A highly sensitive HPV virus detection kit

Abstract

The present invention relates to the technical field of virus detection, and discloses a high-sensitivity HPV virus detection kit. The kit adopts a high-yield magnetic bead for nucleic acid extraction, and the surface of the high-yield magnetic bead is grafted with cysteine-asparagine dipeptide; the cysteine-asparagine dipeptide is grafted on the surface of the high-yield magnetic bead through the carboxyl group in cysteine. The magnetic beads of the present invention can change the electrical properties of the surface of the magnetic beads by changing the pH, so that the magnetic beads can adsorb nucleic acids by electrostatic attraction, and can separate nucleic acids from the magnetic beads by electrostatic repulsion, thereby improving the adsorption and elution effect of nucleic acids, so that nucleic acid extraction has a higher yield, and then improve the detection sensitivity of the kit.

Description

Technical Field

[0001] The present invention relates to the technical field of virus detection, and in particular to a high-sensitivity HPV virus detection kit. Background Art

[0002] Human papillomavirus (HPV) is a spherical DNA virus belonging to the genus Papillomavirus A of the family Papovaviridae. It can infect host cells through micro-injuries of the epithelium, causing proliferation and lesions of squamous epithelial cells of human skin and mucous membranes, and triggering a variety of human benign and malignant tumor diseases. Currently, more than 200 HPV virus genotypes have been discovered, which can be divided into high-risk types (HR-HPV) that mainly cause malignant lesions and low-risk types (LR-HPV) that generally cause benign lesions. Among them, HPV-16 and HPV-18 are the most common carcinogenic types and are also the main factors causing cervical malignancies. At present, the detection of HPV virus usually adopts the PCR method. After the nucleic acid is extracted from the sample to be tested, the target fragment is amplified by PCR to achieve qualitative or quantitative detection.

[0003] The magnetic bead method uses the special properties of the magnetic bead surface to adsorb nucleic acids under certain conditions, and then elutes the nucleic acids from the magnetic beads by changing the conditions, thereby achieving the separation and purification of nucleic acids. It can be automated and operated in large quantities, and has the advantages of simple operation and short time. It is currently one of the main methods for extracting nucleic acids. The magnetic beads used for nucleic acid extraction mainly include silica magnetic beads, carboxyl magnetic beads and positively charged magnetic beads (such as amino magnetic beads and imidazole magnetic beads).

[0004] Positively charged magnetic beads adsorb nucleic acids through electrostatic attraction, and the adsorption of nucleic acids is strong, but the elution is difficult, which will cause a low yield. Due to the existence of the above problems, silica magnetic beads and carboxyl magnetic beads (such as patents CN201511007550.7 and CN202110951242.9) are usually used in the prior art. The surfaces of these two magnetic beads are negatively charged, and the positively charged salt ions in the system are mainly used to form salt bridges between the magnetic beads and the negatively charged nucleic acids, thereby realizing the adsorption of nucleic acids by magnetic beads. This method is easy to elute nucleic acids from magnetic beads (by removing salt ions in the system), but the adsorption effect of magnetic beads on nucleic acids is poor. In the process of washing the magnetic beads to remove impurities such as proteins before elution, nucleic acids are easy to fall off from the magnetic beads, which will also cause a low yield. And high yield is very important for the sensitivity of virus detection, especially in the early stage of the disease, the content of viral nucleic acid in the sample to be tested is usually low. If nucleic acid cannot be extracted with high yield, false negatives are likely to occur, resulting in missed detection. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a highly sensitive HPV virus detection kit. The magnetic beads used in the present invention are grafted with cysteine-asparagine dipeptide in a specific manner, which has good adsorption and elution effects on nucleic acids, can improve the yield of nucleic acid extraction, and thus improve the sensitivity of the HPV virus detection kit.

[0006] The specific technical scheme of the present invention is:

[0007] In a first aspect, the present invention provides high-yield magnetic beads for nucleic acid extraction, wherein the surface of the high-yield magnetic beads is grafted with cysteine-asparagine dipeptide; the cysteine-asparagine dipeptide is grafted on the surface of the high-yield magnetic beads via the carboxyl group in cysteine.

[0008] The present invention grafts cysteine-asparagine dipeptide on the surface of magnetic beads, and can utilize the change of the surface charge of the magnetic beads with the pH to realize the adsorption and elution of nucleic acids. Specifically, in the cysteine-asparagine dipeptide, the α-carboxyl group in the cysteine ​​forms a covalent bond with the magnetic beads, the α-amino group forms an amide bond with the α-carboxyl group in the asparagine, the sulfhydryl group is a free group, and the α-amino group in the asparagine is a free group; the sulfhydryl group in the cysteine ​​is uncharged when the pH is relatively low, and is negatively charged when the pH is greater than a certain value (the pK value of the sulfhydryl group); the α-amino group in the asparagine is positively charged when the pH is relatively low, and is uncharged when the pH is greater than a certain value (the pK value of the amino group). When the pH is controlled within the range of 7 to 8, the α-amino group in asparagine can be positively charged, other groups in the dipeptide (including the sulfhydryl group in cysteine) are uncharged, and the nucleic acid is negatively charged, so electrostatic attraction can be generated between the magnetic beads and the nucleic acid, and the nucleic acid is adsorbed onto the magnetic beads; when the pH is controlled within the range of 9.8 to 10.5, the nucleic acid is still negatively charged, and the sulfhydryl group in cysteine ​​can be negatively charged, and other groups in the dipeptide (including the free α-amino group in asparagine) are uncharged, so electrostatic repulsion can be generated between the nucleic acid and the magnetic beads, which helps to elute the nucleic acid from the magnetic beads. To achieve the above effect, the free groups in the dipeptide must have a suitable pK, so there are restrictions on the types of the two amino acids and the dipeptide connection order.

[0009] In the above process, the magnetic beads of the present invention adsorb nucleic acids through electrostatic attraction, so the adsorption force is strong, the adsorption effect on nucleic acids is good, and the nucleic acids can be prevented from falling off the magnetic beads during the washing process before elution; and the elution effect of nucleic acids can be improved by using electrostatic repulsion; in addition, since cysteine ​​and asparagine are not grafted separately on the magnetic beads, but grafted in the form of dipeptides, this method can reduce the distance between the two amino acids, and then during elution, increase the electrostatic repulsion between the sulfhydryl group and the nucleic acid adsorbed on the magnetic beads, and further improve the elution effect. Therefore, the use of the magnetic beads of the present invention to extract nucleic acids can improve the yield.

[0010] Preferably, the high-yield magnetic beads include a magnetic bead core and a hyperbranched dextran coated outside the magnetic bead core; the cysteine-asparagine dipeptide is grafted onto the hyperbranched dextran via the carboxyl group in cysteine.

[0011] Hyperbranched dextran is a natural hyperbranched polysaccharide with more branched structures in its molecular chain and more hydroxyl groups in each glucose monomer. After it is coated on the surface of magnetic beads to form a cross-linked network, a large number of evenly distributed hydroxyl groups can be introduced on the surface of the magnetic beads. By using the hydroxyl groups to graft cysteine-asparagine dipeptide, the dipeptide can have a higher grafting amount and grafting uniformity, which is beneficial to further improve the efficiency of nucleic acid extraction.

[0012] In a second aspect, the present invention provides a method for preparing the high-yield magnetic beads, comprising the following steps:

[0013] (A) Grafting hyperbranched dextran: hyperbranched dextran and an esterification catalyst are dissolved in water, carboxyl magnetic beads are added, and after being evenly dispersed, an esterification reaction is carried out, followed by magnetic separation, washing, and drying to obtain dextran-magnetic beads;

[0014] In step (A), the carboxyl groups on the surface of the carboxyl magnetic beads are used to react with the hydroxyl groups in the hyperbranched dextran to carry out an esterification reaction, and the hyperbranched dextran is covalently grafted onto the surface of the magnetic beads, thereby introducing a large number of hydroxyl groups on the surface of the magnetic beads.

[0015] (B) Grafting cysteine: Dissolve cysteine ​​with a protective group attached to the thiol group and an esterification catalyst in water, add dextran-magnetic beads, disperse them evenly, and then perform esterification reaction, followed by magnetic separation, washing, and drying to obtain Cys (protecting group)-magnetic beads;

[0016] In step (B), esterification reaction is carried out between the hydroxyl groups on the surface of dextran-magnetic beads and the carboxyl groups in cysteine ​​to graft cysteine ​​onto the magnetic beads;

[0017] (C) Grafting asparagine: dissolving asparagine with protecting groups attached to the α-amino group and the amide group and a condensing agent in an organic solvent, activating the carboxyl group, adding Cys (protecting group)-magnetic beads, dispersing them evenly, and then performing dehydration condensation to obtain Asn (protecting group)-Cys (protecting group)-magnetic beads; removing the protecting group to obtain Asn-Cys-magnetic beads, i.e., high-yield magnetic beads;

[0018] In step (C), the amino group (from cysteine) on the Cys (protecting group)-magnetic beads and the carboxyl group in asparagine are dehydrated and condensed to form an amide bond, and the asparagine is grafted onto the cysteine; in steps (B) and (C), the sulfhydryl group of cysteine ​​and the α-amino group and amide group of asparagine are all connected with protecting groups, which can prevent these groups from reacting in step (C), resulting in too much asparagine grafted on each cysteine, affecting the elution effect.

[0019] In the above process, compared with first synthesizing the dipeptide and then grafting the dipeptide onto the magnetic beads, the present invention adopts a method of sequentially grafting cysteine ​​and asparagine onto the magnetic beads, which is convenient for controlling the free groups in the dipeptide to be the sulfhydryl group on cysteine ​​and the α-amino group on asparagine, rather than the α-carboxyl group on the two amino acids or the α-amino group on cysteine, thereby achieving pH regulation of nucleic acid adsorption and elution.

[0020] Preferably, in step (A), the mass volume ratio of the magnetic beads, hyperbranched dextran and water is 1 g: 1.5-2.5 g: 100-120 mL.

[0021] Preferably, in step (B), the mass volume ratio of the dextran-magnetic beads, the cysteine ​​with a protecting group attached to the thiol group, and water is 1 g: 0.5-1.0 g: 15-25 mL.

[0022] Preferably, in step (C), the mass volume ratio of the Cys (protecting group)-magnetic beads, asparagine with a protecting group attached to the amino group, and the organic solvent is 1 g: 0.8-1.6 g: 15-25 mL.

[0023] In a third aspect, the present invention provides a high-sensitivity HPV virus detection kit, comprising the high-yield magnetic beads.

[0024] The kit of the present invention adopts special magnetic beads, which can improve the yield of nucleic acid extraction, thereby improving the sensitivity of HPV virus detection and reducing the occurrence of false negatives.

[0025] Preferably, the kit comprises a nucleic acid extraction reagent and a PCR amplification reagent; the nucleic acid extraction reagent comprises a magnetic bead suspension, a lysis solution, a cleaning solution A, a cleaning solution B and an elution solution; and the magnetic bead suspension contains the high-yield magnetic beads.

[0026] The use process of the kit is as follows: first, mix the sample to be tested (such as cervical exfoliated cells, skin exfoliated cells, urogenital tract secretions), magnetic bead suspension and lysis solution, control the pH in the range of 7 to 8, lyse the cells and viruses, release the nucleic acid, and adsorb the nucleic acid onto the magnetic beads; after separating the magnetic beads, wash them with cleaning solution A and cleaning solution B in turn; mix the washed magnetic beads with the elution solution, control the pH in the range of 9.8 to 10.5, and elute the nucleic acid from the magnetic beads; then perform PCR amplification on the separated nucleic acid to achieve quantitative or qualitative detection.

[0027] Furthermore, in the magnetic bead suspension, the content of high-yield magnetic beads is 20-30 mg / mL.

[0028] Furthermore, the lysate is a Tris-HCl buffer containing guanidine isothiocyanate and Triton X-100; wherein the concentrations of guanidine isothiocyanate and Tris-HCl are 3-4 mol / L and 10-30 mmol / L, respectively, and the volume concentration of Triton X-100 is 1-3%.

[0029] Furthermore, the nucleic acid extraction reagent also includes a proteinase K solution; in the proteinase K solution, the concentration of proteinase K is 5 to 10 wt%.

[0030] Furthermore, the eluent is a Tris-HCl buffer with a concentration of 10 to 30 mmol / L.

[0031] Furthermore, the cleaning solution A is a mixture of Tris-HCl buffer and ethanol in a volume ratio of 2 to 4:1; in the Tris-HCl buffer, the concentration of Tris-HCl is 10 to 30 mmol / L, and the pH of the Tris-HCl buffer is 7 to 8.

[0032] Furthermore, the cleaning solution B is a mixture of Tris-HCl buffer and ethanol in a volume ratio of 1:3-4; in the Tris-HCl buffer, the concentration of Tris-HCl is 10-30 mmol / L, and the pH of the Tris-HCl buffer is 7-8.

[0033] Furthermore, the PCR amplification reagents include HPV amplification primers, HPV detection Taqman probes, internal standard primers, internal standard Taqman probes, Taq enzyme, dNTPs, Tris-HCl buffer and UNG enzyme.

[0034] Furthermore, the PCR amplification reagent also includes a negative control and a positive control.

[0035] Compared with the prior art, the present invention has the following advantages:

[0036] (1) By grafting cysteine-asparagine dipeptide on the surface of magnetic beads through the carboxyl group in cysteine, the magnetic beads of the present invention can have a higher nucleic acid extraction yield, thereby making the HPV virus detection kit of the present invention have a higher sensitivity;

[0037] (2) By coating the surface of the magnetic beads with hyperbranched dextran and utilizing the hydroxyl groups therein to graft cysteine-asparagine dipeptide, the dipeptide can have a higher grafting amount and grafting uniformity, which is beneficial to further improve the efficiency of nucleic acid extraction and the detection sensitivity of the kit. DETAILED DESCRIPTION

[0038] The present invention will be further described below in conjunction with the embodiments.

[0039] Overall embodiment

[0040] A high-yield magnetic bead for nucleic acid extraction is prepared as follows:

[0041] (A) Grafting hyperbranched dextran: hyperbranched dextran and an esterification catalyst are dissolved in water, carboxyl magnetic beads are added, and after being evenly dispersed, an esterification reaction is carried out, followed by magnetic separation, washing, and drying to obtain dextran-magnetic beads;

[0042] (B) Grafting cysteine: Dissolve cysteine ​​with a protective group attached to the thiol group and an esterification catalyst in water, add dextran-magnetic beads, disperse them evenly, and then perform esterification reaction, followed by magnetic separation, washing, and drying to obtain Cys (protecting group)-magnetic beads;

[0043] (C) Grafting asparagine: Asparagine with protecting groups attached to the α-amino group and the amide group and a condensation agent are dissolved in an organic solvent, and after the carboxyl group is activated, Cys (protecting group)-magnetic beads are added. After being evenly dispersed, dehydration condensation is performed to obtain Asn (protecting group)-Cys (protecting group)-magnetic beads; the protecting group is removed to obtain Asn-Cys-magnetic beads, i.e., high-yield magnetic beads.

[0044] In some preferred embodiments, the specific process of step (A) is as follows: hyperbranched dextran and tungstophosphoric acid are dissolved in water, carboxyl magnetic beads are added, and after uniform dispersion, the mixture is stirred at 75-85° C. for 6-8 hours, and then magnetic separation, washing, and drying are performed in sequence to obtain dextran-magnetic beads.

[0045] Wherein, optionally, the mass volume ratio of the magnetic beads, hyperbranched dextran, tungstophosphoric acid and water is 1g:1.5-2.5g:7-10g:100-120mL.

[0046] In some preferred embodiments, the specific process of step (B) is as follows: S-trityl-L-cysteine ​​and tungstophosphoric acid are dissolved in water, dextran-magnetic beads are added, and after uniform dispersion, the mixture is stirred and reacted at 60-70° C. for 3-5 hours, and magnetic separation, washing, and drying are performed in sequence to obtain Cys (Trt)-magnetic beads.

[0047] Wherein, optionally, the mass volume ratio of the dextran-magnetic beads, S-trityl-L-cysteine, tungstophosphoric acid and water is 1 g: 0.5-1.0 g: 1-3 g: 15-25 mL.

[0048] In some preferred embodiments, the specific process of step (C) is as follows: Fmoc-N-trityl-L-asparagine, condensing agent HBTU and base DIEA are dissolved in solvent DMF, and after stirring and activating at room temperature for 10 to 15 minutes, Cys (protecting group)-magnetic beads are added, and after uniform dispersion, the reaction is stirred at room temperature for 2 to 3 hours, and magnetic separation, washing and drying are performed in sequence to obtain Asn (protecting group)-Cys (protecting group)-magnetic beads; the protecting group is removed to obtain Asn-Cys-magnetic beads, i.e., high-yield magnetic beads.

[0049] Wherein, optionally, the mass ratio of the Cys (protecting group)-magnetic beads, Fmoc-N-trityl-L-asparagine, HBTU, DIEA and DMF is 1g:0.8~1.6g:0.7~1.4g:0.9~1.8g:15~25mL.

[0050] In some preferred embodiments, in step (B), the protecting group connected to the cysteine ​​thiol group is a trityl group; in step (B), the protecting group connected to the asparagine α-amino group is a 9-fluorenylmethoxycarbonyl group, and the protecting group connected to the amide group is a trityl group; in step (C), the specific process of removing the protecting group is as follows: dissolving piperidine in DMF, adding Asn (protecting group) -Cys (protecting group) -magnetic beads, stirring and reacting for 0.5 to 1.5 hours; then adding I2, stirring and reacting for 1 to 2 hours, and then performing magnetic separation, washing, and drying in sequence to obtain Asn-Cys-magnetic beads, i.e., high-yield magnetic beads.

[0051] The magnetic beads are used to prepare a high-sensitivity HPV virus detection kit, which includes a nucleic acid extraction reagent and a PCR amplification reagent; the nucleic acid extraction reagent includes a magnetic bead suspension, a lysis solution, a proteinase K solution, a cleaning solution A, a cleaning solution B and an elution solution; the magnetic bead suspension contains the high-yield magnetic beads.

[0052] Optionally, the content of high-yield magnetic beads in the magnetic bead suspension is 20-30 mg / mL.

[0053] Optionally, the lysate is a Tris-HCl buffer containing guanidine isothiocyanate and Triton X-100; wherein the concentrations of guanidine isothiocyanate and Tris-HCl are 3-4 mol / L and 10-30 mmol / L, respectively, and the volume concentration of Triton X-100 is 1-3%.

[0054] Optionally, the eluent is a Tris-HCl buffer with a concentration of 10 to 30 mmol / L.

[0055] Optionally, the nucleic acid extraction reagent further comprises a proteinase K solution; in the proteinase K solution, the concentration of proteinase K is 5 to 10 wt %.

[0056] Optionally, the cleaning solution A is a mixture of Tris-HCl buffer and ethanol in a volume ratio of 2 to 4:1; in the Tris-HCl buffer, the concentration of Tris-HCl is 10 to 30 mmol / L, and the pH of the Tris-HCl buffer is 7 to 8.

[0057] Optionally, the cleaning solution B is a mixture of Tris-HCl buffer and ethanol in a volume ratio of 1:3-4; in the Tris-HCl buffer, the concentration of Tris-HCl is 10-30 mmol / L, and the pH of the Tris-HCl buffer is 7-8.

[0058] Optionally, the PCR amplification reagents include HPV amplification primers, HPV detection Taqman probes, internal standard primers, internal standard Taqman probes, Taq enzyme, dNTPs, MgCl2, Tris-HCl buffer and UNG enzyme, and also include negative controls and positive controls. Among them, HPV amplification primers can use universal primers for HPV amplification (such as GP5+ / GP6+, SPF1 / GP6++, etc.).

[0059] The method for using the above kit comprises the following steps:

[0060] (1) Mixing the sample to be tested, proteinase K solution, magnetic bead suspension and lysis solution, adjusting the pH to 7.0-8.0 with hydrochloric acid and ammonia water, performing lysis and nucleic acid adsorption, and obtaining magnetic beads adsorbed with nucleic acid after magnetic separation;

[0061] (2) washing the magnetic beads adsorbed with nucleic acid with washing solution A and washing solution B in sequence to obtain washed magnetic beads;

[0062] (3) The washed magnetic beads are mixed with an eluent, and the pH is adjusted to 9.8 to 10.3 with hydrochloric acid and ammonia water, followed by elution. After magnetic separation, an eluent containing nucleic acid is obtained.

[0063] Example 1: Preparation of high-yield magnetic beads

[0064] A high-yield magnetic bead for nucleic acid extraction is prepared as follows:

[0065] (A) Grafting hyperbranched glucan: hyperbranched glucan and tungstophosphoric acid were dissolved in water at a ratio of 1 g:4 g:50 mL, carboxyl magnetic beads were added (the mass ratio to hyperbranched glucan was 1:2), and after uniform dispersion, the mixture was stirred at 80°C for 7 h, followed by magnetic separation, washing with ethanol aqueous solution, and drying to obtain glucan-magnetic beads;

[0066] (B) Grafting cysteine: S-trityl-L-cysteine ​​and tungstophosphoric acid were dissolved in water at a ratio of 0.4 g:1 g:10 mL, dextran-magnetic beads (with a mass ratio of 1:0.8 to S-trityl-L-cysteine) were added, and after uniform dispersion, the mixture was stirred at 65°C for 4 h, and magnetic separation, ethanol aqueous solution washing, and drying were performed in sequence to obtain Cys(Trt)-magnetic beads;

[0067] (C) Grafting asparagine: Fmoc-N-trityl-L-asparagine, condensing agent HBTU and base DIEA were dissolved in solvent DMF in a ratio of 1.2 g:1 g:1.5 g:20 mL, and activated by stirring at room temperature for 10 to 15 min. Cys(Trt)-magnetic beads (with a mass ratio of 1:1.2 to Fmoc-N-trityl-L-asparagine) were added, and after uniform dispersion, the mixture was stirred and reacted at room temperature for 2 to 3 h. Magnetic separation, ethanol and water washing, and drying were performed in sequence to obtain Fmoc-Asn(Trt)-Cys(Trt)-magnetic beads;

[0068] (D) Removal of protecting groups: piperidine and DMF were mixed at a volume ratio of 1:4, and Fmoc-Asn(Trt)-Cys(Trt)-magnetic beads (with a mass volume ratio of 1 g:40 mL to DMF) were added, and the reaction was stirred for 1 h; then I2 (with a mass volume ratio of 1 g:10 mL to DMF) was added, and the reaction was stirred for 1.5 h, followed by magnetic separation, washing, and drying to obtain Asn-Cys-magnetic beads, i.e., high-yield magnetic beads.

[0069] Example 2: HPV virus detection kit

[0070] The HPV virus detection kit of this embodiment is composed of a nucleic acid extraction reagent and a PCR amplification reagent, and the compositions are described in Table 1 and Table 2 respectively.

[0071] Table 1 Nucleic acid extraction reagents

[0072]

[0073] In Table 1, the magnetic beads in the magnetic bead suspension are the high-yield magnetic beads prepared in Example 1.

[0074] Table 2 PCR amplification reagents

[0075]

[0076] In Table 2, the HPV amplification primers are universal primers GP5+ / GP6+, that is, the upstream and downstream primer sequences are 5'-TTTGTTACTGTGGTAGATACYAC-3' and 5'-GAAAAATAAACTGTAAATCATATTC-3', respectively; the HPV detection Taqman probes include a probe for HPV-16 detection and a probe for HPV-18 detection, and the sequences are 5'-CATACACCTCCAGCACCTAA-3' and 5'-GGATGCTGCACCGGTCTGA-3', respectively; the internal standard primers are used to amplify β-Globin in HPV, and the upstream and downstream primer sequences are 5'-GAAGAGCCAAGGACAGGTAC-3' and 5'-CAACTTCATCCACGTTCACC-3', respectively; the sequence of the internal standard Taqman probe is 5'-CCCTAGGGTTGGCCAATCTACTC-3'.

[0077] Example 3: HPV virus detection

[0078] Using the kit in Example 2, nucleic acid extraction is performed on the sample to be tested, and the steps are as follows:

[0079] (1) Add 400 μL of the sample to be tested, 20 μL of magnetic bead suspension, 20 μL of proteinase K solution and 400 μL of lysis solution to a centrifuge tube, mix well, adjust the pH to 8 with hydrochloric acid and ammonia water, and incubate for 10 min; magnetically absorb the magnetic beads to one side of the centrifuge tube and discard the waste liquid;

[0080] (2) Add 600 μL of cleaning solution A to the centrifuge tube and mix for 2 min; magnetically attract the magnetic beads to one side of the centrifuge tube and discard the waste liquid;

[0081] (3) Add 600 μL of cleaning solution B to the centrifuge tube and mix for 2 min; magnetically attract the magnetic beads to one side of the centrifuge tube and discard the waste liquid;

[0082] (4) Add 100 μL of elution solution to the centrifuge tube, adjust the pH to 10 with hydrochloric acid and ammonia water, and mix for 6 minutes; magnetically absorb the magnetic beads to one side of the centrifuge tube, collect the supernatant, discard the magnetic beads, and obtain the nucleic acid sample to be tested;

[0083] (5) Replace the sample to be tested with a negative control and a positive control, and repeat the above process to obtain a negative control nucleic acid sample and a positive control nucleic acid sample.

[0084] The prepared nucleic acid samples (the nucleic acid sample to be tested, the negative control nucleic acid sample and the positive control nucleic acid sample) are respectively subjected to fluorescent quantitative PCR, and the steps are as follows:

[0085] (6) Prepare the PCR reaction system according to Table 3;

[0086] Table 3 PCR reaction system

[0087] Components Sample volume (μL) PCR reaction solution 15 Primer probe solution 3 Taq enzyme solution 1 UNG enzyme solution 0.2 Nucleic acid samples 1 Total volume 20.2

[0088] (7) Perform PCR amplification according to the reaction program in Table 4:

[0089] Table 4 PCR reaction procedure

[0090]

[0091] (8) In step 3 of the PCR amplification process, the fluorescence signal is collected to determine whether HPV-16 and HPV-18 exist in the sample to be tested.

[0092] Comparative Example 1: HPV Virus Detection

[0093] The only difference between this comparative example and Example 3 is that the magnetic beads in the kit are changed, and the high-yield magnetic beads are replaced with carboxyl magnetic beads. The other components in the kit and the HPV virus detection process are the same as those in Example 3.

[0094] Comparative Example 2: HPV Virus Detection

[0095] The difference between this comparative example and Example 3 is that the magnetic beads in the kit are changed, and the other components in the kit and the HPV virus detection process are the same as those in Example 3. The magnetic beads used in this comparative example are prepared according to the method of Example 1, except that steps (B) to (D) are not performed, and the rest of the process is the same as that in Example 1.

[0096] Comparative Example 3: HPV Virus Detection

[0097] The difference between this comparative example and Example 3 is that the magnetic beads in the kit are changed, and the other components in the kit and the HPV virus detection process are the same as those in Example 3. The magnetic beads used in this comparative example are prepared according to the method of Example 1, except that the hyperbranched glucan in step (A) is replaced with glucan, and the rest of the process is the same as that in Example 1.

[0098] Comparative Example 4: HPV Virus Detection

[0099] The difference between this comparative example and Example 3 is that the magnetic beads in the kit are changed, and the other components in the kit and the HPV virus detection process are the same as those in Example 3. The magnetic beads used in this comparative example are prepared according to the method of Example 1, except that the S-trityl-L-cysteine ​​in step (B) is replaced with an equal molar amount of cysteine, and the Fmoc-N-trityl-L-asparagine in step (C) is replaced with an equal molar amount of asparagine, and step (D) is not performed, and the rest of the process is the same as in Example 1.

[0100] Comparative Example 5: HPV Virus Detection

[0101] The difference between this comparative example and Example 3 is that the magnetic beads in the kit are changed, and the pH 10 in step (4) is changed to pH 11. The other components in the kit and the HPV virus detection process are the same as those in Example 3. The magnetic beads used in this comparative example are prepared according to the method of Example 1, except that the S-trityl-L-cysteine ​​in step (B) is replaced by an equal molar amount of N'-(trityl)-L-asparagine, and the Fmoc-N-trityl-L-asparagine in step (C) is replaced by an equal molar amount of Fmoc-S-trityl-L-cysteine, and the rest of the process is the same as that in Example 1.

[0102] Comparative Example 6: HPV Virus Detection

[0103] The difference between this comparative example and Example 3 is that the magnetic beads in the kit are changed, and the other components in the kit and the HPV virus detection process are the same as those in Example 3. The magnetic beads used in this comparative example are prepared according to the method of Example 1, and the only difference is that the Fmoc-N-trityl-L-asparagine in step (C) is added together with S-trityl-L-cysteine ​​in step (B), and step (C) is not performed, and the rest of the process is the same as that in Example 1.

[0104] Test example: Sensitivity

[0105] 60 cervical exfoliated cell samples and 37 urogenital tract secretion samples (dispersed in cell preservation fluid) were tested using the methods of Example 3 and Comparative Examples 1 to 5, and the number of negative and positive results was counted. The results are shown in Table 5.

[0106] Table 5 HPV virus detection results

[0107]

[0108] Data Analysis:

[0109] (1) Compared with Comparative Example 1, the positive detection rate of the two viruses in Example 3 is significantly higher, indicating that compared with ordinary carboxyl magnetic beads, the detection sensitivity of the kit can be improved by sequentially grafting hyperbranched dextran and cysteine-asparagine dipeptide on the surface of the present invention.

[0110] (2) Compared with Comparative Example 2, the positive detection rate of the two viruses in Example 3 is significantly higher, indicating that by grafting cysteine-asparagine dipeptide on the surface of magnetic beads, the detection sensitivity of the test kit can be improved. This is because: by grafting dipeptide on the surface of magnetic beads, the electrical properties of the surface of magnetic beads can be changed by changing the pH, so that the magnetic beads can adsorb nucleic acids through electrostatic attraction, so the adsorption force is strong, the adsorption effect on nucleic acids is better, and the nucleic acids can be prevented from falling off the magnetic beads during the washing process before elution. At the same time, the electrostatic repulsion can be used to separate the nucleic acids from the magnetic beads, which can improve the elution effect of the nucleic acids. In this way, the yield of nucleic acid extraction can be improved, thereby improving the detection sensitivity of the test kit.

[0111] (3) Compared with Comparative Example 3, the positive detection rate of the two viruses in Example 3 is significantly higher, indicating that compared with dextran, the use of hyperbranched dextran grafted magnetic beads can improve the detection sensitivity of the kit. This is because: the hyperbranched dextran molecular chain has more branched structures, which is conducive to the introduction of a large number of evenly distributed hydroxyl groups on the surface of the magnetic beads. The use of the hydroxyl groups therein to graft cysteine-asparagine dipeptide can make the dipeptide have a higher grafting amount and grafting uniformity, which is conducive to further improving the efficiency of nucleic acid extraction, thereby improving the detection sensitivity of the kit.

[0112] (4) Compared with Comparative Example 4, the positive detection rate of the two viruses in Example 3 is significantly higher, indicating that in the process of grafting cysteine ​​and asparagine, the sulfhydryl group of cysteine ​​and the α-amino group and amide group of asparagine are all connected with protective groups, which is beneficial to improve the detection sensitivity of the kit. This is because: the protective groups connected to the sulfhydryl group of cysteine ​​and the α-amino group and amide group of asparagine can prevent these groups from reacting in step (C), resulting in too much asparagine grafted on each cysteine, affecting the elution effect.

[0113] (5) Compared with Comparative Example 5, the positive detection rate of the two viruses in Example 3 is higher, indicating that the grafting order of the two amino acids on the magnetic beads will affect the detection sensitivity of the kit. This is because: when asparagine is grafted first and then cysteine, the free groups in the dipeptide are the α-amino group and the thiol group in cysteine, and the pK of the former is greater than that of the latter. Although when the pH is greater than the α-amino group in cysteine, the amino group can be uncharged and the thiol group can be negatively charged, at too high a pH, DNA is easily denatured, and the resulting single-stranded DNA is more flexible and difficult to be eluted by electrostatic repulsion, which will have an adverse effect on the yield of nucleic acid extraction.

[0114] (6) Compared with Comparative Example 6, the positive detection rate of the two viruses in Example 3 is significantly higher, indicating that compared with the discrete grafting of cysteine ​​and aspartic acid onto the magnetic beads, the sequential grafting is more conducive to improving the detection sensitivity of the kit. This is because: the present invention grafts cysteine ​​onto the magnetic beads through an esterification reaction, and then grafts aspartic acid onto cysteine ​​through dehydration condensation, which can reduce the distance between the two amino acids, and then increase the electrostatic repulsion between the thiol group and the nucleic acid adsorbed on the magnetic beads during elution, further improving the elution effect.

[0115] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0116] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing magnetic beads for nucleic acid extraction, characterized in that the steps include: S1: Dissolve hyperbranched dextran and tungstophosphoric acid in water at a ratio of 1 g:4 g:50 mL, add carboxyl magnetic beads at a mass ratio of 1:2 to hyperbranched dextran, disperse evenly, perform esterification reaction, and sequentially perform magnetic separation, washing, and drying to obtain dextran-magnetic beads; S2: Dissolve S-trityl-L-cysteine ​​and tungstophosphoric acid in water at a ratio of 0.4 g:1 g:10 mL, add the product of step S1 at a mass ratio of 1:0.8 to S-trityl-L-cysteine, disperse evenly, perform esterification reaction, and sequentially perform magnetic separation, washing, and drying to obtain Cys-magnetic beads with protective groups; S3: Fmoc-N-trityl-L-asparagine, condensation agent HBTU and base DIEA were dissolved in an organic solvent according to 1.2g:1g:1.5g:20mL, and after carboxyl activation, the product of step S2 was added in a mass ratio of 1:1.2 to Fmoc-N-trityl-L-asparagine, dispersed evenly, dehydrated and condensed, and then the protecting group was removed to obtain Asn-Cys-magnetic beads.

2. A magnetic bead for nucleic acid extraction prepared by the preparation method according to claim 1.

3. A highly sensitive HPV virus detection kit, characterized in that: It comprises the magnetic beads, nucleic acid extraction reagent and PCR amplification reagent as claimed in claim 2.

4. The kit according to claim 3, characterized in that The nucleic acid extraction reagent comprises a magnetic bead suspension, a lysate, a cleaning solution A, a cleaning solution B and an elution solution; the magnetic bead suspension contains the magnetic beads; the cleaning solution A is a mixture of a Tris-HCl buffer and ethanol in a volume ratio of 2 to 4:1; and the cleaning solution B is a mixture of a Tris-HCl buffer and ethanol in a volume ratio of 1:3 to 4.

5. The kit according to claim 4, characterized in that In the magnetic bead suspension, the content of the magnetic beads is 20-30 mg / mL.

6. The kit according to claim 4, wherein The lysate is a Tris-HCl buffer containing guanidine isothiocyanate and Triton X-100; wherein the concentrations of guanidine isothiocyanate and Tris-HCl are 3-4 mol / L and 10-30 mmol / L, respectively, and the volume concentration of Triton X-100 is 1-3%.

7. The kit according to claim 4, characterized in that The eluent is a Tris-HCl buffer with a concentration of 10-30 mmol / L.

8. The kit according to claim 4, characterized in that The PCR amplification reagent comprises HPV amplification primers, HPV detection Taqman probes, internal standard primers, internal standard Taqman probes, Taq enzyme, dNTPs, MgCl2, Tris-HCl buffer and UNG enzyme.

9. The kit according to claim 8, characterized in that The PCR amplification reagent also includes a negative control and a positive control.

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