Double-stranded DNA adapter and detection method for short DNA fragments

Abstract

A double-stranded DNA adapter and a detection method for short DNA fragments, relating to the technical field of molecular biological detection. The double-stranded DNA adapter comprises: a first adapter as shown in SEQ ID NO: 1, and a second adapter as shown in SEQ ID NO: 2. The provided detection method has high detection accuracy, is time-saving and simple, has a low detection limit, and has universality in detection application. The provided double-stranded DNA adapter has the technical advantage of a wide detection range for short DNA fragments, and has a good detection effect for any exogenous DNA. For detecting exogenous residual DNA, there is no need to design specific primers for specific DNA samples.

Description

A method for detecting double-stranded DNA linkers and short DNA fragments Technical Field

[0001] The present invention relates to the field of molecular biology detection technology, and in particular to a method for detecting double-stranded DNA linkers and short DNA fragments. Background Art

[0002] In recent years, biopharmaceutical companies have become increasingly stringent in their testing for residual exogenous DNA fragments. Exogenous DNA is a process-related impurity in biological products. Testing its content can confirm the effectiveness of the product purification process—that is, whether it effectively removes residual exogenous DNA. It can also confirm whether the impurity content in the product meets regulatory requirements. This test value is a key parameter in biological product quality control.

[0003] The sources of exogenous DNA mainly include host cells, plasmids, etc. In various reagents and consumables, residual exogenous DNA may have adverse effects on subsequent scientific research and industrial production, such as infectious risks, immunogenicity risks, etc. Conventional exogenous DNA detection methods mainly include DNA probe hybridization method, fluorescent probe method, and quantitative PCR method. The current exogenous DNA detection method can only detect residual fragments larger than 100bp, and cannot detect shorter fragments of DNA. Since the DNA sequences in many tubing reagents may be degraded into short fragment sequences, if they cannot be accurately detected, the impact of short fragments (<100bp) on production or scientific research may be ignored.

[0004] In view of this, the present invention is proposed.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide a method for detecting double-stranded DNA linkers and short DNA fragments to solve the above technical problems.

[0007] The present invention is achieved in that:

[0008] In a first aspect, the present invention provides a double-stranded DNA linker for detecting short DNA fragments, comprising: a first linker as shown in SEQ ID NO: 1 and a second linker as shown in SEQ ID NO: 2.

[0009] The present invention is based on the principle of linker library construction and fluorescence quantification, and has developed a double-stranded DNA linker for detecting short fragments. It has been verified that the double-stranded DNA linker provided by the present invention has the technical advantage of a wide range of short DNA fragment detection, and has a good detection effect for any exogenous DNA. In addition, compared with the existing DNA fragment detection method, the double-stranded DNA linker provided by the present invention can achieve the detection of smaller DNA fragments, and can detect short DNA fragments of at least 20bp in length, and can also detect long fragments greater than 100bp. In the future, it can be used for short fragment residue detection in products such as reagents and consumables, and has broad application prospects in exogenous DNA residue detection.

[0010] The sequence of the first linker is as follows:

[0011] 5'-GATCAACGCAGAGTGGCCACAACTTTGTACAAGCGAGTTGT-3' (SEQ ID NO: 1);

[0012] The sequence of the second linker is as follows:

[0013] 5'-CAACTCGCTTGTACAAAGTTGTGGCCACTCTGCGTTGATC-3' (SEQ ID NO: 2).

[0014] In a preferred embodiment of the present invention, the 5' end of the first linker has a modification group. Since the first linker and the second linker are partially complementary, the modification group helps prevent the first linker and the second linker from self-ligating.

[0015] In an optional embodiment, the modification group is selected from any one of biotin, fluorescein isothiocyanate (FITC), carboxyfluorescein (FAM), digoxigenin (DIG) and carboxytetramethylrhodamine (TAMRA).

[0016] In an alternative embodiment, the modifying group is selected from biotin.

[0017] In a second aspect, the present invention provides a reagent or a kit comprising the above-mentioned double-stranded DNA linker.

[0018] In a preferred embodiment of the present invention, the reagent or kit further comprises a detection primer, which comprises an upstream primer and a downstream primer, wherein the upstream primer is selected from at least 27 consecutive bases in the first linker; the downstream primer is selected from at least 27 consecutive bases in the second linker.

[0019] For example, the upstream primer and the downstream primer each include 27-35 consecutive bases, or the upstream primer and the downstream primer each include 27-35 consecutive bases, or the upstream primer and the downstream primer each include 30-35 consecutive bases.

[0020] In an alternative embodiment, the nucleotide sequence of the upstream primer is shown in SEQ ID NO: 3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 3. 5'-TCAACGCAGAGTGGCCACAACTTTGTA-3' (SEQ ID NO: 3).

[0021] In a third aspect, the present invention further provides a method for detecting short DNA fragments, comprising the following steps:

[0022] The DNA sample to be tested is end-repaired and then ligated to the double-stranded DNA adapter to obtain a ligation product. The ligation product is subjected to fluorescent quantitative PCR using the detection primers described above. The difference in Ct values ​​between the negative control and the tested DNA sample is used to determine whether short DNA fragments are present in the DNA sample to be tested.

[0023] The classic DNA structure is double-stranded, with flat ends at both ends, i.e., flat ends. However, DNA molecules under natural conditions may not have such an ideal structure as above. Because DNA molecules are degraded to varying degrees in the natural environment, their residual fragments may be shorter (less than 50bp), and the ends may not be flat ends, but sticky ends with several protruding bases at the single-stranded ends. At the same time, naturally occurring short-fragment DNA molecules may have fragments of various lengths mixed together, and the length cannot be accurately determined, so it is difficult to directly make a quantitative determination. The short DNA detection method provided by the present invention includes performing end repair on the DNA sample to be tested, and then connecting the above-mentioned double-stranded DNA linker, and performing fluorescent quantitative PCR on the connection product, which can quantitatively detect naturally occurring DNA fragments or artificially synthesized DNA fragments in the natural environment. By comparing with the negative control, the difference in Ct value can be used to judge whether there are short DNA fragments (or whether there are short DNA fragments remaining) in the DNA sample to be tested.

[0024] In a preferred embodiment of the present invention, the method for determining whether short DNA fragments are present in the DNA sample to be tested based on the difference in Ct values ​​between the negative control and the tested DNA sample includes: if the Ct value difference between the negative control and the tested DNA sample is significant, and it is determined that short DNA fragments are present in the tested DNA sample.

[0025] If the Ct values ​​of the negative control and the tested DNA sample are significantly different, or the melting curve peak shapes are the same, it is determined that there are no short DNA fragments in the tested DNA sample.

[0026] In a preferred embodiment of the present invention, the negative control is a control group to which a double-stranded DNA linker is added.

[0027] In a preferred embodiment of the present invention, the DNA sample to be tested is an artificially synthesized random DNA short fragment or a natural DNA short fragment.

[0028] In an alternative embodiment, the natural short DNA fragments are from environmental samples, tubes, equipment, cultures, tissues, serum or blood;

[0029] In an optional embodiment, random short DNA fragments or natural short DNA fragments refer to short DNA fragments with a length of less than 100 bp.

[0030] In a preferred embodiment of the present invention, the reaction system of fluorescent quantitative PCR includes: SYBR Mix, detection primers and ligation products;

[0031] In an alternative embodiment, the final concentration of the detection primer is 0.4-0.5 μM;

[0032] In an optional embodiment, the reaction conditions of fluorescent quantitative PCR include: 95° C., 1-5 min; 94° C., 5-20 s, 60° C., 30 s-35 s, 35-45 cycles; and then performing melting curve analysis.

[0033] In a preferred embodiment of the present invention, end repair refers to: filling the ends of the fragments of the DNA sample to be tested, phosphorylating the 5' end of the DNA sample to be tested, and introducing an A base at the 3' end of the DNA sample to be tested.

[0034] Double-stranded DNA molecules with overhanging ends cannot be directly ligated. The present invention uses DNA end repair to repair the overhanging ends of double-stranded DNA molecules and add an A base to the 3' end of each single strand to facilitate the next step of ligation. End repair can be performed using a commercially available rapid end repair / A-tailing kit. The result is a double-stranded DNA molecule with repaired ends and A-tailing. The structure of the repaired DNA molecule is shown in Figure 13.

[0035] In an optional embodiment, the reaction conditions of end repair include: 95-105°C, 2-10 min; 25-30°C, 20 min; 72°C, 1-20 min.

[0036] The schematic diagram of the double-stranded structure linker is shown in FIG13 . The linker structure is configured to perform T4 ligation reaction with the prepared double-stranded DNA via phosphate bonds according to the A-T pairing principle.

[0037] In a fourth aspect, the present invention also provides the use of a double-stranded DNA adaptor or the aforementioned reagent or kit for detecting residual short DNA fragments, where the purpose is not to diagnose a disease. Such applications include, but are not limited to, detection of imported or exported foreign organisms, forensic or judicial bioassays, and residual DNA detection in test tubes, EP tubes, cellulose membranes, and the like.

[0038] The present invention has the following beneficial effects:

[0039] The double-stranded DNA linker and detection method provided by the present invention have the following advantages:

[0040] (1) High detection accuracy. The detection method provided by the present invention can detect residual double-stranded DNA fragments in reagents and consumables, and can absolutely quantitatively determine the content of the ligation product by dye method, with high accuracy.

[0041] (2) Time-saving and simple. In the present invention, a specific linker is used to build a library for connection and a fluorescence quantitative method is used to determine the double-stranded structure of a short fragment. The test experiment takes about 4-6 hours, which can save test time.

[0042] (3) Low detection limit. The present invention can detect double-stranded fragments as small as 20 bp, and the lower limit of the detection range is even lower.

[0043] (4) Universality of detection applications: The double-stranded DNA adapter provided by the present invention has the technical advantage of a wide range of detection for short DNA fragments, and has good detection results for any exogenous DNA. When detecting exogenous residual DNA, there is no need to design specific primers for a specific DNA sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0045] FIG1 is an electrophoretic analysis of the preparation of the double-stranded linker in Example 1; wherein lanes 1-3 shown in FIG1A are electrophoretic images of AF (single-stranded linker 1), AR (single-strand complementary to single-stranded linker 1), and an equal volume mixture of AF and AF (prepared double-stranded linker); FIG1B shows an electrophoretic image of an E. coli gene fragment (lane 1) and a honeybee EFB gene fragment (lane 2); and FIG1C shows an electrophoretic image of an E. coli gene fragment (lane 1) and an EFB gene fragment (lane 2) after linker ligation.

[0046] FIG2 is a fluorescence amplification curve in Example 1; wherein A is a ligation product based on a double-stranded DNA template having a length of 268 bp, and B is a ligation product based on a double-stranded DNA template having a length of 642 bp;

[0047] FIG3 is a fluorescent quantitative melting curve in Example 1; wherein A is a ligation product based on a double-stranded DNA template having a length of 268 bp, and B is a ligation product based on a double-stranded DNA template having a length of 642 bp;

[0048] Figure 4-1 is a diagram showing the alignment of the ligation product of fragment A amplified using the linker sequence as a primer in Example 1 after sequencing, and the alignment with the linker primer sequence (sequencing comparison can confirm that the ligation product is connected to the double-ended linker);

[0049] Figure 4-2 is a comparison diagram of the sequence of the linker primers after sequencing of the ligation product of the B segment amplified using the linker sequence as a primer in Example 1;

[0050] FIG5 is a fluorescence amplification curve in Example 2; wherein C is a ligation product with a double-stranded DNA of about 100 bp in length as a template, and D is a ligation product with a double-stranded DNA of about 110 bp in length as a template;

[0051] FIG6 is a fluorescent quantitative melting curve in Example 2; wherein C is a ligation product using a double-stranded DNA template of about 100 bp in length, and D is a ligation product using a double-stranded DNA template of about 110 bp in length;

[0052] FIG7 is a fluorescence quantitative amplification curve in Example 3; wherein E is based on a double-stranded DNA ligation product of about 40 bp in length as a template, and F is based on a double-stranded DNA ligation product of about 50 bp in length as a template;

[0053] FIG8 is a fluorescent quantitative melting curve in Example 3; wherein E is a ligation product using a double-stranded DNA template of about 40 bp in length, and F is a ligation product using a double-stranded DNA template of about 50 bp in length;

[0054] FIG9 is a fluorescence quantitative amplification curve in Example 4; wherein G is a double-stranded DNA ligation product of about 20 bp in length as a template;

[0055] FIG10 is a fluorescence quantitative melting curve in Example 4; wherein G is a double-stranded DNA ligation product of about 20 bp in length as a template.

[0056] Figure 11 is a fluorescence quantitative amplification curve in Example 5; wherein H is a ligation product of human 293T cell DNA interrupted by ultrasound as a template, and I is a ligation product of soil bacterial DNA interrupted by ultrasound as a template;

[0057] Figure 12 is a fluorescent quantitative melting curve in Example 5; wherein H is a ligation product of human 293T cell DNA interrupted by ultrasound as a template, and I is a ligation product of soil bacterial DNA interrupted by ultrasound as a template;

[0058] Figure 13 is a schematic diagram of the detection principle;

[0059] FIG14 is a fluorescence quantitative curve related to the method accuracy experiment;

[0060] Figure 15 shows the melting curve. DETAILED DESCRIPTION

[0061] Reference will now be made in detail to embodiments of the present invention, one or more examples of which are described below. Each example is provided to illustrate, not to limit, the present invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment may be used in another embodiment to produce further embodiments.

[0062] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a person skilled in the art. The technique is fully explained in the literature, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); and PCR: The Polymerase Chain Reaction. Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0063] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0064] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0065] Example 1

[0066] This example provides a method for detecting short DNA fragments:

[0067] (1) A double-stranded DNA linker for short DNA fragment detection was synthesized, comprising a first linker as shown in SEQ ID NO: 1 and a second linker as shown in SEQ ID NO: 2. Furthermore, a detection primer as shown in SEQ ID NO: 3 was synthesized for subsequent fluorescent quantitative PCR.

[0068] Preparation of double-stranded DNA adapters includes: synthesizing a single-stranded adapter sequence and diluting it with sterile water to a final concentration of 10 μM. Denaturing and annealing the double strands, taking 50 μL of each of the two single-stranded sequences, mixing equal volumes, and setting the following reaction temperature conditions on the PCR instrument: 95°C (5 min), 85°C (30 s), 80°C (30 s), 75°C (30 s), 70°C (30 s), 65°C (30 s), 60°C (30 s). The mixture was allowed to stand until the temperature dropped to room temperature and was then used.

[0069] The electrophoresis results of the double-stranded connector are shown in FIG1 , and the results show that the double-stranded connector was successfully prepared.

[0070] (2) Preparation of PCR products of specific length. Taq enzyme and specific primers were used to amplify two bands, where fragment A was based on E. coli DNA as a template and fragment B was based on the honey bee genome as a template. The following are the two primers.

[0071] Primer F for fragment A: 5′-GTGCCAGCMGCCGCGGTAA-3′; R: 5′-GGCGTGGACTTCCAGGGTATCT-3′;

[0072] Fragment B primer F: 5'-CTCGTCAATATGGCATTCATTCG-3'; R: 5'-GTGTCACTACTTGTTCTTCTGTTG-3'.

[0073] The PCR conditions and system are:

[0074] The reaction system is as follows:

[0075] The reaction procedure is as follows:

[0076] The PCR products were gel-recovered using the SanPrep DNA gel recovery kit (Sangon Biotechnology, B518131). This yielded 268 bp of double-stranded E. coli DNA and 642 bp of double-stranded honeybee DNA (EFB gene) for end-repair and A addition.

[0077] (3) Fragment end repair and A tailing. This step can fill the ends of the DNA fragments, phosphorylate the 5' end, and add an A tail to the 3' end. Prepare the following reaction system in a sterile PCR tube:

[0078] The end repair and A-tailing reaction system is as follows, where the reagent components are derived from the EzyNGS DNA library construction kit (Cat. No. N608380) from Sangon Biotechnology Co., Ltd.:

[0079] Use a pipette to mix thoroughly, centrifuge briefly, and then place the tube in a PCR instrument and set the following program:

[0080] Repair reaction program

[0081] (4) Use T4 ligase to ligate the synthesized adapter to the repaired fragment to be tested. Prepare the following ligation system in a 0.2 mL PCR tube, mix thoroughly, and incubate at 16°C overnight (12-24 hours). Set aside the ligation product.

[0082] Ligation reaction system

[0083] The electrophoretic diagrams of the E. coli gene fragment and the honeybee EFB gene fragment before and after ligation with the linker are shown in B and C in FIG1 .

[0084] (5) Fluorescence quantitative detection: The ligation products were detected using the detection primers shown in SEQ ID NO: 3, and the fluorescence amplification curves, melting curves, and Ct values ​​of each group were compared.

[0085] The dye-based fluorescence quantitative detection was performed using the following reaction system and conditions (the template in the NTC control group was ddH2O, and the negative control group was a control group with only the linker but no test fragment added):

[0086] The fluorescence quantitative reaction system is as follows:

[0087] PCR reaction procedure

[0088] Analyze and compare the differences in Ct values ​​between the NTC control group, negative control group, and test sample group. Analyze and compare the differences in fluorescence amplification curves, melting curves, and Ct values ​​between the control group and the test group.

[0089] If the NTC control group does not produce a peak, the melting curve of the test group is different from that of the negative control group, and the Ct value is significantly different, it means that double-stranded DNA fragments are present in the sample; if the NTC control group does not produce a peak, the melting curve of the test group is the same as that of the negative control group, it means that no double-stranded DNA fragments are detectable in the sample.

[0090] Specifically in this example, if the NTC control group does not produce a peak, the melting curve of the test group differs from that of the negative control group, and the Ct value is significantly different, then double-stranded DNA fragments are present in the E. coli or honey bee sample. If the NTC control group does not produce a peak, and the melting curve of the E. coli or honey bee sample is the same as that of the negative control group, then no double-stranded DNA fragments are detectable in the sample.

[0091] The fluorescence amplification curve is shown in Figure 2, and the fluorescence quantitative melting curve is shown in Figure 3. Comparison shows that compared with the negative control, ligation product A (E. coli) and ligation product B (honey bee) have significantly different Ct values. The melting curves show that: while the NTC control and negative control groups have no peaks, ligation products A and B do have peaks. The peak times and peak shapes of ligation products A (E. coli) and B (honey bee) differ significantly. Therefore, the detection method provided by the present invention is highly capable of detecting residual DNA from different sample sources.

[0092] The ligation products A and B were sequenced separately. The results are shown in Figures 4-1 and 4-2. The TA cloning sequencing results showed that both ends of the fragment to be tested were connected to the adapter sequence, indicating that the adapter was successfully connected to both ends of the fragment to be tested.

[0093] Example 2

[0094] The only difference between this embodiment and embodiment 1 is that the detection objects are different. In this embodiment, the detection objects are double-stranded DNA fragment C with an initial length of 100 bp and double-stranded DNA fragment D with a length of 110 bp.

[0095] (1) Preparation of double-stranded adapters: Double-stranded adapters were prepared according to the steps in Example 1.

[0096] (2) Preparation of PCR products of specific length. Taq enzyme and specific primers were used to amplify two bands, where fragment C was based on human 293T cell DNA and fragment D was based on E. coli DNA. The following are the two primers.

[0097] Primer F for fragment C: 5′-TGGTAGTCTGGAACACCGTAAGAGT-3′; R: 5′-CATATGGCAGGCTTTAGGTACCC-3′;

[0098] D fragment primer F: 5'-CAAGGCTAAATACTCCTGAC-3'; R: 5'-CACTCCCCTCGCCGGGGTTC-3'.

[0099] (3) Repair the fragment ends and add A according to the steps of Example 1.

[0100] (4) Ligation was performed using T4 DNA ligase according to the procedure of Example 1.

[0101] (5) Fluorescence quantitative detection: Detect the ligation products using adapter primers and compare the fluorescence amplification curves, melting curves, and Ct values ​​of each group.

[0102] The fluorescence quantification curve and melting curve are shown in Figures 5 and 6, respectively. The amplification curves of samples C and D have peaks, while the NTC and negative control groups have no peaks, and the melting curve peaks are different. This shows that the linker and detection method provided in this example can be used to detect samples C and D.

[0103] Example 3

[0104] The only difference between this embodiment and embodiment 1 is that the detection objects are different. In this embodiment, the detection objects are artificially synthesized double-stranded DNA fragment E (40 bp) and double-stranded DNA fragment F (50 bp).

[0105] (1) Preparation of double-stranded adapters: Double-stranded adapters were prepared according to the steps in Example 1.

[0106] (2) Preparation of double-stranded short DNA fragments. The double-stranded DNA fragment preparation steps in Example 1 were followed. The complementary sequence of fragment E is:

[0107] F:

[0108] 5'-GGGTTTGATGGGGACGTTTAATTAATGGTGTAAAAATGTTG-3';

[0109] R:

[0110] 5'-ACATTTTTACACCATTAATTAAACGTCCCATCAAACCC-3';

[0111] The sequence of fragment F is:

[0112] 5'-AACTCGTCTCCGTCCGCCCCAGAAAATTACGGTACACATGGCTTTAA-3';

[0113] R:

[0114] 5'-AAAGCCATGTGTACCGTAATTTTCTGTGGGGGCCGGACGGAGACGAGTT-3'.

[0115] (3) Repair the fragment ends by adding A, according to the steps described in Example 1.

[0116] (4) Ligation was performed using T4 DNA ligase according to the procedure described in Example 1.

[0117] (5) Fluorescence quantitative detection: Detect the ligation products using adapter primers and compare the fluorescence amplification curves, melting curves, and Ct values ​​of each group.

[0118] The results are shown in Figures 7 and 8 . The amplification curves of samples E and F have peaks, while the NTC and negative control groups do not, and the melting curves have different peaks. This indicates that the linker and detection method provided in this example can be used to detect artificially synthesized samples E and F.

[0119] Example 4

[0120] The only difference between this embodiment and embodiment 1 is that the detection object is different. The detection object in this embodiment is the artificially synthesized double-stranded DNA fragment G (20 bp).

[0121] (1) Preparation of double-stranded adapters: Double-stranded adapters were prepared according to the steps in Example 1.

[0122] (2) Preparation of double-stranded products of specific length: Double-stranded DNA was prepared according to the steps in Example 1, wherein the G segment primer F: 5'-ACATGGAGGCTTCAGACATCG-3'; R: 5'-ACGATGTCTGAAGCCTCCATG-3'.

[0123] (3) Repair the fragment ends and add A according to the steps in Example 1.

[0124] (4) Ligation was performed using T4 DNA ligase according to the steps in Example 1.

[0125] (5) Fluorescence quantitative detection: Detect the ligation products using adapter primers and compare the fluorescence amplification curves, melting curves, and Ct values ​​of each group.

[0126] The results are shown in Figures 9 and 10. The amplification curve of sample G has a peak, while the NTC and negative control groups have no peak, and the melting curves have different peaks, indicating that the adapter and detection method provided in this example can be used to detect sample G.

[0127] Example 5

[0128] The only difference between this embodiment and embodiment 1 is that the detection objects are different. The detection objects of this embodiment are human DNA and bacterial DNA interrupted by ultrasound.

[0129] (1) Preparation of double-stranded adapters: Double-stranded adapters were prepared according to the steps in Example 1.

[0130] (2) Ultrasonic fragmentation for preparation of human and bacterial DNA. 293T cell DNA and soil bacterial DNA were extracted and the sample concentrations were determined. 30 μL of the sample was ultrasonically fragmented for 8 minutes at 750W and 20 kHz.

[0131] (3) The fragmented samples were repaired and A-terminated according to the steps in Example 1.

[0132] (4) Ligation was performed using T4 DNA ligase according to the steps in Example 1.

[0133] (5) Fluorescence quantitative detection: Take 1 μL as template and use adapter primers to detect the ligation product. Compare the fluorescence amplification curve, melting curve and Ct value of each group.

[0134] The results are shown in Figures 11 and 12. The amplification curves of the H and I samples have peaks, while the NTC and negative control groups do not, and the melting curves have different peaks. This indicates that the adapter and detection method provided in this example can be used to detect both H and I samples.

[0135] Example 6

[0136] The accuracy of the method was verified by the following experimental method:

[0137] (1) Preparation of double-stranded adapters: Double-stranded adapters were prepared according to the steps in Example 1.

[0138] (2) Preparation of PCR products of specific lengths. A total of 6 aliquots were prepared. The bands were amplified using Taq enzyme and specific primers. Fragment C was based on human 293T cell DNA as a template. The primers are as follows.

[0139] C fragment primer F: 5'-TGGTAGTCTGGAACACCGTAAGAGT-3'; R: 5'-CATATGGCAGGCTTTAGGTACCC-3'.

[0140] (3) Repair the fragment ends and add A according to the steps of Example 1.

[0141] (4) Ligation was performed using T4 DNA ligase according to the procedure of Example 1.

[0142] (5) Fluorescence quantitative detection: Detect the ligation products using adapter primers and compare the fluorescence amplification curves and Ct values ​​of each group.

[0143] The fluorescence quantitative curve and the melting curve are shown in Figures 14 and 15 respectively. The fluorescence amplification curve trends of the 6 samples are consistent, the Ct values ​​are close, and the melting curve is a single peak. The Ct values ​​of each group of samples are 23.26, 24.95, 25.59, 24.90, 22.42, and 22.89, respectively, with a mean of 24.00, a standard deviation of 1.31, a standard error of 0.53, and a relative standard deviation of 5.5%. The relative standard deviation (RSD) is the value obtained by dividing the standard deviation by the corresponding mean value and multiplying it by 100%. It is generally used to evaluate the precision and repeatability of the method in the validation of the analytical method. The smaller the RSD value, the higher the precision and the better the repeatability. As can be seen from this, the repeatability of this method is good. This shows that the joint and detection method provided in this embodiment have good accuracy.

[0144] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A double-stranded DNA adapter for short DNA fragment detection, characterized in that: It includes: The first linker is shown as SEQ ID NO:1 and the second linker is shown as SEQ ID NO:

2.

2. The double-stranded DNA adaptor according to claim 1, characterized in that The 5' end of the first linker has a modification group; Preferably, the modification group is selected from any one of biotin, fluorescein isothiocyanate (FITC), carboxyfluorescein (FAM), digoxigenin (DIG) and carboxytetramethylrhodamine (TAMRA); Preferably, the modifying group is selected from biotin.

3. A reagent or a kit, characterized in that: It comprises the double-stranded DNA linker according to any one of claims 1 to 2.

4. The reagent or kit according to claim 3, characterized in that The reagent further comprises a detection primer, wherein the detection primer comprises an upstream primer and a downstream primer, wherein the upstream primer is selected from at least 27 consecutive bases in the first linker; and the downstream primer is selected from at least 27 consecutive bases in the second linker; Preferably, the nucleotide sequence of the upstream primer is as shown in SEQ ID NO:3, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO:

3.

5. A method for detecting short DNA fragments, characterized in that: It includes the following steps: The DNA sample to be tested is end-repaired, and then connected to the double-stranded DNA adapter described in any one of claims 1-2 to obtain a connection product, and the connection product is subjected to fluorescent quantitative PCR using the detection primer described in claim 4; and the presence of short DNA fragments in the DNA sample to be tested is determined based on the difference in Ct values ​​between the negative control and the DNA sample to be tested.

6. The detection method according to claim 5, characterized in that: The method for judging whether there is a short DNA fragment in the DNA sample to be tested according to the difference in Ct values ​​between the negative control and the DNA sample to be tested comprises: if the Ct values ​​of the negative control and the DNA sample to be tested are significantly different, and the peak shapes of the melting curves are different, then it is judged that the DNA sample to be tested has a short DNA fragment; If the Ct value difference between the negative control and the tested DNA sample is not significant, or the melting curve peak shape is the same, it is judged that there are no short DNA fragments in the tested DNA sample.

7. The detection method according to claim 6, characterized in that: The negative control is a control group to which the double-stranded DNA adapter is added.

8. The detection method according to claim 5, characterized in that: The DNA sample to be tested is an artificially synthesized random DNA short fragment or a natural DNA short fragment; Preferably, the natural short DNA fragments are from environmental samples, tubes, equipment, cultures, tissues, serum or blood; Preferably, the random short DNA fragments or natural short DNA fragments refer to short DNA fragments with a length of less than 100 bp.

9. The detection method according to claim 5, characterized in that: The reaction system of the fluorescent quantitative PCR comprises: SYBR Mix, the detection primer and the ligation product; Preferably, the final concentration of the detection primer is 0.4-0.5 μM; Preferably, the reaction conditions of the fluorescent quantitative PCR include: 95°C, 1-5min; 94°C, 5-20s, 60°C, 30s-35s, 35-45 cycles; and then performing melting curve analysis; Preferably, the end repair refers to: filling the ends of the fragments of the DNA sample to be tested, phosphorylating the 5' end of the DNA sample to be tested, and introducing an A base at the 3' end of the DNA sample to be tested; Preferably, the reaction conditions for the end repair include: 95-105°C, 2-10 min; 25-30°C, 20 min; 72°C, 1-20 min.

10. Use of the double-stranded DNA adaptor according to any one of claims 1 to 2 or the reagent or kit according to any one of claims 3 to 4 in the detection of residual short DNA fragments, wherein the use is not for the purpose of disease diagnosis.

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