Method for detecting peripheral blood free eccDNA based on nested PCR combined with CRISPR / Cas12a

Abstract

The invention discloses a method for detecting free eccDNA based on nested PCR combined with CRISPR-CAS12a. The method comprises the following steps: step 1, extracting free DNA in a serum sample; 2, linear DNA in the free DNA is removed through enzyme digestion, and eccDNA is reserved; 3, by taking the eccDNA-1 and the eccDNA-2 as target genes, carrying out a first round of PCR amplification by using the long primer pair, and carrying out a second round of PCR amplification by using the short primer pair; and 4, detecting the target eccDNA by adopting a CRISPR-CAS12a (clustered regularly interspaced short palindromic repeats) detection system. According to the invention, two pairs of PCR primers and crRNA are respectively designed aiming at Junction sites of eccDNA, the sensitivity and the specificity of target sequence recognition are enhanced through two rounds of PCR reactions, and meanwhile, the detection time is shortened.

Description

Technical Field

[0001] The present invention relates to the field of gene detection technology, and in particular to a method for detecting free eccDNA based on nested PCR combined with CRISPR / Cas12a. Background Art

[0002] DNA molecules in eukaryotic organisms have long been thought to exist mainly in linear form in the cell nucleus, until the discovery of a circular DNA molecule that exists outside chromosomes - extrachromosomal circular DNA (eccDNA) (hereinafter referred to as "eccDNA") in 1965. eccDNA separates or falls off from the normal genome and is free outside the chromosome genome. The size of eccDNA ranges from 100 bp to millions of bp. eccDNA is ubiquitous in both normal cells and tumor cells, and participates in physiological or pathological processes in a special way.

[0003] Compared with linear chromosomal DNA, eccDNA has higher chromatin accessibility (open chromatin), carries active histone modifications, can mediate ultra-long-range interactions between genes, and can greatly enhance the transcriptional activity of genes. In addition, eccDNA has no telomeres and centromeres, and cannot be evenly separated and distributed to daughter cells, but follows a random distribution or Gaussian distribution. This uneven separation will cause eccDNA molecules with oncogenes to accumulate in a certain type of cell subpopulation, accelerate tumor progression, and increase tumor heterogeneity with daughter cells containing an increasing number of oncogene copies. Moreover, due to the high copy number of eccDNA, they can transcribe a large number of gene products, thereby affecting the phenotype and function of cells. These characteristics enable eccDNA to drive malignant transformation and promote tumor evolution; at the same time, due to the circular closed structure of eccDNA, it is nuclease-resistant, and cell-free eccDNA is not easily destroyed by physical factors and enzymes, thus serving as a potential biomarker for tumor diagnosis and prognosis. Therefore, further exploration of its genetic information will help provide new ideas for the diagnosis, treatment and prediction of cancer.

[0004] At present, compared with genomic DNA in tumor tissues, the amount of eccDNA is very small, and there is still a lack of mature, stable, simple and efficient identification methods for tumor cell eccDNA in this field. Therefore, it is urgent to establish a micro-eccDNA enrichment detection technology with high sensitivity, high specificity and good repeatability with practical application value to achieve the detection of micro-eccDNA contained in tissue samples or other body fluid samples. Summary of the invention

[0005] The purpose of the present invention is to provide a method for detecting free eccDNA based on Nested PCR and CRISPR / Cas12a (NPCC) in response to the deficiencies in the prior art.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] A method for detecting free eccDNA based on nested PCR and CRISPR / Cas12a is provided, comprising the following steps:

[0008] Step 1, taking a serum sample and extracting free DNA in the serum sample;

[0009] Step 2: using enzyme digestion to remove the linear DNA in the free DNA in step 1 and retain the eccDNA;

[0010] Step 3: Using eccDNA-1 and eccDNA-2 as target genes, amplification was performed using nested PCR, using a long primer pair for the first round of PCR amplification, and then using a short primer pair for the second round of PCR amplification;

[0011] Step 4: Use the CRISPR / Cas12a detection system to detect the target eccDNA, wherein the crRNA sequence targeting the target eccDNA-1 is shown in SEQ ID NO.5, and the crRNA sequence targeting the target eccDNA-2 is shown in SEQ ID NO.10; then the fluorescence intensity of the reaction system is detected.

[0012] Furthermore, the physical position of the eccDNA-1 is Chr17:64682690-64683063; the physical position of the eccDNA-2 is Chr8:100632795-100633127.

[0013] Furthermore, the long primer pair includes a first long primer pair for eccDNA-1, whose sequences are shown in SEQ ID NO.1 and SEQ ID NO.2; and a second long primer pair for eccDNA-2, whose sequences are shown in SEQ ID NO.3 and SEQID NO.4.

[0014] Furthermore, the short primer pair includes a first short primer pair for eccDNA-1, whose sequences are shown in SEQ ID NO.6 and SEQ ID NO.7; and a second short primer pair for eccDNA-2, whose sequences are shown in SEQ ID NO.8 and SEQID NO.9.

[0015] Furthermore, the program of the first round of PCR amplification was: pre-denaturation at 95°C for 10 min, followed by denaturation at 95°C for 15 s, annealing at 45°C for 30 s, and extension at 72°C for 30 s. After 20 cycles, the product was briefly stored at 4°C.

[0016] Furthermore, the procedure for the second round of PCR amplification was: pre-denaturation at 95°C for 10 min, followed by denaturation at 95°C for 15 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. After 40 cycles, the product was briefly stored at 4°C.

[0017] Furthermore, the detection of fluorescence intensity is a real-time fluorescence quantitative detection method.

[0018] The present invention also provides a nucleic acid molecule combination for the above method, comprising: a first long primer pair as shown in sequences SEQ ID NO.1 and SEQ ID NO.2, a second long primer pair as shown in sequences SEQ ID NO.3 and SEQ ID NO.4, a first short primer pair as shown in sequences SEQ ID NO.6 and SEQ ID NO.7, a second short primer pair as shown in sequences SEQ ID NO.8 and SEQ ID NO.9, and crRNA as shown in sequences SEQ ID NO.5 and SEQ ID NO.10.

[0019] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0020] The present invention designs two pairs of PCR primers and crRNA respectively for the junction site of eccDNA, and enhances the sensitivity and specificity of target sequence recognition through nested PCR reaction, while reducing the detection time.

[0021] The present invention combines nested PCR and CRISPR / Cas12a detection technology to achieve rapid detection of free eccDNA, which can provide effective information for precision medical programs such as medication and prognosis for tumor patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the experimental operation of the detection method of the present invention.

[0023] Figure 2 This is a quality control chart for validating the detection method of the present invention using circular DNA of known concentration.

[0024] Figure 3 This is a result diagram to verify the specificity of crRNA recognition.

[0025] Figure 4 This is a graph showing the eccDNA-1 detection results in Example 4.

[0026] Figure 5 This is a graph showing the eccDNA-2 detection results in Example 4. DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but is not intended to be limiting of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention may be combined with each other without conflict.

[0028] The method flow and detection principle of detecting free eccDNA disclosed in the present invention:

[0029] like Figure 1 As shown, 1) a serum sample is taken and free DNA in the serum sample is extracted; linear DNA in the free DNA in step one is removed by enzyme cutting, and eccDNA is retained; 2) a first round of PCR amplification and a second round of PCR amplification are performed; 3) a CRISPR / Cas12a detection system is used to detect the target eccDNA, and then the fluorescence intensity of the reaction system is detected.

[0030] Example 1 Construction of CRISPR / Cas12a Detection Platform

[0031] 1. Sequence Design

[0032] PCR primers and crRNA sequences were designed according to the target genes eccDNA-1 and eccDNA-2 (wherein the physical location of eccDNA-1 is Chr17: 64682690-64683063; the physical location of eccDNA-2 is Chr8: 100632795-100633127); specifically as shown in Table 1 below:

[0033] Table 1

[0034]

[0035] 2. eccDNA Extraction

[0036] 1) Take serum samples and use a free nucleic acid extraction kit (QIAamp CircμLating Nucleic Acid Kit, Qiagen, 55114) to extract free nucleic acids in the serum samples. The extracted DNA is quantified using a Nanodrop spectrophotometer (Thermo).

[0037] 2) Add Plasmid safeATP-dependent DNase (Epicenter, E3110K) to the total DNA to remove linear DNA. The digestion system is: 500ng total DNA, 1μL (10U) DNase, 2μL (25mM) ATP, 5μL 10× reaction buffer, and sterile deionized water to 50μL. Place the above digestion system in a 37℃ water bath for 24h, and then inactivate DNase at 70℃ for 30min. Add 100μL magnetic beads (VAHTSDNA Clean Beads, Norvegian, N411-01) to the above reaction system in proportion to purify the DNA after linear digestion, and then use 30μL sterile deionized water to dissolve the purified DNA.

[0038] 3. Nested PCR Amplification

[0039] First round of PCR amplification:

[0040] The 20μL PCR system was: 2μL purified product, 1μL (10μM) upstream primer, 1μL (10μM) downstream primer, 10μL 2× PCR premix, 6μL sterile deionized water; pre-denaturation at 95°C for 10 min, followed by denaturation at 95°C for 15s, annealing at 45°C for 30s, and extension at 72°C for 30s. After 20 cycles, the product was briefly stored at 4°C.

[0041] Second round of PCR amplification:

[0042] The first round PCR reaction product was used as a template and the 20 μL reaction system was: 8 μL template, 1 μL (10 μM) upstream primer, 1 μL (10 μM) downstream primer, 10 μL 2× PCR premix. Pre-denaturation at 95°C for 10 min, followed by denaturation at 95°C for 15 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. After 40 cycles, the product was briefly stored at 4°C.

[0043] 4. CRISPR / Cas12a detection

[0044] The crRNA targeting the target eccDNA was synthesized by an in vitro transcription kit (Cas12a High Yield crRNA Synthesis and Purification Kit, TOLOBIO, #31903). The target eccDNA was then detected by HOLMES nucleic acid rapid detection technology (CRISPR / Cas12a). The 20μL reaction system was: 2μL PCR reaction product, 0.5μL (10μM) Lbcas12a, 0.5μL (10μM) crRNA, 0.5μL (10μM) ssDNA reporter, 10×HOLMES buffer, and deionized water was added to 20μL. The fluorescence signal was detected using a fluorescent quantitative PCR instrument, and the reaction procedure was 37°C, 20min, where the fluorescence signal value was detected every 30s.

[0045] Example 2: Verification of the feasibility of the method

[0046] The feasibility of this method was verified by using a circular DNA sample of known concentration as a positive quality control. The results showed that ( Figure 2 ), the detection method of the present invention can fully detect the target eccDNA, and the resulting fluorescence signal value is positively correlated with the initial concentration of the sample.

[0047] Example 3 Verification of crRNA recognition specificity

[0048] The crRNA transcribed DNA template was used as a positive control, a garbled DNA of the same length was used as a verification object, and a blank was used as a negative control. CRISPR / Cas12a was further used for testing, and the results showed that ( Figure 3 ), the DNA transcription template can produce an obvious positive signal, while the scrambled DNA and negative control do not produce a positive fluorescence signal. This result shows that crRNA can specifically recognize the target DNA sequence.

[0049] Example 4 Detection of clinical samples

[0050] Twenty patients with liver cancer pathologically diagnosed from Shanghai Tongji Hospital were tested. The results showed that ( Figure 4 and Figure 5 ), eccDNA-1 was detected in 10 of 20 serum samples of liver cancer patients, with a detection rate of 50%; eccDNA-2 was detected in 9 of liver cancer patients, with a detection rate of 45%. This result shows that the method of the present invention can fully detect eccDNA in clinical samples, and also shows the random distribution of eccDNA in clinical samples. The results are in line with expectations.

[0051] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the specification and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting free eccDNA based on nested PCR combined with CRISPR-CAS12a, characterized in that: The steps include: Step 1, taking a serum sample and extracting free DNA in the serum sample; Step 2: using enzyme digestion to remove the linear DNA in the free DNA in step 1 and retain the eccDNA; Step 3: Using eccDNA-1 and eccDNA-2 as target genes, a long primer pair was used for the first round of PCR amplification, and then a short primer pair was used for the second round of PCR amplification; Step 4: Use the CRISPR-CAS12a detection system to detect the target eccDNA, wherein the crRNA sequence targeting the target eccDNA-1 is shown in SEQ ID NO.5, and the crRNA sequence targeting the target eccDNA-2 is shown in SEQ ID NO.10; then the fluorescence intensity of the reaction system is detected.

2. The method for detecting free eccDNA based on nested PCR combined with CRISPR-CAS12a according to claim 1, characterized in that: The physical position of the eccDNA-1 is Chr17:64682690-64683063; the physical position of the eccDNA-2 is Chr8:100632795-100633127.

3. The method for detecting free eccDNA based on nested PCR combined with CRISPR-CAS12a according to claim 1, characterized in that: The long primer pair includes a first long primer pair for eccDNA-1, whose sequences are shown in SEQ ID NO.1 and SEQ ID NO.2; and a second long primer pair for eccDNA-2, whose sequences are shown in SEQ ID NO.3 and SEQ ID NO.

4.

4. The method for detecting free eccDNA based on nested PCR combined with CRISPR-CAS12a according to claim 1, characterized in that: The short primer pair includes a first short primer pair for eccDNA-1, whose sequences are shown in SEQ ID NO.6 and SEQ ID NO.7; and a second short primer pair for eccDNA-2, whose sequences are shown in SEQ ID NO.8 and SEQ ID NO.

9.

5. The method for detecting free eccDNA based on nested PCR combined with CRISPR-CAS12a according to claim 1, characterized in that: The program for the first round of PCR amplification was: pre-denaturation at 95°C for 10 min, followed by denaturation at 95°C for 15 s, annealing at 45°C for 30 s, and extension at 72°C for 30 s. After 20 cycles, the product was briefly stored at 4°C.

6. The method for detecting free eccDNA based on nested PCR combined with CRISPR-CAS12a according to claim 1, characterized in that: The program for the second round of PCR amplification was: pre-denaturation at 95°C for 10 min, followed by denaturation at 95°C for 15 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. After 40 cycles, the product was briefly stored at 4°C.

7. The method for detecting free eccDNA based on nested PCR combined with CRISPR-CAS12a according to claim 1, characterized in that: The detection of fluorescence intensity is a real-time fluorescence quantitative detection method.

8. A nucleic acid molecule combination for use in the method of claim 1, characterized in that: include: A first long primer pair as shown in sequences SEQ ID NO.1 and SEQ ID NO.2, a second long primer pair as shown in sequences SEQ ID NO.3 and SEQ ID NO.4, a first short primer pair as shown in sequences SEQ ID NO.6 and SEQ ID NO.7, a second short primer pair as shown in sequences SEQ ID NO.8 and SEQ ID NO.9, and crRNA as shown in sequences SEQ ID NO.5 and SEQ ID NO.10.

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