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CN117070509A - Pretreatment kit for host cell residual DNA detection sample and use method thereof - Google Patents

Pretreatment kit for host cell residual DNA detection sample and use method thereof Download PDF

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CN117070509A
CN117070509A CN202311161090.8A CN202311161090A CN117070509A CN 117070509 A CN117070509 A CN 117070509A CN 202311161090 A CN202311161090 A CN 202311161090A CN 117070509 A CN117070509 A CN 117070509A
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lysate
centrifuge tube
pretreatment kit
centrifuging
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许金超
栗红建
张丹
李永锋
刘丽美
张伟男
蔡宇卿
郭彬彬
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Jiangsu Puxin Biomedical Co ltd
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    • C12N15/09—Recombinant DNA-technology
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    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
    • C—CHEMISTRY; METALLURGY
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Abstract

The application relates to the field of molecular biology, and particularly provides a pretreatment kit for detecting residual DNA of a host cell, which comprises the following components: lysate, wash, eluent, bead suspension, proteinase K, glycogen, yeast tRNA; the lysate comprises the following components in concentration: 18-25mmol/L Tris-HCl, 125-145mmol/L phosphatase inhibitor, 16-23mmol/L thiols, 1-4mmol/L Na 3 VO 4 Aprotin 0.05-2.5ug/mL, leucetin 0.5-2ug/mL, PMSF 0.5-2 mmol/L; the magnetic beads are hydroxyl modified magnetic beads; the kit can obtain purer and more reliable DNA samples, and has strong applicability and flexibility; and the operation is convenient, the specificity is strong, and the performance is reliable.

Description

Pretreatment kit for host cell residual DNA detection sample and use method thereof
Technical Field
The application relates to the technical field of molecular biology, in particular to a pretreatment kit for a host cell residual DNA detection sample and a use method thereof.
Background
The amount of DNA remaining in the host cell determines the impurity content, safety, etc. of the host cell biological product, and it is very important how to accurately and efficiently quantify the amount of DNA remaining in the host cell. The analysis of trace DNA in biological products not only ensures the removal of matrix interferents of the sample itself, but also avoids cross contamination during operation and ensures the accuracy and precision of the extraction method.
The existing pretreatment method for the residual DNA sample has a plurality of problems, such as (1) high requirements on the number and the quality of the sample, (2) large difference between the length and the quality of the DNA fragment, low accuracy of subsequent repeated experiments, (3) long operation time, complicated operation process, high pretreatment cost and the like. Chinese patent CN109957561a discloses a method for extracting residual DNA, in which a lysate, glycogen, sodium iodide, isopropanol, etc. are used to treat DNA, in which an organic reagent washing step is not involved, which reduces environmental pollution and solves the problem of high cost of a kit. However, the purity of DNA extracted by this method is still not high.
The prior art still has a plurality of defects in the aspect of DNA extraction, so that improvement in aspects of improving extraction efficiency and purity, simplifying operation steps, reducing safety risks and the like are needed to meet wider requirements.
Disclosure of Invention
In order to solve the above technical problems, the present application firstly provides a pretreatment kit for detecting residual DNA of host cells, the kit comprising: lysates, washes, eluates, bead suspensions, proteinase K, glycogen, yeast tRNA.
Further, the lysate comprises the following concentration components: 15-30mmol/L Tris-HCl, 110-150mmol/L phosphatase inhibitor, 15-30mmol/L mercaptan substance, 0.5-5mmol/L Na 3 VO 4 。
Further, the phosphatase inhibitor is selected from the group consisting of NaCl,NaF、Na 4 P 2 O 7 At least one of beta-glycerophosphate.
Preferably, the phosphatase inhibitor is NaCl and/or NaF.
Further, the lysate also includes a protease inhibitor.
Further, the protease inhibitor is selected from at least one of Aprotinin, leupetin, bestatin, pepstatin, PMSF.
Preferably, the protease inhibitors are Aprotinin, leupetin and PMSF.
Further, the components of the lysate also comprise Aprotin of 0.05-4ug/mL, leucetin of 0.5-3ug/mL and PMSF of 0.5-3 mmol/L.
Preferably, the lysate comprises the following concentrations of components: 18-25mmol/L Tris-HCl, 125-145mmol/L phosphatase inhibitor, 16-23mmol/L thiols, 1-4mmol/L Na 3 VO 4 Aprotin 0.05-2.5ug/mL, leucetin 0.5-2ug/mL, PMSF 0.5-2 mmol/L.
In a preferred embodiment, the lysate comprises the following concentrations of components: 20mmol/L Tris-HCl, 137mmol/L phosphatase inhibitor, 20mmol/L mercaptan substance, 2mmol/L Na 3 VO 4 Aprotin 1ug/mL, leucetin 1ug/mL, PMSF 1 mmol/L.
Further, the mercaptan substance is at least one of beta-mercaptoethanol, dithiothreitol and dithioerythritol.
Preferably, the thiol is dithiothreitol.
Further, the lysate also comprises NP-40, and the mass fraction of the NP-40 in the lysate is 0.5-3%, preferably 1%.
In a more preferred embodiment, the lysate comprises the following concentrations of components: 20mmol/L Tris-HCl, 137mmol/L NaCl, 20mmol/L dithiothreitol, 2mmol/L Na 3 VO 4 Aprotin 1ug/mL, leucetin 1ug/mL, PMSF 1mmol/L, NP-40 with mass fraction of 1%. Wherein mmol/L, ug/mL and 1% refer to the corresponding componentsMolar concentration, mass concentration and mass fraction in the lysate.
In the lysate of the application, na 3 VO 4 And NaCl can inhibit dephosphorylation of the phosphorylated protein, and maintain the phosphorylation state of the protein; aprotinin, leupetin and PMSF are effective in inhibiting proteases produced during cell lysis to keep the proteins from being degraded, but different protease inhibitors have different sensitivities to the proteins, and only when 0.05-2.5ug/mL of Aprotin, 0.5-2ug/mL of leupeptin and 0.5-2mmol/L of PMSF are used in the lysate and matched with 0.5-3% mass fraction of NP-40, the protease activity can be reduced and disappeared most effectively, and the protease is not denatured; dithiothreitol can reduce disulfide bond in DNA, inhibit DNA from forming dimer, raise the fixing efficiency of magnetic bead to DNA, and promote proteinase K digestion of tissue. The kit has higher extraction efficiency of DNA and better purity of the obtained DNA by optimizing the components and the concentration of the lysate.
Further, the washing liquid is Na of 0.2-3.0mol/L 2 HPO 4 The solution is preferably Na in an amount of 0.4 to 1.0mol/L 2 HPO 4 A solution; more preferably 0.5mol/L Na 2 HPO 4 A solution.
Further, the eluent includes, but is not limited to, at least one of TE buffer and PB buffer.
Further, the eluent is TE buffer; the TE buffer refers to Tris-EDTA buffer solution, wherein the concentration of Tris is 10mmol/L, the concentration of EDTA is 1mmol/L, and the pH is 7.9-8.1.
Further, the concentration of the magnetic beads in the magnetic bead suspension is 0.8-2mg/mL, preferably 1mg/mL.
Further, the magnetic beads are hydroxyl modified magnetic beads, and are purchased from HiMat and are available in the model number of hydro 01-200.
Further, in the kit, the lysis solution, the washing solution, the eluent, the magnetic bead suspension and the proteinase K need to be stored in an environment of 2-8 ℃, and the glycogen and the yeast tRNA need to be stored in an environment of below-20 ℃.
Secondly, the application also provides a use method of the pretreatment kit for the host cell residual DNA detection sample, which comprises the following steps:
s1, adding a sample to be treated into the mixture in a volume ratio of (1-3.5): 1 and proteinase K, centrifuging after vortex oscillation in a centrifuge tube, and incubating for 10-30min at 50-70 ℃;
s2, centrifuging the mixed system after the incubation of the S1, sequentially adding glycogen, yeast tRNA, lysate, isopropanol and magnetic bead suspension, and rapidly centrifuging after vortex oscillation; adding a washing liquid into the centrifuge tube after removing the supernatant, dispersing the magnetic beads by vortex vibration, and rapidly centrifuging; adding 75-85wt% ethanol into the centrifuge tube after removing the supernatant again, vortex vibrating, rapidly centrifuging, standing for 0.5-2min, and removing the supernatant;
s3, carrying out rapid centrifugal treatment on the mixed system after the S2 is centrifuged again, and drying the residual substances in the centrifuge tube after sucking out the residual ethanol;
s4, adding eluent into the centrifuge tube of the S3, and after vortex shaking, incubating for 3-12min at 65-80 ℃ and uniformly mixing every 2-3min in the incubation period;
s5, centrifuging the centrifuge tube of the S4 at a high speed, and collecting supernatant.
Further, the sample to be tested is liquid or dry powder. When the sample to be tested is liquid and contains higher DNA content, PBS can be used for diluting the sample and then treating the sample, and the dilution times can be adjusted according to practical conditions, such as 100 times and 1000 times. When the sample to be tested is dry powder, the sample to be tested is diluted to 10mg/mL-100mg/mL, and then pretreatment operation is carried out.
Further, the pH of the sample to be treated is 6.0-8.0.
Further, the volume of the sample to be treated in S1 is 50-200. Mu.L, preferably 100. Mu.L.
Further, the volume ratio of the lysate to proteinase K is 2.5:1.
further, the S1 specifically is: 100 μl of the sample to be treated was added to a volume ratio of 2.5:1, 20-40 mu L of proteinase K, centrifuging after vortex shaking in a centrifuge tube, and incubating for 12-20min at 60-68 ℃.
In a more preferred embodiment, the S1 is specifically: 100. Mu.L of the sample to be treated was added to a mixture of 25. Mu.L of lysate and 10. Mu.L of proteinase K, centrifuged after vortexing in a centrifuge tube, and incubated at 65℃for 15min.
Further, the volume ratio of glycogen, yeast tRNA, lysate, isopropanol and magnetic bead suspension in the S2 is (7-10): (0.1-0.5): (40-60): (120-180): (15-25).
Preferably, the volume ratio of glycogen, yeast tRNA, lysate, isopropanol, and magnetic bead suspension in S2 is (8.5-9.5): (0.1-0.3): (45-55): (140-160): (18-22).
More preferably, the volume ratio of glycogen, yeast tRNA, lysate, isopropanol, and magnetic bead suspension in S2 is 9.0:0.2:50:150:20.
further, the volume of the magnetic bead suspension is 1/3-1/6, preferably 1/5 of the volume of the sample to be detected.
Further, the volume of the washing liquid and the ethanol is 3-8 times, preferably 5 times, the volume of the sample to be detected.
Further, the S2 specifically is: centrifuging the mixed system after the incubation of S1, sequentially adding 9.0 mu L glycogen and 0.2 mu L yeast tRNA mixed solution, 50 mu L lysate, 150 mu L isopropanol and 20 mu L magnetic bead suspension, and rapidly centrifuging for 8-15S after vortex oscillation; after removing the supernatant, adding 500 mu L of washing liquid into the centrifuge tube, dispersing the magnetic beads by vortex vibration, and rapidly centrifuging; after removing the supernatant again, 500. Mu.L of 75-85wt% ethanol is added into the centrifuge tube, vortex shaking and rapid centrifugation are performed, and after standing for 0.5-2min, the supernatant is removed.
Further, the method for removing the supernatant liquid adopted by the application is as follows: placing the centrifuge tube on a magnetic rack, slightly rotating left and right, keeping the centrifuge tube fixed on the magnetic rack after the magnetic beads are gathered on the pipe wall close to the magnetic rack, and sucking the supernatant by a pipetting gun, wherein the magnetic beads cannot be touched in the process.
Further, the drying of S3 adopts room temperature drying or air drying, the drying time can be adaptively adjusted, but the overweight drying of the magnetic beads is avoided.
Further, when the drying of the S3 is room temperature drying, the drying time is 2-6min; if the forced air drying is adopted, the drying time is 1-3min.
Further, the addition volume of the eluent in the step S4 is 0.8-3 times, preferably 1-1.5 times, of the volume of the sample to be treated; preferably 1 time.
In a preferred embodiment, the S4 is specifically: 100 mu L of eluent is added into the centrifuge tube of the S3, after vortex shaking for 0.5-2min, the mixture is incubated for 7min at 70 ℃, and vortex mixing is carried out every 2-3min during incubation.
Further, the step S5 specifically includes: centrifuging the centrifuge tube of the S4 at a high speed for 0.5-2min, standing on a magnetic rack, and transferring the supernatant to a new reagent tube after the magnetic beads are separated.
Further, in the S1-S5, the centrifugal rotating speed is 1000-5000r/min.
Further, the kit is used in the process of extracting and purifying DNA in CHO cells, E.coli cells, vero cells, human cells, plasmids and the like.
Advantageous effects
1. Compared with the traditional DNA extraction method, the DNA sample pretreatment kit greatly simplifies experimental operation, avoids complicated steps and complicated operation procedures, and reduces experimental difficulty;
2. the DNA sample pretreatment kit utilizes the magnetic beads with the surface modified to carry out specific combination on DNA molecules, improves the efficiency of DNA extraction, and ensures that the extracted DNA is higher in quantity and more stable; further provided that lysates with specific composition and concentration are used, and that glycogen, yeast tRNA, lysates, isopropanol, and magnetic bead suspension are used in a volume ratio of (7-10): (0.1-0.5): (40-60): (120-180): (15-25) effectively reducing DNA loss and sample residue, improving purity, thereby obtaining purer and more reliable DNA samples;
3. the DNA sample pretreatment kit can be suitable for various samples such as blood, tissues, body fluid, saliva, cell culture solution and the like, and has strong applicability and flexibility; and the detection is quick, the specificity is strong, and the performance is reliable.
Detailed Description
Examples
Example 1
The embodiment provides a pretreatment kit for detecting residual DNA of host cells, which comprises the following components: lysate, wash, eluent, bead suspension, proteinase K, glycogen, yeast tRNA;
the lysate comprises the following components in concentration: 20mmol/L Tris-HCl, 137mmol/L NaCl, 20mmol/L dithiothreitol, 2mmol/L Na 3 VO 4 Aprotin 1ug/mL, leucetin 1ug/mL, PMSF 1mmol/L, NP-40 with mass fraction of 1%. Wherein mmol/L, ug/mL and 1% refer to the molar concentration, mass concentration and mass fraction of the corresponding components in the lysate.
The washing liquid is Na of 0.5mol/L 2 HPO 4 A solution.
The eluent is TE buffer; wherein the concentration of Tris is 10mmol/L, the concentration of EDTA is 1mmol/L, and the pH is 7.9-8.1.
The mass concentration of the magnetic beads in the magnetic bead suspension is 1mg/mL, the magnetic beads are hydroxyl modified magnetic beads, the hydroxyl group content is 600-1200nmol/mg, and the magnetic beads are purchased from HiMat and are available in the model of 01-200.
Example 2
The embodiment provides a method for using the host cell residual DNA sample pretreatment kit described in the embodiment 1, which specifically comprises the following steps:
s1, adding 100 mu L of a sample to be treated into 25 mu L of a mixed solution of lysate and 10 mu L of proteinase K, centrifuging after vortex oscillation for 30S in a centrifuge tube, and incubating for 15min at 65 ℃;
s2, centrifuging the mixed system after the incubation of S1, sequentially adding 9.0 mu L glycogen and 0.2 mu L yeast tRNA mixed solution, 50 mu L lysate, 150 mu L isopropanol and 20 mu L magnetic bead suspension, and rapidly centrifuging for 10S after vortex oscillation for 5min; adding 500 mu L of washing liquid into the centrifuge tube after removing the supernatant, carrying out vortex vibration for 30s to ensure the dispersion of magnetic beads, and carrying out rapid centrifugation for 10s; after removing the supernatant again, adding 500 mu L of 75-85wt% ethanol into the centrifuge tube, carrying out vortex shaking for 30s, carrying out rapid centrifugation for 10s, standing for 1min, and removing the supernatant;
s3, rapidly centrifuging the mixed system after the centrifugation of S2 for 10 seconds again, sucking out residual ethanol, and drying the residual substances in the centrifuge tube at room temperature for 3-5 minutes;
s4, adding 100 mu L of eluent into the centrifuge tube of the S3, performing vortex oscillation for 1min, incubating for 7min at 70 ℃, and uniformly mixing every 2-3min in the incubation period;
s5, centrifuging the centrifuge tube of the S4 at a high speed for 1min, standing on a magnetic rack, and transferring out supernatant into a new reagent tube after magnetic beads are separated.
In the above S1-S5, the centrifugal rotation speed is 3000r/min; the supernatant was removed/aspirated/transferred in the following manner: placing the centrifuge tube on a magnetic rack, slightly rotating left and right, keeping the centrifuge tube fixed on the magnetic rack after the magnetic beads are gathered on the pipe wall close to the magnetic rack, and sucking the supernatant by a pipetting gun, wherein the magnetic beads cannot be touched in the process.
Comparative example 1
Substantially identical to example 1, except that: the lysate comprises the following components in concentration: 20mmol/L Tris-HCl, 137mmol/L NaCl, 20mmol/L dithiothreitol, 2mmol/L Na 3 VO 4 Aprotin 2ug/mL, PMSF 1mmol/L, NP-40 with mass fraction of 1%. The method of use was the same as in example 1.
Comparative example 2
Substantially identical to example 1, except that: the lysate comprises the following components in concentration: 20mmol/L Tris-HCl, 137mmol/L NaCl, 20mmol/L dithiothreitol, 2mmol/L Na 3 VO 4 Aprotin 1ug/mL, leucetin 1ug/mL, PMSF 1mmol/L, NP-40 with mass fraction of 5%. The method of use was the same as in example 1.
Comparative example 3
Substantially identical to example 1, except that: the lysate comprises the following components in concentration: 20mmol/L Tris-HCl, 137mmol/L NaCl, 20mmol/L dithiothreitol, 2mmol/L sodium molybdate, 1ug/mL Aprotin, 1ug/mL leucetin, 1mmol/L PMSF, and 1% NP-40 by mass. The method of use was the same as in example 1.
The performance test method comprises the following steps:
the recovery of the final samples was measured by carrying out the carrying and mechanical extraction treatments respectively on DNA samples of different host cells using the sample pretreatment cartridges in examples and comparative examples, and the results are shown in Table 1.
TABLE 1
Summarizing: the pretreatment box of the embodiment 1 carries out portable and mechanical extraction on DNA samples of different host types respectively, and the final sample recovery rate is 70% -130%, which is better than 50% -150% required by pharmacopoeia.

Claims (10)

1. A pretreatment kit for detecting residual DNA of a host cell, the kit comprising: lysate, wash, eluent, bead suspension, proteinase K, glycogen, yeast tRNA;
the lysate comprises the following components in concentration: 15-30mmol/L Tris-HCl, 110-150mmol/L phosphatase inhibitor, 15-30mmol/L mercaptan substance, 0.5-5mmol/L Na 3 VO 4 。
2. The pretreatment kit of claim 1, wherein the phosphatase inhibitor is selected from NaCl, naF, na 4 P 2 O 7 At least one of beta-glycerophosphate.
3. The pretreatment kit of claim 1, wherein the lysate further comprises a protease inhibitor; the protease inhibitor is selected from at least one of Aprotinin, leupetin, bestatin, pepstatin, PMSF.
4. A pretreatment kit according to claim 3, wherein the lysate comprises the following concentrations of components: 18-25mmoL/L Tris-HCl, 125-145mmol/L phosphatase inhibitor, 16-23mmol/L thiol, 1-4mmol/L Na 3 VO 4 Aprotin 0.05-2.5ug/mL, leucetin 0.5-2ug/mL, PMSF 0.5-2 mmol/L.
5. The pretreatment kit according to claim 4, wherein the lysate further comprises NP-40, and the mass fraction of NP-40 in the lysate is 0.5-3%.
6. The pretreatment kit according to any one of claims 1 to 5, wherein the thiol compound is dithiothreitol.
7. The pretreatment kit according to any one of claims 1 to 5, wherein the washing solution is 0.2 to 3.0mol/L of Na 2 HPO 4 A solution.
8. The method of using a pretreatment kit according to any one of claims 1 to 7, comprising the steps of:
s1, adding a sample to be treated into the mixture in a volume ratio of (1-3.5): 1 and proteinase K, centrifuging after vortex oscillation in a centrifuge tube, and incubating for 10-30min at 50-70 ℃;
s2, centrifuging the mixed system after the incubation of the S1, sequentially adding glycogen, yeast tRNA, lysate, isopropanol and magnetic bead suspension, and rapidly centrifuging after vortex oscillation; adding a washing liquid into the centrifuge tube after removing the supernatant, dispersing the magnetic beads by vortex vibration, and rapidly centrifuging; adding 75-85wt% ethanol into the centrifuge tube after removing the supernatant again, vortex vibrating, rapidly centrifuging, standing for 0.5-2min, and removing the supernatant;
s3, carrying out rapid centrifugal treatment on the mixed system after the S2 is centrifuged again, and drying the residual substances in the centrifuge tube after sucking out the residual ethanol;
s4, adding eluent into the centrifuge tube of the S3, and after vortex shaking, incubating for 3-12min at 65-80 ℃ and uniformly mixing every 2-3min in the incubation period;
s5, centrifuging the centrifuge tube of the S4 at a high speed, and collecting supernatant.
9. The method of claim 8, wherein the volume ratio of glycogen, yeast tRNA, lysate, isopropanol, bead suspension in S2 is (7-10): (0.1-0.5): (40-60): (120-180): (15-25), the volume of the magnetic bead suspension is 1/3-1/6 of the volume of the sample to be detected.
10. The method of claim 8, wherein the washing solution and ethanol are used in a volume 3-8 times the volume of the sample to be measured.
CN202311161090.8A 2023-09-11 2023-09-11 Pretreatment kit for host cell residual DNA detection sample and use method thereof Pending CN117070509A (en)

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