⅰ 5 μL; 2.5 mM dNTPs 4μL; TransTaq HiFi DNA Polymerase 1 μL; dd H2O up to 50 μL.

[0028] The reaction conditions were as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 sec, 56℃ annealing for 30 sec, 72℃ extension for 30 sec, 32 cycles; 72℃ post-extension for 5 min.

[0029] TransStartFastPfu The DNA Polymerase reaction system is as follows: Template 1 μL; Forward Primer (10 μM) 1 μL, Reverse Primer (10 μM) 1 μL, 5× TransStart FastPfu buffer 10 μL; 2.5 mM dNTPs 4 μL, TransStart FastPfu DNA Polymerase 1 μL; dd H2O up to 50 μL The reaction conditions were as follows: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 20 sec, 56℃ annealing for 20 sec, 72℃ extension for 20 sec, 32 cycles; 72℃ extension for 5 min.

[0030] Agarose gel electrophoresis: 0.3 g agarose was dissolved in 30 mL of 1× TAE solution, heated to boiling twice in a microwave oven, 3 μL of nucleic acid dye was added, mixed well, poured into a mold, and loaded after the gel solidified. Electrophoresis was performed at 130 V for 30 min. The electrophoresis image of the amplified products is shown below. Figure 1As shown.

[0031] from Figure 1 As can be seen, the product amplified by primer ND5 is of the correct size and has no non-specific bands. Moreover, compared with other primer groups, the bands are brighter, indicating that ND5 has higher amplification efficiency and sensitivity.

[0032] (3) Detection of the lowest detection concentration PCR amplification and agarose gel electrophoresis: The agarose gel extracted in Example 1 was then subjected to PCR amplification and agarose gel electrophoresis. MHC-GAL4 ×W 1118 -1 genomic DNA was used as a template for the PCR reaction. The template was diluted with ddH2O to reduce its concentration to 100 ng / μL, 10 ng / μL, 1 ng / μL, 100 pg / μL, and 10 pg / μL, respectively. ND5-F / ND5-R primers were used. TransStartFastPfu PCR amplification was performed using DNA polymerase, and the PCR products were then subjected to agarose gel electrophoresis. The electrophoresis results are shown in the image below. Figure 2 As shown.

[0033] from Figure 2 As can be seen, the lowest detectable concentration is 100 pg / μL, meaning the sensitivity of primer ND5 is 100 pg / μL.

[0034] (3) qPCR detection The qPCR reaction system is as follows: 2×SYBR Green Pro Taq HS Premix (Blue) 5 μL 40×Dilution Buffer (Yellow) 0.25 μL Forward Primer (10 μM) 0.5 μL Reverse Primer (10 μM) 0.5 μL Template 1 μL dd H2O up to 10 μL The reaction procedure was as follows: pre-denaturation at 95℃ for 3 min; denaturation at 95℃ for 10 sec; annealing at 60℃ for 15 sec; 40 cycles.

[0035] During the melting curve analysis stage, the temperature was increased from 60-95℃ at a rate of 1℃ / sec, and the changes in fluorescence signal were detected to verify the specificity of the amplified product.

[0036] Fruit flies extracted using Example 1 MHC-GAL4 × W 1118-1 Genomic DNA was used as a template for qPCR. The template was serially diluted (five-fold). The high-concentration template was gradually diluted into a series of lower-concentration templates according to a certain ratio. This can avoid the errors caused by transferring a very small amount of stock solution or adding a large amount of solvent when diluting a large number of times at once.

[0037] The initial concentration of the template was 87.5 ng / μL. It was first diluted to 50 ng / μL with dd H2O, and then serially diluted five-fold with dd H2O to obtain six templates with concentrations of 50 ng / μL, 10 ng / μL, 2 ng / μL, 0.4 ng / μL, 0.08 ng / μL, and 0.016 ng / μL, respectively. These six templates were subjected to qPCR reaction, and the average dCt value of each group is shown in Table 3.

[0038] Table 3. dCt values ​​obtained from serial dilutions of the same template with different mtDNA primers According to the results in Table 3, primer ND5 had the fewest cycles to reach the threshold in the qPCR reaction of each concentration template. The smaller the Ct value, the earlier the amplification started, suggesting that primer ND5 has higher amplification efficiency and higher sensitivity compared with other primers.

[0039] To further verify this result, the standard curve and coefficient of determination (R²) were calculated based on the dCT values ​​above, and the standard curve was plotted. The standard curve was obtained by performing linear regression with the logarithm of the template concentration (log10) as the X-axis and the measured Ct value as the Y-axis.

[0040] In the standard curve, the slope (m) represents the change in Ct value for every 10-fold change in template concentration (i.e., 1 log10 unit). Figure 2 The standard curve for primer ND5 can be seen as y = 3.36x + 13.74, and the amplification efficiency is given by the formula: E = (10... -1 / m The amplification efficiency of primer ND5 was calculated to be 98.4% by multiplying the result by 1)×100%.

[0041] R², the coefficient of determination, is a crucial statistical indicator that describes the "goodness of linear fit" of the standard curve, i.e., the degree of agreement between the data points and the fitted straight line. The closer R² is to 1, the better the linear relationship of the standard curve, the more reliable the data, and the higher the accuracy of the primer. Figure 2 As can be seen, the R² of primer ND5 is 0.9995, which is the highest among the four primer groups.

[0042] The results above show that primer ND5 has excellent amplification efficiency and perfect linearity. Compared with other qPCR-mtDNA primers, the ND5-F / ND5-R primers designed in this invention have higher amplification efficiency and are more accurate.

[0043] Example 3. Practical Application of the Drosophila melanogaster mtDNA Detection Kit WT, ... were extracted according to the Drosophila melanogaster genomic DNA extraction kit of this invention. Polg1RNAi Three groups of Drosophila were used to create a Drosophila colon cancer model, with three biological replicates in each group. The concentrations of the replicates were leveled and diluted to 1.5 ng / μL to serve as templates for qPCR reactions. ND5, designed in this invention, was used as the mtDNA primer, and RPL32 as the nuclear DNA primer. qPCR detection was performed according to the reaction system and procedure described in this specification. The obtained dCT values ​​were processed, and the relative mtDNA copy number for each group was calculated using the formula for calculating the relative mtDNA copy number, as shown in Tables 4-5. A t-test was then performed, and the results are as follows: Figure 3 As shown.

[0044] Table 4. Relative copy number of mtDNA Table 5. Relative copy number of mtDNA from Figure 3 As can be seen from this, compared with the control group, Polg1RNAi The mtDNA levels in the Drosophila colon cancer model showed a significant decrease compared to the control group; however, the relative copy number of mtDNA in the model was significantly increased. These results are consistent with those in the literature, thus confirming the feasibility of this kit.

[0045] The embodiments described above are merely preferred embodiments for fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A kit for detecting the number of copies of mtDNA in Drosophila melanogaster, characterized by: The kit includes a Drosophila genomic DNA extraction reagent and Drosophila mtDNA amplification primers; The Drosophila genomic DNA extraction reagent includes Buffer 1, Buffer 2, and Washing Buffer; each 25 ml of Buffer 1 contains 25 mL of 1 mol / L, pH 8.0 Tris·HCl, 250 μL of 0.5 mol / L, pH 8.0 EDTA, 250 μL of 5 mol / L NaCl, 500 μL of 10% SDS, 250 μL of β-mercaptoethanol, 25 ml of 20 mg / mL proteinase K, and the remainder is water; Each 25ml of Buffer 1 contains 12.5ml of anhydrous ethanol, 250μL of 5 mol / L NaCl, 1 mol / L pH8.0 Tris.HCl, and the remainder is water; Each 200ml of the Washing Buffer contains 150ml of anhydrous ethanol, 2ml of 1mol / L pH8.0 Tris.HCl, and 2ml of 5mol / L NaCl; The Drosophila mtDNA amplification primers are shown in SEQ ID NO.7 and SEQ ID NO.

8.

2. A method for detecting the copy number of mtDNA of Drosophila melanogaster for non-diagnostic purposes, characterized by, Includes the following steps: a) Extracting genomic DNA from Drosophila melanogaster using the Drosophila genomic DNA extraction reagent of claim 1; b) Using the DNA extracted in step a as a template, amplify the Drosophila mtDNA using the described Drosophila mtDNA amplification primers via real-time quantitative PCR; c) Calculate the relative copy number of mtDNA based on the Ct value obtained in step b.

3. The method according to claim 2, characterized in that, The reaction system for qPCR in step b is as follows: 5 μL of 2×SYBRGreen Pro Taq HS Premix, 0.25 μL of 40×Dilution Buffer, 0.5 μL of 10 μM forward primer, 0.5 μL of 10 μM reverse primer, 1 μL of template DNA, and dd H2O to a final volume of 10 μL.

4. The method according to claim 2, characterized in that, The qPCR reaction program in step b is as follows: 95℃ for 3 minutes; then perform 40 cycles of 95℃ for 10 seconds and 60℃ for 15 seconds; finally, perform melting curve analysis.

5. Use of the kit of claim 1 in the preparation of a product for detecting the relative copy number of mitochondrial DNA in Drosophila melanogaster.

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