Primer composition, primer probe composition and kit for detecting cfDNA integrity and application of primer composition, primer probe composition and kit

Abstract

The invention provides a primer composition, a primer probe composition and a kit for detecting cfDNA integrity and application of the primer composition, the primer probe composition and the kit. The kit comprises a primer composition and a primer probe composition, the primer composition comprises an ALU115 upstream primer, an ALU115 downstream primer, an ALU247 upstream primer and an ALU247 downstream primer, the primer probe composition comprises an ALU115 primer probe composition and an ALU247 primer probe composition, and the kit comprises the ALU115 primer probe composition and the ALU247 primer probe composition and further comprises an internal control gene primer probe composition. The invention also provides application of the primer composition, the primer probe composition and the kit in preparation of a product for detecting cfDNA integrity. The invention provides a specific primer and probe composition aiming at ALU115 and ALU247 genes, non-specific amplification is not generated, and high specificity and high sensitivity of detection are achieved. The detection of cfDNA integrity by using the specific primer and probe composition has the advantages of short detection time and low cost, and can be used for rapid detection of cfDNA level detection.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and more specifically to a primer composition, a primer probe composition, a kit and applications thereof for detecting cfDNA integrity. Technical Background

[0002] Free nucleic acid (cfDNA) exists outside cells, in plasma and various other biological fluids, including DNA, mRNA, mitochondrial DNA, etc. Free nucleic acid DNA (cfDNA) is composed of nuclear DNA fragments in the blood and originates from various cells in the body. The release of cfDNA is caused by apoptosis, necrosis or inflammatory cell secretion. The presence of cfDNA and the integrity of cfDNA can reflect different physiological or pathological states of the body. The DNA fragments released from apoptotic cells are usually 185 to 200 base pairs (bp) in length. This uniformly truncated DNA is produced by programmed enzymatic cleavage during apoptosis. In contrast, cfDNA fragments from necrotic cells or tumor cells are longer, indicating more disordered decomposition of DNA. Therefore, high levels of long DNA fragments can serve as potential markers of disease. The ratio of long DNA fragments from necrosis (ALU247) to short apoptotic fragments (ALU115) representing total cfDNA represents the level of cfDNA integrity. The higher the ratio, the more conducive to the occurrence and development of the disease.

[0003] The prior art has not disclosed a suitable ALU115, ALU247 primer and probe combination for detecting cfDNA integrity. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a primer composition, a primer probe composition, a kit and applications thereof for detecting cfDNA integrity.

[0005] On the one hand, the present invention provides a primer composition for detecting the integrity of cfDNA, comprising an ALU115 upstream primer, an ALU115 downstream primer, an ALU247 upstream primer, and an ALU247 downstream primer; the nucleotide sequence of the ALU115 upstream primer is shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 or SEQ ID No.4, and the nucleotide sequence of the ALU115 downstream primer is shown in SEQ ID No.5, SEQ ID No.6, SEQ ID No.7 or SEQ ID No.8.

[0006] On the other hand, the present invention provides a primer-probe composition for detecting cfDNA integrity, comprising an ALU115 primer-probe composition and an ALU247 primer-probe composition, wherein the ALU115 primer-probe composition comprises the ALU115 upstream primer, the ALU115 downstream primer and the ALU115 probe, and the ALU247 primer-probe composition comprises the ALU247 upstream primer, the ALU247 downstream primer and the ALU247 probe; the nucleotide sequence of the ALU115 probe is shown in SEQ ID No. 9 or SEQ ID No. 10, and the nucleotide sequence of the ALU247 probe is shown in SEQ ID No. 19 or SEQ ID No. 20.

[0007] In another aspect, the present invention provides a kit for detecting the integrity of cfDNA, comprising the ALU115 primer-probe composition and the ALU247 primer-probe composition, and also comprising an internal control gene primer-probe composition, namely, an internal control gene upstream primer, an internal control gene downstream primer and an internal control gene probe, wherein the nucleotide sequence of the internal control gene upstream primer is shown in SEQ ID No.21 or SEQ ID No.22, the nucleotide sequence of the internal control gene downstream primer is shown in SEQ ID No.23 or SEQ ID No.24, and the nucleotide sequence of the internal control gene probe is shown in SEQ ID No.25 or SEQ ID No.26;

[0008] Preferably, the 5' end of the probe is labeled with a fluorescent reporter group.

[0009] Further preferably, the fluorescent reporter group is at least one of FAM, HEX, VIC, ROX, TAMRA or CY5;

[0010] More preferably, the 5' end of the probe ALU115 and the ALU247 probe is labeled with a FAM fluorescent group, and the 5' end of the internal control gene probe is labeled with a HEX fluorescent group;

[0011] Preferably, the 3' end of the probe is labeled with a quencher group;

[0012] Further preferably, the quenching group at the 3' end of the probe is at least one of TAMRA, BHQ1, BHQ2, MGB, DABCYL or BHQ3;

[0013] More preferably, the 3' end of the probe ALU115 and the probe ALU247 is labeled with an MGB quenching group, and the 3' end of the internal control gene probe is labeled with a BHQ1 quenching group.

[0014] Preferably, the ALU115 primer-probe combination is one of the following combinations:

[0015] 1) Primer probe combination for detecting the level of ALU115 gene position 1: SSEQ ID No. 1, SEQ ID No. 5 and SEQ ID No. 9;

[0016] 2) Primer probe combination for detecting the level of ALU115 gene position 2: SEQ ID No. 1, SEQ ID No. 7 and SEQ ID No. 10;

[0017] 3) Primer probe combination for detecting the level of ALU115 gene position 3: SEQ ID No. 2, SEQ ID No. 6 and SEQ ID No. 10;

[0018] 4) Primer probe combination for detecting the level of position 4 of the ALU115 gene: SEQ ID No. 2, SEQ ID No. 8 and SEQ ID No. 9;

[0019] 5) Primer probe combination for detecting the level of position 5 of the ALU115 gene: SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No. 10;

[0020] 6) Primer probe combination for detecting the level of position 6 of the ALU115 gene: SEQ ID No. 4, SEQ ID No. 6 and SEQ ID No. 9;

[0021] Preferably, the ALU247 primer-probe combination is one of the following combinations:

[0022] 1) Primer probe combination for detecting the level of ALU247 gene position 1: SEQ ID No. 11, SEQ ID No. 16 and SEQ ID No. 19;

[0023] 2) Primer probe combination for detecting the level of ALU247 gene position 2: SEQ ID No. 11, SEQ ID No. 17 and SEQ ID No. 20;

[0024] 3) Primer probe combination for detecting the level of ALU247 gene position 3: SEQ ID No. 12, SEQ ID No. 15 and SEQ ID No. 20;

[0025] 4) Primer probe combination for detecting the level of position 4 of the ALU247 gene: SEQ ID No. 12, SEQ ID No. 18 and SEQ ID No. 19;

[0026] 5) Primer probe combination for detecting the level of position 5 of the ALU247 gene: SEQ ID No. 13, SEQ ID No. 15 and SEQ ID No. 19;

[0027] 6) Primer probe combination for detecting the level of ALU247 gene position 6: SEQ ID No. 14, SEQ ID No. 18 and SEQ ID No. 20;

[0028] Preferably, the internal control gene primer-probe combination is one of the following combinations:

[0029] 1) Primer probe combination for detecting the level of Arabidopsis thaliana internal control gene position 1: SEQ ID No. 21, SEQ ID No. 23 and SEQ ID No. 25;

[0030] 2) Primer probe combination for detecting the level of Arabidopsis internal control gene position 2: SEQ ID No. 22, SEQ ID No. 24 and SEQ ID No. 25;

[0031] 3) Primer probe combination for detecting the level of position 3 of the internal control gene of Arabidopsis thaliana: SEQ ID No.22, SEQ ID No.24 and SEQ ID No.26.

[0032] Preferably, the nucleotide sequences of ALU115 and ALU247 are shown in SEQ ID No.27.

[0033] Preferably, the nucleotide sequence of the internal control gene is shown as SEQ ID No.28.

[0034] Preferably, the ALU115 gene position 1 is at the 64th-178th base of SEQ ID No.27, the ALU115 gene position 2 is at the 64th-187th base of SEQ ID No.27, the ALU115 gene position 3 is at the 63rd-176th base of SEQ ID No.27, and the ALU115 gene position 4 is at the 63rd-188th base of SEQ ID No.27; the ALU115 gene position 5 is at the 81st-178th base of SEQ ID No.27, and the ALU115 gene position 6 is at the 74th-176th base of SEQ ID No.27; the ALU247 gene position 1 is at the 12th-275th base of SEQ ID No.27, the ALU247 gene position 2 is at the 12th-280th base of SEQ ID No.27, and the ALU247 gene position 3 is at the 12th-280th base of SEQ ID No.27. No.27 at the 18th-258th base, ALU247 gene position 4 at the 18th-253rd base of SEQ ID No.27, ALU247 gene position 5 at the 19th-258th base of SEQ ID No.27; ALU247 gene position 6 at the 19th-253rd base of SEQ ID No.27; internal control gene position 1 at the 338th-440th base of SEQ ID No.28, internal control gene position 2 at the 352nd-441st base of SEQ ID No.28, and internal control gene position 3 at the 352nd-441st base of SEQ ID No.28.

[0035] Furthermore, the kit includes, in addition to the aforementioned primer-probe combination, PCR buffer (20mMTris-SO4, 8mM MgCl2, 50mM K2SO4, 0.2mM dNTP, 5% glycerol, 0.1% Tween, 1M betaine, 10mMTMAC), Taq enzyme and an internal control plasmid, the nucleotide sequence of the internal control plasmid being shown in SEQ ID NO.28.

[0036] Finally, the present invention provides the use of the aforementioned primer composition, primer probe composition, and kit in the preparation of a product for detecting cfDNA integrity.

[0037] Specifically, the product includes but is not limited to reagents or kits for detecting the level of cfDNA integrity.

[0038] Furthermore, a method for detecting the integrity level of cfDNA using the product comprises the following steps:

[0039] 1) Extract human genomic DNA;

[0040] Human peripheral blood DNA was extracted using a healthy human serum / plasma circulating DNA extraction kit. After the peripheral blood DNA was detected by UV spectrophotometer, the DNA concentration was required to be no less than 40 ng / μL and the OD260 / OD280 ratio was no less than 1.80;

[0041] 2) The sample DNA was subjected to PCR amplification of ALU115 and ALU247 genes using the aforementioned primer-probe combination, and the Ct value of the ALU115 gene was measured by FAM, the Ct value of the ALU247 gene was measured by FAM, and the Ct value of the internal control gene was measured by HEX.

[0042] 3) The cfDNA of the extracted sample and the human genomic DNA standard substance from leukocytes were serially diluted and subjected to PCR reaction by PCR reagents, and the concentrations of ALU115 and AUL247 were calculated by establishing corresponding standard curves. The integrity of cfDNA was evaluated by the ratio of AUL247 to ALU115 concentrations.

[0043] Preferably, in steps 2 and 3), the detection of ALU115 and ALU247 genes is performed by two tubes for independent amplification detection (i.e., ALU115 and ALU247 are amplified independently), and the reaction system for PCR amplification of ALU115 and ALU247 genes is as follows:

[0044]

[0045] Further preferably, the reaction system for PCR amplification of ALU115 and ALU247 genes in steps 2 and 3) is reaction system 2, and the amount of each reagent added is as follows:

[0046]

[0047]

[0048] Preferably, in steps 2, 3) the ALU115 and ALU247 gene amplification procedures are as follows:

[0049]

[0050] Furthermore, the above detection method also includes an internal control, by adding an internal control primer probe composition, performing an internal control detection during specific real-time quantitative fluorescence PCR detection, and when the Ct value of the fluorescence channel where the internal control probe is located is less than the set value and the amplification curve is normal, the sample detection is judged to be normal. This method uses specific primer probes designed for the ALU115 and ALU247 genes, and selects Arabidopsis as an internal control. The entire detection method has the advantages of simple operation process, short detection time, and high specificity.

[0051] The beneficial effects of the present invention are as follows: the present invention provides a specific primer and probe combination for ALU115 and ALU247 genes, which does not produce nonspecific amplification of other alleles, thereby achieving high specificity and high sensitivity of detection. The detection of cfDNA integrity using a specific primer and probe combination has the advantages of short detection time and low cost, and can be used for rapid detection of cfDNA levels. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 It is a specific amplification graph of primer probe combination screening, wherein AR represents ALU115 gene position 1 primer probe combination, ALU115 gene position 2 primer probe combination, ALU115 gene position 3 primer probe combination, ALU115 gene position 4 primer probe combination, ALU115 gene position 5 primer probe combination, ALU115 gene position 6 primer probe combination, ALU115 other primer probe combination 1, ALU115 other primer probe combination 2, ALU115 other primer probe combination 3, ALU247 gene position 1 primer probe combination, ALU247 gene position 2 primer probe combination, ALU247 gene position 3 primer probe combination, ALU247 gene position 4 primer probe combination, ALU247 gene position 5 primer probe combination, ALU247 gene position 6 primer probe combination, ALU247 other primer probe combination 1, ALU247 other primer probe combination 2, ALU247 other primer probe combination 3.

[0053] Figure 2 The sensitivity amplification diagram of the primer-probe combination screening, where AR represents the primer-probe combination at position 1 of the ALU115 gene (sample dilution factor: 10 5 ), ALU115 gene position 2 primer probe combination (sample dilution factor: 10 4 ), ALU115 gene position 3 primer probe combination (sample dilution factor: 10 4 ), ALU115 gene position 4 primer probe combination (sample dilution factor: 10 4 ), ALU115 gene position 5 primer probe combination (sample dilution factor: 10 4 ), ALU115 gene position 6 primer probe combination (sample dilution factor: 10 4 ), ALU115 other primer probe combination 1 (sample dilution factor: 10 3 ), ALU115 other primer probe combination 2 (sample dilution factor: 10 3 ), ALU115 other primer probe combination 3 (sample dilution factor: 10 3 , ALU247 gene position 1 primer probe combination (sample dilution factor: 105 ), ALU247 gene position 2 primer probe combination (sample dilution factor: 10 4 ), ALU247 gene position 3 primer probe combination (sample dilution factor: 10 4 ), ALU247 gene position 4 primer probe combination (sample dilution factor: 10 4 ), ALU247 gene position 5 primer probe combination (sample dilution factor: 10 4 ), ALU247 gene position 6 primer probe combination (sample dilution factor: 10 4 ), ALU247 other primer probe combination 1 (sample dilution factor: 10 3 ), ALU247 other primer probe combination 2 (sample dilution factor: 10 3 ), ALU247 other primer probe combination 3 (sample dilution factor: 10 3 ).

[0054] Figure 3 This is the cross-reaction result diagram between the internal standard Arabidopsis gene and the target gene ALU.

[0055] Figure 4 It is the map of internal control plasmid and ALU plasmid and the plate map of successful transformation of internal control plasmid and ALU plasmid into Escherichia coli, where A and B represent Arabidopsis internal control colony and ALU colony, respectively.

[0056] Figure 5 AF represents ALU115 reaction solution 1, ALU115 reaction solution 2, ALU115 reaction solution 2, ALU247 reaction solution 1, ALU247 reaction solution 2, and ALU247 reaction solution 3, respectively.

[0057] Figure 6 Figure 3. Differences in cfDNA integrity between Parkinson's disease patients and healthy controls. DETAILED DESCRIPTION

[0058] The present invention is further described in detail in conjunction with the following specific examples, which are not intended to limit the present invention but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0059] Example 1 Design and screening of primer probes for detecting cfDNA integrity levels

[0060] The main principle of the present invention is: in the fluorescent probe PCR reaction system, as the PCR amplifies, the Taq enzyme encounters the probe that specifically binds to the template during chain extension, and the 3'-5' exonuclease activity of the Taq enzyme cuts off the probe, and the fluorescent group moves away from the quenching group, so that the instrument can detect a specific fluorescent signal. The Ct value of the fluorescent PCR reaction is linearly correlated with the logarithmic value of the initial sample concentration. The higher the concentration, the smaller the Ct value.

[0061] The extracted candidate primers were screened according to the following requirements: ① probe length (L) between 20-30bp, ② Tm value between 58-61℃; ③ GC% between 40-75%; ④ poLyN≤4bp; ⑤ Hairpin≤4bp; ⑥ amplification product length controlled between 50-200bp; ⑦ primer Tm value is about 10℃ lower than probe Tm value;

[0062] It should be noted that the detection in the present invention is performed in two tubes for independent amplification detection (i.e., ALU115 and ALU247 are amplified independently), so the probes of ALU115 and ALU247 can use the same fluorescent signal. The 5' end of probe ALU115 is labeled with a FAM fluorescent group, and the 3' end is labeled with an MGB quenching group. The 5' end of probe ALU247 is labeled with a FAM fluorescent group, and the 3' end is labeled with an MGB quenching group.

[0063] In this example, the ALU sequence was used as a template, and the primer probe related sequences screened were shown in Tables 2 and 3. The strains in Table 1 were amplified to determine the specificity of the primer probe. The three strains were cultured in the corresponding appropriate culture medium, and the culture solution was counted by the dilution plate method. The concentration of each strain in the sample was diluted to 1×10 4 CFU / μL, and PCR amplification was performed. The ALU sequence template was diluted to a certain extent to determine the sensitivity of the primer-probe combination.

[0064] Table 1 Bacterial species information

[0065] Species name serial number source Escherichia coli CMCC(B)44102 China Food and Drug Inspection Institute Staphylococcus aureus CMCC(B)26003 China Food and Drug Inspection Institute Candida albicans CMCC(F)98001 China Food and Drug Inspection Institute

[0066] Table 2 Primer probe sequence information

[0067]

[0068]

[0069] Table 3 Other primer-probe combinations

[0070]

[0071] The primer-probe combinations in Table 2 and Table 3 were screened by specific amplification test. The experimental results are shown in Figure 1 As shown, the primer and probe combinations selected in Table 2 can achieve high specificity of detection and do not produce non-specificity compared with other primer and probe combinations in Table 3. It can be seen that the primer and probe combinations ALU115 and ALU2476 in Table 2 are both superior.

[0072] The primer-probe combinations in Table 2 and Table 3 were screened by sensitivity amplification. 2 , 10 3 , 10 4 , 10 5 The primer-probe combination is amplified at 100 times the original amount. Figure 2 As shown, the sensitivity of the six combinations of primers and probes of the ALU115 gene (Table 2) is better than that of the other groups of primers and probes (Table 3), among which the sample concentration is the lowest, the sensitivity is the best, and the primer and probe combination is the best when the ALU115 gene is at position 1. The sensitivity of the six combinations of primers and probes of the ALU247 gene is better than that of the other groups of primers and probes, among which the sample concentration is the lowest, the sensitivity is the best, and the primer and probe combination is the best when the ALU247 gene is at position 1.

[0073] The final selected primer-probe combinations are as follows:

[0074] 1) Primer probe combination for detecting the level of ALU115 gene position 1: SEQ ID No. 1, SEQ ID No. 5 and SEQ ID No. 9;

[0075] 2) Primer probe combination for detecting the level of ALU115 gene position 2: SEQ ID No. 1, SEQ ID No. 7 and SEQ ID No. 10;

[0076] 3) Primer probe combination for detecting the level of ALU115 gene position 3: SEQ ID No. 2, SEQ ID No. 6 and SEQ ID No. 10;

[0077] 4) Primer probe combination for detecting the level of position 4 of the ALU115 gene: SEQ ID No. 2, SEQ ID No. 8 and SEQ ID No. 9;

[0078] 5) Primer probe combination for detecting the level of position 5 of the ALU115 gene: SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No. 10;

[0079] 6) Primer probe combination for detecting the level of position 6 of the ALU115 gene: SEQ ID No. 4, SEQ ID No. 6 and SEQ ID No. 9;

[0080] 7) Primer probe combination for detecting the level of ALU247 gene position 1: SEQ ID No. 11, SEQ ID No. 16 and SEQ ID No. 19;

[0081] 8) Primer probe combination for detecting the level of ALU247 gene position 2: SEQ ID No. 11, SEQ ID No. 17 and SEQ ID No. 20;

[0082] 9) Primer probe combination for detecting the level of ALU247 gene position 3: SEQ ID No. 12, SEQ ID No. 15 and SEQ ID No. 20;

[0083] 10) Primer probe combination for detecting the level of ALU247 gene position 4: SEQ ID No. 12, SEQ ID No. 18 and SEQ ID No. 19;

[0084] 11) Primer probe combination for detecting the level of position 5 of the ALU247 gene: SEQ ID No. 13, SEQ ID No. 15 and SEQ ID No. 19;

[0085] 12) Primer probe combination for detecting the level of ALU247 gene position 6: SEQ ID No. 14, SEQ ID No. 18 and SEQ ID No. 20;

[0086] 13) Primer probe combination for detecting the level of Arabidopsis thaliana internal control gene position 1: SEQ ID No. 21, SEQ ID No. 23 and SEQ ID No. 25;

[0087] 14) Primer probe combination for detecting the level of Arabidopsis thaliana internal control gene position 2: SEQ ID No. 22, SEQ ID No. 24 and SEQ ID No. 25;

[0088] 15) Primer probe combination for detecting the level of position 3 of the internal control gene of Arabidopsis thaliana: SEQ ID No.22, SEQ ID No.24 and SEQ ID No.26.

[0089] Example 2 Plasmid Design and Plasmid Transformation of Arabidopsis Internal Control Plasmid and ALU

[0090] First of all, Arabidopsis thaliana is widely present in nature. Compared with endogenous internal standards, the selection of exogenous internal standards Arabidopsis thaliana has its unique significance in nucleic acid detection, which is mainly reflected in the wider monitoring range: exogenous internal standards usually use artificial synthetic plasmids. These non-human genes are added to the sample before extraction, which can more comprehensively monitor the entire process from nucleic acid extraction to product analysis. In contrast, although endogenous internal standards can monitor the sampling and amplification processes, they cannot fully monitor problems in the extraction process, such as amplification efficiency or amplification inhibition. The exogenous internal standard Arabidopsis thaliana has poor homology with the target gene, which reduces cross-reactions.

[0091] The results are as follows Figure 3 As shown, the Arabidopsis gene and the ALU gene sequence were compared and found to have poor homology. By using a set of primer-probe combinations finally screened in Example 1, the Arabidopsis plasmid was amplified with the primer-probe combination for detecting the level of position 1 of the ALU115 gene: SEQ ID No. 1, SEQ ID No. 5 and SEQ ID No. 9 and the primer-probe combination for detecting the level of position 1 of the ALU247 gene: SEQ ID No. 11, SEQ ID No. 16 and SEQ ID No. 19, and it was found that no cross reaction was generated, as shown in FIG. Figure 3 shown.

[0092] As a commonly used gene carrier, plasmid plays a vital role in the construction of PCR diagnostic kits. By amplifying specific gene fragments through PCR technology and cloning them into plasmids, it is easy to accurately obtain, store and subsequently analyze the gene sequence. Compared with plasmids containing internal control genes, external internal control plasmids have a series of advantages. These advantages are mainly reflected in the stability, reliability, accuracy and flexibility of the experiment.

[0093] First, the target fragment of the internal control plasmid was artificially synthesized, and the sequence was shown in SEQ ID NO.28.

[0094] Secondly, the target fragment was transferred into the pUC57 plasmid. The plasmid map is shown in Figure 4 The constructed plasmid was used as the internal control plasmid of the reaction system.

[0095] Finally, the constructed plasmid described above was successfully transformed into the corresponding E. coli competent cells through heat stress. The specific steps are as follows:

[0096] 1) Light an alcohol lamp in a clean bench, take 100 μl of competent cell suspension into a sterilized pre-cooled 1.5 mL centrifuge tube next to the flame, and place it on an ice box for 5 minutes to thaw;

[0097] 2) Add the internal control and ALU plasmid solutions to the centrifuge tubes respectively, shake gently to mix. Place on ice for 30 minutes;

[0098] 3) Place the centrifuge tube in a metal bath, heat shock at 42°C for 90 seconds, and then quickly cool on ice for 3 minutes

[0099] 4) Add 1 ml of LB liquid culture medium to the centrifuge tube, mix well and place in a constant temperature water bath shaker at 37°C.

[0100] The culture was shaken at 220 rpm for 1 h, centrifuged at 5000 rpm for 10 min, 900 μl of supernatant was aspirated and discarded, and the remaining bacterial solution was mixed.

[0101] 5) Spread the mixed bacterial solution onto LB solid screening medium using a spreading rod, and invert it in a biochemical incubator at 37°C for 16-24 hours.

[0102] The results are as follows Figure 4 As shown, the constructed plasmid was successfully transformed into the corresponding Escherichia coli competent cells through heat stress.

[0103] Example 3 Real-time fluorescence PCR detection method for cfDNA integrity level using the screened primer probe combination

[0104] (1) Extraction of cfDNA from peripheral blood samples

[0105] A healthy human serum / plasma circulating DNA extraction kit (Jiangsu Provi Biotechnology Co., Ltd.) was used to extract human peripheral blood DNA. After the peripheral blood DNA was detected by UV spectrophotometer, the DNA concentration was required to be no less than 40 ng / μL and the OD260 / OD280 ratio was required to be no less than 1.80;

[0106] (2) Optimization of genomic DNA-specific real-time fluorescence PCR detection system

[0107] Optimize the components of primers, probes and enzyme reagents to determine the optimal system.

[0108] 1) Amplification effects of different PCR reaction systems for ALU115 gene level detection Figure 5 The amount of each reagent added in the system is shown in Table 4.

[0109] Table 4 Reaction system for ALU115 gene level detection

[0110]

[0111]

[0112] The 5' end of the ALU115 fluorescent probe was modified with a FAM fluorescent group and the 3' end was modified with an MGB group, while the 5' end of the internal control gene fluorescent probe was modified with a HEX fluorescent group and the 3' end was modified with a BHQ1 group.

[0113] 2) Amplification effects of different PCR reaction systems for ALU247 gene level detection Figure 5 The amount of each reagent added in the system is shown in Table 5.

[0114] Table 5 Reaction system for ALU247 gene level detection

[0115]

[0116]

[0117] The 5' end of the ALU247 fluorescent probe was modified with a FAM fluorescent group and the 3' end was modified with an MGB group, while the 5' end of the internal control gene fluorescent probe was modified with a HEX fluorescent group and the 3' end was modified with a BHQ1 group.

[0118] 3) The ALU115 and ALU247 gene amplification procedures are shown in Table 6.

[0119] Table 6 Gene amplification procedures

[0120]

[0121] 4) The selected primer-probe combination is suitable for amplification in reaction system 2, wherein the primer-probe combination for detecting the level of ALU115 gene position 1: SEQ ID No. 1, SEQ ID No. 5 and SEQ ID No. 9 and the primer-probe combination for detecting the level of ALU247 gene position 1: SEQ ID No. 11, SEQ ID No. 16 and SEQ ID No. 19 amplification results are as follows: Figure 5 The experimental results show that the selected primer and probe combination can efficiently amplify ALU115 and ALU247 genes under reaction system 2 and the above reaction procedure, without producing nonspecific amplification of other alleles, and can achieve high specificity and high amplification efficiency of detection.

[0122] Example 4 cfDNA integrity level detection

[0123] According to the optimized detection method provided in Example 3, 30 healthy subjects and 30 Parkinson's disease patients from the Xuanwu Hospital of Capital Medical University were tested for peripheral blood circulating free nucleic acid DNA, each repeated 3 times, and the concentration ratios of ALU247 and ALU115 were calculated by fluorescent quantitative PCR to determine the cfDNA integrity level.

[0124] The results are shown in Table 7. The primer-probe combination for detecting the level of ALU115 gene position 1: SEQ ID No. 1, SEQ ID No. 5 and SEQ ID No. 9 and the primer-probe combination for detecting the level of ALU247 gene position 1: SEQ ID No. 11, SEQ ID No. 16 and SEQ ID No. 19 were used for amplification, and the cfDNA integrity level can be stably and accurately detected, as shown in Table 7. Figure 6 As shown, the primer-probe combination detected significantly greater cfDNA integrity in Parkinson's patients than in healthy subjects (p < 0.001), providing a better primer-probe combination for accurate diagnosis of neurological diseases.

[0125] Table 7 cfDNA integrity level detection

[0126]

[0127]

Claims

1. A primer composition for detecting cfDNA integrity, Features: It includes an ALU115 upstream primer, an ALU115 downstream primer, an ALU247 upstream primer, and an ALU247 downstream primer; the nucleotide sequence of the ALU115 upstream primer is shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 or SEQ ID No.4, and the nucleotide sequence of the ALU115 downstream primer is shown in SEQ ID No.5, SEQ ID No.6, SEQ ID No.7 or SEQ ID No.

8.

2. A primer-probe combination for detecting cfDNA integrity, Features: It comprises an ALU115 primer-probe composition and an ALU247 primer-probe composition, wherein the ALU115 primer-probe composition comprises the ALU115 upstream primer, the ALU115 downstream primer and the ALU115 probe in the primer composition according to claim 1, and the ALU247 primer-probe composition comprises the ALU247 upstream primer, the ALU247 downstream primer and the ALU247 probe in the primer composition according to claim 1; the nucleotide sequence of the ALU115 probe is shown in SEQ ID No.9 or SEQ ID No.10, and the nucleotide sequence of the ALU247 probe is shown in SEQ ID No.19 or SEQ ID No.

20.

3. The primer-probe composition according to claim 2, Features: The ALU115 primer-probe combination is one of the following sequence combinations: (1) SEQ ID No.1, SEQ ID No.5 and SEQ ID No.9; (2) SEQ ID No.1, SEQ ID No.7 and SEQ ID No.10; (3) SEQ ID No.2, SEQ ID No.6 and SEQ ID No.10; (4) SEQ ID No.2, SEQ ID No.8 and SEQ ID No.9; (5) SEQ ID No.3, SEQ ID No.5 and SEQ ID No.10; (6) SEQ ID No.4, SEQ ID No.6 and SEQ ID No.9; The ALU247 primer-probe combination is one of the following combinations: (1) SEQ ID No.11, SEQ ID No.16 and SEQ ID No.19; (2) SEQ ID No.11, SEQ ID No.17 and SEQ ID No.20; (3) SEQ ID No.12, SEQ ID No.15 and SEQ ID No.20; (4) SEQ ID No.12, SEQ ID No.18 and SEQ ID No.19; (5) SEQ ID No.13, SEQ ID No.15 and SEQ ID No.19; (6) SEQ ID No. 14, SEQ ID No. 18 and SEQ ID No.

20.

4. A kit for detecting the integrity of cfDNA, Features: The method comprises the ALU115 primer-probe composition and the ALU247 primer-probe composition in the primer-probe composition described in claim 2 or 3, and also comprises an internal control gene primer-probe composition, namely, an internal control gene upstream primer, an internal control gene downstream primer and an internal control gene probe, wherein the nucleotide sequence of the internal control gene upstream primer is shown in SEQ ID No.21 or SEQ IDNo.22, the nucleotide sequence of the internal control gene downstream primer is shown in SEQ ID No.23 or SEQ IDNo.24, and the nucleotide sequence of the internal control gene probe is shown in SEQ IDNo.25 or SEQ IDNo.

26.

5. The kit according to claim 4, Features: The internal control gene primer probe composition is one of the following combinations: (1) SEQ ID No. 21, SEQ ID No. 23 and SEQ ID No. 25; (2) SEQ ID No. 22, SEQ ID No. 24 and SEQ ID No. 25; (3) SEQ ID No. 22, SEQ ID No. 24 and SEQ ID No.

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6. The kit according to claim 4, Features: The 5' end of the ALU115 probe is modified with a FAM fluorescent group, and the 3' end is modified with an MGB group. The 5' end of the ALU247 probe is modified with a FAM fluorescent group, and the 3' end is modified with an MGB group. The 5' end of the internal control gene probe is modified with a HEX fluorescent group, and the 3' end is modified with a BHQ1 group.

7. The kit according to claim 4, Features: The invention also comprises a PCR reaction solution and an internal control plasmid, wherein the PCR reaction solution comprises a reaction buffer and a Taq enzyme.

8. Use of the primer composition according to claim 1, the primer-probe composition according to claim 2, and the kit according to claim 4 in the preparation of a product for detecting cfDNA integrity.

9. The use according to claim 8, Features: The method for detecting the integrity of cfDNA by the product comprises the following steps: (1) Extraction of cfDNA from peripheral blood samples; (2) Genomic DNA-specific real-time fluorescence PCR amplification detection; (3) Detection of cfDNA integrity level.

10. The use according to claim 9, Features: In step (2), ALU115 and ALU247 are amplified independently, and the amplification reaction system is: 12.5 μl of 2×PCR reaction buffer, 0.5 μl of 5U / μL Taq enzyme, 1 μl of ALU115 / ALU247 probe, 1 μL of 10 μM ALU115 / ALU247 upstream primer, 1 μL of 10 μM ALU115 / ALU247 downstream primer, 0.5 μL of 10 μM internal control gene probe, 0.8 μL of 10 μM internal control gene upstream primer, 0.8 μL of 10 μM internal control gene downstream primer, 2 μL of internal control plasmid, 2 μL of genomic DNA sample, and the volume is made up to 25 μL with water; The amplification reaction program was: 95°C for 1 min; 95°C for 30 s, 62°C for 30 s, 72°C for 1 min, 35 cycles; the fluorescence signal was collected at 62°C.

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