CN110373450B - Kit and method for nondestructive extraction of cervical mucus sample DNA - Google Patents
Kit and method for nondestructive extraction of cervical mucus sample DNA Download PDFInfo
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Abstract
The invention provides a nondestructive extraction kit and an extraction method for cervical mucus sample DNA. The kit comprises proteinase K, lysis solution, binding solution, absolute ethyl alcohol and magnetic beads. The method adopts the dry method to preserve the cervical mucus sample, can preserve the DNA in the mucus sample for a long time, has no damage to the DNA, does not lose DNA loci, improves the DNA extraction efficiency of the cervical mucus sample, and is particularly suitable for the extraction of the cervical mucus sample DNA which is preserved for a long time and is transported for a long distance.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a cervical mucus sample DNA nondestructive extraction kit and an extraction method.
Background
Cervical cancer is one of the most common gynecological malignancies, and screening for cervical cancer has been of widespread concern. In remote areas such as Tibet, Xinjiang and the like in China, medical facilities and services are weak, and cervical mucus samples are often required to be mailed to developed cities in the east for detection. It usually takes several months to collect a batch of specimens, and in this process, DNA is easily degraded and lost, and missed detection and false negative are easily caused for samples collected at an early stage. At present, the collected cervical swabs are usually put in a preservation solution, and the preservation tube is easy to be damaged or leaked in the transportation process, so that the sample is polluted. Sample degradation, leakage and pollution caused by long-term storage and long-distance transportation of samples in remote areas generally have difficulty in obtaining reliable results capable of repeated detection.
Disclosure of Invention
The invention aims to provide a kit for nondestructive extraction of cervical mucus sample DNA.
The invention also aims to provide a method for improving the extraction efficiency of the DNA of the cervical mucus sample, so as to solve the problem of low extraction efficiency of the DNA of the cervical mucus sample stored for a long time and transported for a long distance.
In order to achieve the purpose of the invention, in a first aspect, the invention provides a kit for nondestructive extraction of cervical mucus sample DNA, which comprises proteinase K, a lysis solution, a binding solution, absolute ethyl alcohol and magnetic beads;
wherein the lysis solution is: Tris-HCl solution containing 70-150mM EDTA, pH 8.0.
The binding solution is as follows: 15-30% w/v PEG solution (MW 5000-10000) containing 10-20% sodium chloride.
In the present invention, the magnetic beads are silanized.
In a second aspect, the invention provides an application of the kit in nondestructive extraction of cervical mucus sample DNA.
In a third aspect, the present invention provides a method for improving DNA extraction efficiency of a cervical mucus sample, comprising the following steps:
a. adding lysis solution and proteinase K into the cervical mucus sample, and oscillating at high speed vortex at 800-;
b, oscillating at low speed for 4-12h at the temperature of 56 ℃ and the speed of 200-300rpm to obtain a mixture I;
c. incubating the mixture I at 90-100 ℃ for 5-8min, centrifuging at 13000rpm for 4min, and taking the supernatant;
d. adding the binding solution, absolute ethyl alcohol and a proper amount of magnetic beads into the supernatant obtained in the step c, blowing, uniformly mixing, standing at room temperature for 5-15min, adsorbing by a magnetic rack, discarding the supernatant, and leaving the magnetic beads (DNA is adsorbed on the magnetic beads);
e. adding 50-80% v/v ethanol solution into the magnetic beads, cleaning, blowing, uniformly mixing, adsorbing by a magnetic frame, discarding the supernatant, leaving the magnetic beads, performing instantaneous centrifugation, and discarding the residual liquid;
f. adding ddH into the magnetic beads obtained in the step e2And O, uniformly mixing by blowing, incubating at 70-80 ℃ for 10min, adsorbing magnetic beads by a magnetic frame to obtain supernatant, namely DNA solution, transferring the supernatant to a new tube, and storing for later use.
The method described above, step a, is to add 1000. mu.L of lysate and 0.5-2mg of proteinase K to the cervical mucus sample.
The cervical mucus sample is a swab sample from a subject, swab specification model: CH 2. The C-shaped swab consists of a brush rod and a brush head: the brush rod is made of polypropylene (PP), the H-shaped brush head is made of nylon wires and 0cr18Ni9 stainless steel wires, and the bead of the H-shaped brush head is made of epoxy resin. The brush head is 20mm long and 5-12mm wide; the brush rod is 170mm long and 25mm wide.
In the method, 400 mu L of binding solution, 80-160 mu L of absolute ethyl alcohol and 1.5-3mg of magnetic beads are added into the supernatant in the step d.
The method as described above, wherein the cervical mucus sample obtained in step a is obtained from the cervix of a subject, and the specific method is as follows: inserting the swab into the cervix of a subject, rotating for 3-8 circles in the same direction, then placing the swab adsorbed with the cervical mucus into a sample storage tube, screwing down a bottle cap, and storing at normal temperature (dry method).
In one embodiment of the present invention, the method for improving the efficiency of extracting DNA from a cervical mucus sample comprises the following steps:
1) inserting the swab into the cervix of a subject, rotating for 6 circles in the same direction, then putting the swab adsorbed with cervical mucus into a sample storage tube, screwing down a bottle cap, and storing at normal temperature;
2) adding 1000 μ L of the lysate and 1mg of proteinase K into the swab sample adsorbed with cervical mucus, and performing high-speed vortex oscillation at room temperature at 1000rpm for 10 min;
3) oscillating at low speed for 4h at 56 ℃ and 200rpm to obtain a mixture I;
4) incubating 350 μ L of mixture I at 100 deg.C for 8min, centrifuging at 13000rpm for 4min, and collecting supernatant;
5) adding 400 mu L of the binding solution, 160 mu L of absolute ethyl alcohol and 2mg of magnetic beads into the supernatant obtained in the step 4), uniformly blowing, standing at room temperature for 15min, adsorbing by a magnetic frame, discarding the supernatant, and keeping the magnetic beads;
6) adding 80% v/v ethanol solution into the magnetic beads, cleaning for 2 times, wherein the amount of the 80% v/v ethanol solution used for cleaning is 200 μ L each time, blowing, uniformly mixing, adsorbing by a magnetic rack, discarding the supernatant, leaving the magnetic beads, centrifuging at 13000rpm for 10s, and discarding the residual liquid;
7) adding 50 mu L of ddH into the magnetic beads obtained in the step 6)2And O, uniformly mixing by blowing, incubating at 80 ℃ for 10min, adsorbing magnetic beads by a magnetic frame to obtain supernatant, namely DNA solution, transferring the supernatant to a new tube, and storing for later use.
Wherein the lysis solution is: Tris-HCl solution containing 100mM EDTA, pH 8.0;
the binding solution is as follows: 20% w/v PEG solution containing 15% sodium chloride.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the invention adopts dry method to preserve cervical mucus sample, can preserve DNA in the mucus sample for a long time, and has no damage to DNA. The conventional cell preservation solution has short preservation time for DNA in mucus, has loss on DNA, and is easy to cause that the DNA cannot be extracted from a positive specimen.
The DNA extraction method provided by the invention improves the DNA extraction efficiency of the cervical mucus sample, does not use guanidinium lysate, has no damage to DNA, does not lose gene loci, and is particularly suitable for extracting the DNA of the cervical mucus sample which is stored for a long time and transported for a long distance.
And thirdly, the cervical mucus DNA extracted by the method can be directly used for subsequent operations such as DNA detection and the like.
Drawings
FIG. 1 shows the amplification result of the DNA of the cervical mucus sample extracted in example 1 of the present invention.
FIG. 2 is a comparison of amplification profiles of DNA extracted by the dry method storage and the solution storage in example 2 of the present invention.
FIG. 3 is a comparison of amplification patterns of DNA extracted at different times by high-speed vortex oscillation in example 3 of the present invention. The vortex oscillation mixing at 1000rpm for 10min is obviously different from 0min, 5min, 20min and 30 min.
FIG. 4 is a comparison of the amplification patterns of DNA extracted by different low-speed oscillation and rotation speeds during the protease digestion in example 4 of the present invention. The rotation speed of 200rpm is obviously different from 0rpm, 300rpm and 400 rpm.
FIG. 5 is a comparison of amplification profiles of DNAs extracted by protease digestion time in example 5 of the present invention. The heating time at 56 ℃ and 200rpm is 4h or more, which is obviously different from 1h, 2h and 3 h.
FIG. 6 is a comparison of amplification profiles of DNA extracted from different binding solutions according to example 6 of the present invention. The concentration of sodium chloride in the binding solution is 15 percent, 10 percent, 20 percent and 30 percent which have obvious difference.
FIG. 7 shows the results of stability and reproducibility tests of the method of the present invention in example 7 of the present invention.
FIG. 8 shows the human genome DNA composite STR amplification map extracted by the dry preservation (A) and the solution preservation (B) of the present invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available.
The magnetic beads used in the following examples were silanized magnetic beads having a diameter of 30nm and purchased from Beijing Biotechnology research institute No. OH-MNP, Inc. of Java technologies, Inc. of Java, Wuhan. The swab specification and model: CH2, available from ningbo hua lais medical devices, ltd. The C-shaped swab consists of a brush rod and a brush head: the brush rod is made of polypropylene (PP), the H-shaped brush head is made of nylon wires and 0cr18Ni9 stainless steel wires, and the bead of the H-shaped brush head is made of epoxy resin. The brush head is 20mm long and 5-12mm wide; the brush rod is 170mm long and 25mm wide. The protease K is purchased from Beijing Kogyo Honda biotechnology limited.
The lysis solution used in the following examples was a Tris-HCl solution containing 100mM EDTA, pH 8.0.
The percent in the present invention means mass percent unless otherwise specified; however, the percent of the solution, unless otherwise specified, refers to the grams of solute contained in 100mL of the solution.
Example 1 method for improving extraction efficiency of DNA from cervical mucus sample
1. Specimen collection and dry preservation
Inserting the cervical swab into the cervix of a subject with known positive human papilloma virus DNA, rotating for 6 circles in the same direction, then putting the cervical swab into a sample storage tube, breaking and retaining the cervical swab in the sample storage tube, screwing down a bottle cap, and storing for one month at normal temperature.
2. Method for extracting DNA (deoxyribonucleic acid) of cervical mucus sample
The method comprises the following steps:
adding 1000 microliter of lysate and 1mg of proteinase K into the swab sample adsorbed with cervical mucus, and carrying out vortex oscillation at room temperature at 1000rpm for 10 min;
2) oscillating for 4 hours at 56 ℃ and 200rpm by using a metal constant temperature oscillator to obtain a mixture I;
3) taking 350 mu L of the mixture I, incubating for 8min at 100 ℃, then centrifuging for 4min at 13000rpm, and taking supernatant;
4) adding 400 mu L of binding solution (15% sodium chloride solution containing 20% w/v PEG), 160 mu L of absolute ethyl alcohol and 2mg of magnetic beads into the supernatant obtained in the step 3), blowing and uniformly mixing, standing at room temperature for 15min, adsorbing by a magnetic frame, discarding the supernatant, and retaining the magnetic beads (DNA is adsorbed on the magnetic beads);
5) adding 80% v/v ethanol solution into the magnetic beads, cleaning for 2 times, wherein the amount of the 80% v/v ethanol solution used for cleaning is 200 μ L each time, blowing, uniformly mixing, adsorbing by a magnetic rack, discarding the supernatant, leaving the magnetic beads, centrifuging at 13000rpm for 10s, and discarding the residual liquid;
6) adding 50 mu L of ddH into the magnetic beads obtained in the step 5)2And O, uniformly mixing by blowing, incubating at 80 ℃ for 10min, adsorbing magnetic beads by a magnetic frame to obtain supernatant, namely DNA solution, transferring the supernatant to a new tube, and storing for later use.
3. PCR reaction system and amplification conditions
The PCR amplification reagent is TB of BAO bioengineering (Dalian) Co., Ltd
Premix Ex Taq II (TliRNaseH Plus) reagent. The primers were synthesized by Biotechnology engineering (Shanghai) Inc.
Primer design reference: multichannel real-time fluorescent quantitative PCR detection of HPV DNA results analysis [ J ] university report of johnsons: medical edition, 2013,34(5),31-35.
The reaction system is shown in Table 1:
TABLE 120 μ L amounts of the respective components of the reaction system reagents
The amplification procedure is shown in table 2:
TABLE 2
The detection equipment is a SLAN-96P type detector of Shanghai Hongshi medical science and technology Limited.
The results are shown in FIG. 1, with a Ct value of 25.30 (experimental group). As can be seen from FIG. 1, the sample is a positive specimen, and the method can successfully extract DNA. Wherein, water is used as a control group.
Example 2 comparison of the DNA extraction efficiency of samples stored in Dry and solution
Sampling a cervical swab of the same known HPV positive subject twice (inserting the cervical swab into the cervix of the subject, rotating for 6 circles in the same direction), putting one cervical swab into a sample storage tube, breaking and retaining the cervical swab in the sample storage tube, screwing a bottle cap, and storing the cervical swab at normal temperature for one month; the other part of the cervical swab is put into a preservation tube filled with 1mL of preservation solution (purchased from the bioenergy technology (Shenzhen) Co., Ltd.), the bottle cap is screwed down, and the cervical swab is preserved for one month at normal temperature. Two subjects (1#, 2#) were sampled separately.
1.1 the dry preservation and the method for extracting DNA from cervical mucus comprise the following steps:
1.1.1 Add 1000. mu.L lysate and 1mg proteinase K to cervical mucus sample (swab sample) and vortex at 1000rpm for 10min at room temperature;
1.1.256 ℃, oscillating for 4h at 200rpm to obtain a mixture I;
1.1.3 the rest of the procedure is as in example 1.
1.2 solution preservation, the method for extracting DNA from cervical mucus comprises the following steps:
1.2.1 adding 1mg proteinase K into 1000. mu.L of sample solution, blowing, beating and uniformly mixing, and then incubating at 56 ℃ for 4h with the oscillation speed of 200rpm to obtain a mixture I.
1.2.2 mu.L of the mixture I is incubated for 8min at 100 ℃ and then centrifuged for 4min at 13000rpm, and the supernatant is taken;
1.2.3 the rest of the procedure is as in example 1.
FIG. 2 is an amplification map of DNA extracted by two methods of dry preservation and solution preservation. A1, B1 indicate taking cervical mucus from test # 1 women, and a2, B2 indicate taking cervical mucus from test # 2 women. A1 and A2 refer to two samples which are stored by dry method and extracted with DNA. B1 and B2 refer to DNA extracted from two samples by adopting preservation solution.
Ct values of A1, A2, B1 and B2 are 23.46, 23.64, 28.01 and No.Ct (No.Ct represents index value greater than 30), respectively. As can be seen from FIG. 1, the threshold of the amplification curve A1 is higher than that of the amplification curve B1, and the threshold of the amplification curve A2 is higher than that of the amplification curve B2, which indicates that the dry storage effect is better than the solution storage effect when the sample is stored for one month at normal temperature. The Ct values of A1 and A2 were 4.6 or more lower than those of B1 and B2, respectively, indicating that the amplification effect of the DNA was better under the dry storage condition.
Example 3 optimization of high-speed vortex oscillation time
1. Inserting the cervical swab into the cervix of a subject with known positive human papilloma virus DNA, rotating for 6 circles in the same direction, then putting the cervical swab into a sample storage tube, breaking and retaining the cervical swab in the storage tube, and screwing down a bottle cap. The swabs of the collected subjects were divided into 5 portions on average and stored at room temperature for one month.
2. Adding 1000 μ L lysate and 1mg proteinase K into cervical mucus sample, and performing vortex oscillation at room temperature at 1000rpm for 0min, 5min, 10min, 20min and 30 min.
3. The rest of the procedure was the same as in example 1.
FIG. 3 shows the amplification patterns of DNA extracted by different vortex oscillation times. Ct values of No.Ct, 26.89, 25.92, 28.12 and 29.68 when the vortex oscillation time is 0min, 5min, 10min, 20min and 30min respectively (No.Ct represents that the index value is more than 30). As can be seen from FIG. 2, the vortex oscillation time is longer from 0min to 10min, and the value is smaller, which shows that the extracted DNA is better as the time is longer. The larger the value of the vortex oscillation time from 10min to 30min with time, the worse the effect of DNA extraction with time. When the vortex oscillation time is 10min, the amplification curve firstly moves upwards, and the numerical value is the minimum. The best results were shown when the vortex oscillation time was 10 min.
Example 4 optimization of the protease digestion stage, Low Oscillating rotational speed
1. Inserting the cervical swab into the cervix of a subject with known positive human papilloma virus DNA, rotating for 6 circles in the same direction, then putting the cervical swab into a sample storage tube, breaking and retaining the cervical swab in the storage tube, and screwing down a bottle cap. The swabs of the collected subjects were divided into 4 portions on average and stored at room temperature for one month.
2. Adding 1000 microliter of lysate and 1mg of proteinase K into the cervical mucus sample, and whirling at 1000rpm at room temperature for 10 min;
3. incubating at 56 deg.C for 4h, with oscillation rotation speed of 0rpm, 200rpm, 300rpm, 400rpm respectively to obtain mixture I;
4. the rest of the procedure was the same as in example 1.
FIG. 4 shows the DNA amplification maps extracted at different oscillation speeds. Ct values at 0rpm, 200rpm, 300rpm and 400rpm of the oscillation were 28.58, 25.48, 26.29 and 26.57, respectively. As can be seen from FIG. 3, the values are larger as the rotation speed increases from 200rpm to 400rpm, indicating that the effect of extracting DNA is worse as the rotation speed increases. The amplification curve first passed the threshold at 200rpm with the lowest value. The best results are shown for an oscillation speed of 200 rpm.
Example 5 optimization of protease digestion time
1. Inserting the cervical swab into the cervix of a subject with known positive human papilloma virus DNA, rotating for 6 circles in the same direction, then putting the cervical swab into a sample storage tube, breaking and retaining the cervical swab in the storage tube, and screwing down a bottle cap. The swabs of the collected subjects were divided into 5 parts on average and stored at room temperature for three months.
2. To the cervical mucus sample, 1000. mu.L of lysate and 1mg proteinase K were added and vortexed at 1000rpm for 10min at room temperature.
3. Oscillating at 56 deg.C and 200rpm, and incubating for 1 hr, 2 hr, 3 hr, 4 hr, and 4 hr to obtain mixture I.
4. The rest of the procedure was the same as in example 1.
FIG. 5 shows the amplification profiles of DNA extracted at different digestion times with protease. The Ct values of 1h, 2h, 3h, 4h and more than 4h are No.Ct, 29.05, 26.04 and 26.06 respectively (No.Ct represents that the index value is more than 30). As can be seen from FIG. 5, the longer the incubation time is from 1 to 4 hours, the smaller the numerical value is, indicating that the better the effect of DNA extraction is as the incubation time is longer. The incubation time is 4h and the incubation time is more than 4h, the numerical values are similar, the incubation time is 4h and the incubation time is more than 4h, and the extracted DNA effect is the same.
EXAMPLE 6 optimization of sodium chloride concentration in binding solution
1. Inserting the cervical swab into the cervix of a subject with known positive human papilloma virus DNA, rotating for 6 circles in the same direction, then putting the cervical swab into a sample storage tube, breaking and retaining the cervical swab in the storage tube, and screwing down a bottle cap. The swabs of the collected subjects were divided into 3 portions on average and stored at room temperature for one month.
2. To the cervical mucus sample, 1000. mu.L of lysate and 1mg proteinase K were added and vortexed at 1000rpm for 10min at room temperature.
3. Shaking at 56 deg.C and 200rpm for 4h to obtain mixture I.
4. mu.L of the mixture I was incubated at 100 ℃ for 8min and then centrifuged at 13000rpm for 4min, and the supernatant was taken.
5. And (3) adding 400 mu L of binding solution, 160 mu L of absolute ethyl alcohol and 2mg of magnetic beads into the supernatant obtained in the step (3) (wherein the binding solution is 20% w/v PEG, 10%, 15%, 20% and 30% NaCl are respectively added into the PEG), blowing, uniformly mixing, standing at room temperature for 15min, adsorbing by a magnetic rack, discarding the supernatant, and keeping the magnetic beads (DNA is adsorbed on the magnetic beads).
6. The rest of the procedure was the same as in example 1.
FIG. 6 shows the amplification profiles of DNA extracted at different NaCl concentrations. The Ct values of the sodium chloride concentrations of 10%, 15%, 20% and 30% are respectively 28.60, 26.57, 24.86 and 27.15. As can be seen from FIG. 6, the higher the concentration of 0-15% NaCl, the lower the value, indicating that the higher the effect of DNA extraction with increasing NaCl concentration. The higher the sodium chloride concentration of 15 to 30%, the higher the value, indicating that the effect of DNA extraction is rather worse as the sodium chloride concentration increases. The amplification curve first goes up with a minimum value at a sodium chloride concentration of 15%. The best results were demonstrated at a sodium chloride concentration of 15%.
Example 7 stability and reproducibility test of the method
The cervical swabs were inserted into the cervix uteri of subject a, subject B and subject C, respectively, who had known positive human papillomavirus DNA, rotated 6 turns in the same direction, then the cervical swabs were placed in a sample storage tube, broken and left in the storage tube, and the cap was tightened. Dividing the swabs of the collecting subject A into 3 parts with numbers of A1, A2 and A3 on average; dividing the swab of the collecting subject B into 3 parts on average, wherein the numbers of the parts are B1, B2 and B3; the swabs of the collecting subject C were divided equally into 3 parts, numbered C1, C2, C3. Storing at normal temperature for one month.
1. Adding 1000 microliter of lysate and 1mg of proteinase K into the cervical mucus sample, and carrying out vortex oscillation at room temperature at 1000rpm for 10 min;
2. oscillating at 56 ℃ and 200rpm for 4h to obtain a mixture I;
the rest of the procedure was the same as in example 1.
FIG. 7 is an amplification map of DNA extracted multiple times for the same sample. The results for a1, a2, A3 were 26.42, 26.39, 26.29, respectively. Results for B1, B2, B3 were 27.05, 27.08, 27.00, respectively. The results for C1, C2, C3 were 27.34, 27.44, 27.37, respectively. As can be seen from fig. 7, the values of a1, a2, and A3 are almost the same, the values of B1, B2, and B3 are almost the same, and the values of C1, C2, and C3 are almost the same, indicating that the method has high stability and good reproducibility.
Example 8 extraction of genomic DNA
Purpose of the experiment: the dry preservation and the solution preservation are different from the detection of the human genome DNA locus in the cervical mucus.
The experimental method comprises the following steps: sampling a cervical swab of the same subject for 2 times, putting one cervical swab into a sample storage tube, breaking and storing the cervical swab in the sample storage tube, screwing a bottle cap, and storing the cervical swab for six months at normal temperature; the other part of the cervical swab is put into a preservation tube filled with 1mL of preservation solution (purchased from the bioenergy technology (Shenzhen) Co., Ltd.), the bottle cap is screwed down, and the cervical swab is preserved for six months at normal temperature.
1.1 dry preservation, the method for extracting the human genome DNA from cervical mucus comprises the following steps:
1.1.1 adding 1000 mul of lysate and 1mg of proteinase K into a cervical mucus sample, and carrying out vortex oscillation at room temperature at 1000rpm for 10 min;
1.1.256 ℃, oscillating for 4h under the condition of 200rpm to obtain a mixture I;
1.1.3 taking 350 mu L of the mixture I, incubating for 8min at 100 ℃, then centrifuging for 4min at 13000rpm, and taking supernatant;
1.1.4 adding 400 μ L of binding solution (15% sodium chloride solution containing 20% w/v PEG), 160 μ L of absolute ethanol and 2mg of magnetic beads into the supernatant obtained in the step 1.1.3, uniformly blowing, standing at room temperature for 15min, adsorbing by a magnetic frame, discarding the supernatant, and leaving the magnetic beads (DNA is adsorbed on the magnetic beads);
1.1.5 adding 200L of cleaning solution (80% v/v ethanol solution) into the magnetic beads, cleaning for 2 times, blowing and uniformly mixing, adsorbing by a magnetic frame, discarding the supernatant, leaving the magnetic beads, centrifuging instantaneously, and discarding the residual liquid;
1.1.6 to the beads obtained in step 1.1.5, 50. mu.L of ddH was added2And O, uniformly mixing by blowing, incubating at 80 ℃ for 10min, adsorbing magnetic beads by a magnetic frame to obtain supernatant, namely DNA solution, transferring the supernatant to a new tube, and storing for later use.
1.2 solution preservation, the method for extracting the human genome DNA from cervical mucus comprises the following steps:
1.2.1 adding 1mg of proteinase K into 1000 mu L of sample solution, blowing, beating and uniformly mixing, then incubating for 4 hours at 56 ℃, and obtaining a mixture I with the oscillation rotation speed of 200rpm respectively;
1.2.2 the rest steps are the same as 1.14-1.1.6.
1.3 PCR amplification and electrophoresis
1.3.1 the amplification kit is the sinofiler kit from ABI.
1.3.2 amplification System: 7L MIX (GF600) +3L DNA template.
1.3.3 amplification reaction System and amplification program
The reaction system is shown in Table 1.
The amplification procedure was as follows:
1.3.4 the electrophoresis apparatus is a 3130xl sequencer from ABI.
1.3.5 Loading System: mu.L of amplification product + 15. mu.L of formamide (Liz added).
1.3.6 other steps: the procedure was performed exactly as described in 3130xl sequencer.
1.4 results of the experiment
1.4.1 Dry preservation and solution preservation methods for extracting the human genome DNA composite STR amplification map are shown in FIG. 8(A and B). As can be seen from the figure, compared with the solution preservation, the dry preservation can well meet the requirements of STR locus multiplex amplification, and reduce the phenomena of peak loss and amplification imbalance.
Example 9 comparison of the effects of the present method and commercial kit on the extraction of human genomic DNA
Human genomic DNA was extracted using Qiagen and TIANGEN kits, respectively.
The experimental method comprises the following steps: inserting the swab into the cervix of a subject, rotating for 6 circles in the same direction, then placing the swab adsorbed with cervical mucus into a sample storage tube, breaking off and retaining the swab in the storage tube, and screwing down a bottle cap. A total of 25 human samples were taken (25 subjects). The cervical swabs of each subject were divided into 3 parts on average and stored at room temperature for six months.
1. Method for extracting human genome DNA
1.1 adding 1000 microliter of lysate and 1mg of proteinase K into the swab sample adsorbed with cervical mucus, and carrying out vortex oscillation at room temperature at 1000rpm for 10 min;
1.2 oscillating the mixture for 4 hours at 56 ℃ and 200rpm by a metal constant temperature oscillator to obtain a mixture I;
1.3 incubating 350 μ L of the mixture I at 100 deg.C for 8min, then centrifuging at 13000rpm for 4min, and taking the supernatant;
1.4 adding 20% w/v PEG, 400 μ L of 15% sodium chloride solution, 160 μ L of absolute ethanol and 2mg of magnetic beads into the supernatant obtained in the step 1.3, blowing and uniformly mixing, standing at room temperature for 15min, adsorbing by a magnetic frame, discarding the supernatant, and leaving the magnetic beads (DNA is adsorbed on the magnetic beads);
1.5 adding 80% v/v ethanol solution into the magnetic beads for cleaning for 2 times, wherein the dosage of the 80% v/v ethanol solution for each cleaning is 200 mu L, uniformly blowing, adsorbing by a magnetic frame, discarding the supernatant, leaving the magnetic beads, centrifuging at 13000rpm for 10s, and discarding the residual liquid;
1.6 Add 50. mu.L of ddH to the beads obtained in step 1.52O, blowing, beating and mixing uniformly, incubating for 10min at 80 ℃, adsorbing magnetic beads by a magnetic frame to obtain supernatant, namely DNA solution, transferring the supernatant to a new tubeAnd storing for later use.
2. Extraction of human genomic DNA from German Qiagen kit
2.1 the extraction procedure was performed exactly as described in the MagAttrack DNA Mini M48 Kit.
Extraction of human genomic DNA from TIANGEN kit (from Tiangen Biotechnology Co., Ltd., Beijing)
3.1 the extraction procedure is strictly performed according to the instruction of the universal genomic DNA extraction kit by the paramagnetic particle method.
PCR amplification and electrophoresis
4.1 the amplification kit is the sinofiler kit from ABI.
4.2 amplification System: mu.L of MIX (GF600) + 3. mu.L of DNA template.
4.3 amplification procedure as follows:
4.4 the electrophoresis apparatus is a 3130xl sequencer from ABI.
4.5 loading system: 1.5uL amplification product +15uL formamide (Liz added).
4.6 other steps: the procedure was performed exactly as described in 3130xl sequencer.
5. The results are shown in Table 3.
TABLE 3 comparison of DNA results obtained by different methods
| Method of producing a composite material | Obtaining the total number of fluorescence peaks | Peak height average (RFU) |
| M48 | 391 | 353.38 |
| TIANGEN | 386 | 305.10 |
| Method for producing a composite material | 400 | 430.02 |
*: the total number of fluorescence peaks obtained was the sum of 2 parallel amplification results, totaling 400 per method.
As can be seen from Table 3, the method obtains all 400 fluorescence peaks, while M48 and TIANGEN obtain incomplete fluorescence peaks and have the phenomenon of missing peaks. Therefore, in several different DNA extraction methods, the method is lossless for extracting human genome DNA, and STR-DNA sites are not lost.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (1)
1. A method for improving the extraction efficiency of cervical mucus sample DNA is characterized by comprising the following steps:
1) inserting the swab into the cervix of a subject, rotating for 6 circles in the same direction, then putting the swab adsorbed with cervical mucus into a sample storage tube, screwing down a bottle cap, and storing at normal temperature;
2) adding 1000 μ L of lysate and proteinase K1mg into the swab sample adsorbed with cervical mucus, and performing high-speed vortex oscillation at room temperature at 1000rpm for 10 min;
3) oscillating at low speed for 4h at 56 ℃ and 200rpm to obtain a mixture I;
4) incubating 350 μ L of mixture I at 100 deg.C for 8min, centrifuging at 13000rpm for 4min, and collecting supernatant;
5) adding 400 mu L of binding solution, 160 mu L of absolute ethyl alcohol and 2mg of magnetic beads into the supernatant obtained in the step 4), uniformly blowing, standing at room temperature for 15min, adsorbing by a magnetic frame, discarding the supernatant, and keeping the magnetic beads;
6) adding 80% v/v ethanol solution into the magnetic beads, cleaning for 2 times, wherein the amount of the 80% v/v ethanol solution used for cleaning is 200 μ L each time, blowing, uniformly mixing, adsorbing by a magnetic rack, discarding the supernatant, leaving the magnetic beads, centrifuging at 13000rpm for 10s, and discarding the residual liquid;
7) adding 50 mu L of ddH into the magnetic beads obtained in the step 6)2O, uniformly mixing by blowing, incubating at 80 ℃ for 10min, adsorbing magnetic beads by a magnetic frame to obtain supernatant, namely DNA solution, transferring the supernatant to a new tube, and storing for later use;
wherein, the lysis solution in the step 2) is Tris-HCl solution containing 100mM EDTA, and the pH value is 8.0;
the binding solution is as follows: 20% w/v PEG solution containing 15% sodium chloride.
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Citations (4)
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| CN105368817A (en) * | 2015-12-08 | 2016-03-02 | 苏州国科闻普生物科技有限公司 | Cervical cell preservation and DNA fast extraction integrated kit and extraction method |
| CN105368820A (en) * | 2015-12-22 | 2016-03-02 | 南京先进激光技术研究院 | Whole blood DNA (deoxyribonucleic acid) extraction kit based on paramagnetic particle method and application of extraction kit |
| CN105441421A (en) * | 2014-09-30 | 2016-03-30 | 深圳华大基因研究院 | Cell lysis method |
| CN106620681A (en) * | 2017-01-12 | 2017-05-10 | 南京佰泰克生物技术有限公司 | Cell lysis solution, kit and application of cell lysis solution to preparation of tumor whole cell antigen loaded DC tumor vaccine |
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| CN105441421A (en) * | 2014-09-30 | 2016-03-30 | 深圳华大基因研究院 | Cell lysis method |
| CN105368817A (en) * | 2015-12-08 | 2016-03-02 | 苏州国科闻普生物科技有限公司 | Cervical cell preservation and DNA fast extraction integrated kit and extraction method |
| CN105368820A (en) * | 2015-12-22 | 2016-03-02 | 南京先进激光技术研究院 | Whole blood DNA (deoxyribonucleic acid) extraction kit based on paramagnetic particle method and application of extraction kit |
| CN106620681A (en) * | 2017-01-12 | 2017-05-10 | 南京佰泰克生物技术有限公司 | Cell lysis solution, kit and application of cell lysis solution to preparation of tumor whole cell antigen loaded DC tumor vaccine |
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