Fish egg cell lysate and rapid PCR amplification sequencing detection method
阅读说明：本技术 一种鱼卵细胞裂解液及快速pcr扩增测序检测方法 (Fish egg cell lysate and rapid PCR amplification sequencing detection method ) 是由 张钦 刘俊 张建军 刘肖瑶 樊梓龙 于 2021-09-07 设计创作，主要内容包括：本发明提供一种鱼卵细胞裂解液及快速PCR扩增测序检测方法。包括：异硫氰酸胍、表面活性剂以及EDTA。本发明的有益效果在于：采用含有异硫氰酸胍、表面活性剂以及EDTA的细胞裂解液,可以一次性进行组织消化、细胞裂解,充分释放基因组,释放后的上清液可直接进行PCR反应,可实现PCR快速扩增,不需要在额外使用其余试剂以及后续进行DNA提取,增加了样品的安全性,减短了测试流程,提高了测试效率。(The invention provides a fish egg cell lysate and a rapid PCR amplification sequencing detection method. The method comprises the following steps: guanidine isothiocyanate, surfactant and EDTA. The invention has the beneficial effects that: by adopting the cell lysate containing guanidine isothiocyanate, a surfactant and EDTA, tissue digestion and cell lysis can be carried out at one time, the genome is fully released, the released supernatant can be directly subjected to PCR reaction, PCR rapid amplification can be realized, other reagents are not required to be additionally used, and subsequent DNA extraction is not required, so that the safety of a sample is improved, the test flow is shortened, and the test efficiency is improved.)
1. A fish egg cell lysate, comprising:
guanidine isothiocyanate, surfactant and EDTA.
2. The fish egg cell lysate according to claim 1, wherein the surfactant comprises Tris-HCl and triton x-100.
3. The fish egg cell lysate according to claim 1 or 2, wherein the following components are present in amounts:
4.0 mol/L-5.0 mol/L guanidinium isothiocyanate;
Tris-HCl of 40 mmol/L-50 mol/L;
triton 100 with the volume percentage of 1 to 1.5 percent;
and EDTA 15 mmol/L-25 mmol/L.
4. A rapid PCR amplification sequencing detection method for fish eggs is characterized by comprising the following steps:
step S1, using the cell lysis solution of any claim 1 to 3 to the roe sample, and then breaking cell walls;
step S2, heating and standing the mixture until the cells are fully cracked to obtain a crude product;
step S3, carrying out solid-liquid separation on the crude product, and collecting the liquid;
and step S4, adding amplification primers for PCR amplification.
5. The method for rapid PCR amplification sequencing detection of fish eggs according to claim 4, wherein the sample is fixed by centrifugation after cell lysis solution is added to break cell walls.
6. The method for rapid PCR amplification sequencing detection of fish eggs according to claim 4, wherein in the step S2, the fish eggs are kept standing at 75-85 ℃ for 25-35 min.
7. The method for rapid PCR amplification sequencing detection of fish eggs according to claim 4, wherein in the step S3, solid-liquid separation is performed by centrifugation at 8000r/min to 10000r/min for 40S to 90S.
8. The method for rapid PCR amplification sequencing detection of fish eggs according to claim 4, wherein in step S4, the universal primers L14724 and H15915 are used for PCR amplification.
Technical Field
The invention belongs to the field of DNA (deoxyribonucleic acid) testing, and particularly relates to a fish egg cell lysate and a rapid PCR (polymerase chain reaction) amplification sequencing detection method.
Background
Roe belongs to eukaryotic cell tissue, the cell wall of roe contains protein, carbohydrate, etc., which causes difficult wall breaking, and the improper treatment of genome release can cause PCR amplification failure.
When fish eggs are subjected to PCR amplification, the traditional method is to extract the total genome of the fish eggs and then perform PCR amplification, the method can obtain the genome with relatively high purity to perform PCR amplification, the amplification effect is ideal, however, the extraction of the genome not only consumes a large amount of time and increases the experiment cost, but also causes cross contamination among samples due to a small amount of careless operation in the process of genome extraction, and troubles are brought to subsequent experiments.
Chinese patent application No. 201110027643.1 discloses a DNA extraction method sequentially by enzymatic digestion, cell lysis and cell precipitation. The method has long operation procedures, increased sample pollution during operation and low detection efficiency.
Disclosure of Invention
In order to solve the technical problems, the invention provides a fish egg cell lysate and a rapid PCR amplification sequencing detection method.
The specific technical scheme comprises the following steps:
a fish egg cell lysate, which is different from the fish egg cell lysate and comprises:
guanidine isothiocyanate, surfactant and EDTA.
Further, the surfactant includes Tris-HCl and triton x-100.
Further, the following components in percentage by weight:
4.0 mol/L-5.0 mol/L guanidinium isothiocyanate;
Tris-HCl of 40 mmol/L-50 mol/L;
triton 100 with the volume percentage of 1 to 1.5 percent;
and EDTA 15 mmol/L-25 mmol/L.
A rapid PCR amplification sequencing detection method for fish eggs is characterized by comprising the following steps:
step S1, breaking cell wall of roe sample with the cell lysate;
step S2, heating and standing the mixture until the cells are fully cracked to obtain a crude product;
step S3, carrying out solid-liquid separation on the crude product, and collecting the liquid;
and step S4, adding amplification primers for PCR amplification.
Further, after cell lysis solution is added to break cell walls, centrifugation is adopted to deposit and fix the sample.
Further, in the step S2, the mixture is allowed to stand at 75 to 85 ℃ for 25 to 35 min.
Further, in the step S3, solid-liquid separation is performed by centrifugation at 2500r/min to 3500r/min for 40S to 90S.
Further, in step S4, PCR amplification is performed using the universal primers L14724 and H15915.
Compared with the prior art, the invention has the beneficial effects that:
(1) by adopting the cell lysate containing guanidine isothiocyanate, a surfactant and EDTA, tissue digestion and cell lysis can be carried out at one time, the genome is fully released, the released supernatant can be directly subjected to PCR reaction, PCR rapid amplification can be realized, other reagents are not required to be additionally used, and subsequent DNA extraction is not required, so that the safety of a sample is improved, the test flow is shortened, and the test efficiency is improved.
(2) The cell lysate is adopted for one-step operation, so that the cross contamination can be reduced, and the test result is ensured to have no false positive.
Drawings
FIG. 1 is a gum running chart of example 5;
FIG. 2 is a peak sequencing chart of example 5.
Detailed Description
The present invention is further described in detail below with reference to specific examples so that those skilled in the art can more clearly understand the present invention.
The following examples are provided only for illustrating the present invention and are not intended to limit the scope of the present invention. All other embodiments obtained by a person skilled in the art based on the specific embodiments of the present invention without any inventive step are within the scope of the present invention.
In the examples of the present invention, all the raw material components are commercially available products well known to those skilled in the art, unless otherwise specified; in the examples of the present invention, unless otherwise specified, all technical means used are conventional means well known to those skilled in the art.
In the examples of the present invention, the raw materials used were all conventional commercially available products.
Example 1
The embodiment provides a fish egg cell lysate, which specifically comprises the following components in parts by weight:
4.7mol/L of guanidinium isothiocyanate;
46mol/L Tris-HCl;
triton x-100 with volume percentage of 1.2 percent;
and 20mmol/L EDTA.
Example 2
The embodiment provides a fish egg cell lysate, which specifically comprises the following components in parts by weight:
4.0mol/L of guanidinium isothiocyanate;
40mmol/L Tris-HCl;
triton x-100 with the volume percentage of 1 percent;
and 15mmol/L EDTA.
Example 3
5.0mol/L of guanidinium isothiocyanate;
50mol/L Tris-HCl;
triton x-100 with the volume percentage of 1.5 percent;
and 25mmol/L EDTA
Example 4
The embodiment provides a fish egg cell lysate, which specifically comprises the following components in parts by weight:
4.5mol/L of guanidinium isothiocyanate;
45mol/L Tris-HCl;
triton x-100 with volume percentage of 1.3 percent;
and 22mmol/L EDTA.
Example 5
The embodiment provides a fish egg PCR amplification sequencing method, which comprises the following specific operations:
1. roe was put into a 1.5ML centrifuge tube, 50ul of the cell lysate of example 1 was added, and the cell wall was disrupted by crushing with a tip.
2. 50ul of lysis solution was added to wash the pipette tip and the wash solution was centrifuged.
3.3000r/min, and depositing roe sample by centrifuging for 1 min.
4. Soaking in 80 deg.C hot water for 30min, centrifuging at 10000r/min for 1min
5. Taking 1ul of supernatant, 1ul of each of universal primers L14724 GACTTGAAAAACCACCGTTG and H15915 CTCCGATCTCCGGATTACAAG AC and ddH2O7 ul, MIX 10ul, for amplification
And (3) amplification procedure: 96 ℃ for 5min
96°30S
55°30S
72 deg. 1min 30 cycles
72°5min
6. The sample is printed and run on agarose electrophoresis, the amplification result is recorded, the PCR product is recovered, 11 groups of different samples (P1-P11) are tested by the method, the running result is shown in figure 1, and the sequencing peak is shown in figure 2(P1 sample). From the glue running result, the sequencing product of the invention is pure, and the results are consistent after multiple tests, thus ensuring the accuracy of the test results.
Example 6
The embodiment provides a fish egg PCR amplification sequencing method, which comprises the following specific operations:
1. roe was put into a 1.5ML centrifuge tube, 50ul of the cell lysate of example 2 was added, and the cell wall was disrupted by crushing with a tip.
2. 50ul of lysis solution was added to wash the pipette tip and the wash solution was centrifuged.
3.3000r/min, and depositing roe sample by centrifuging for 1 min.
4. Soaking in 80 deg.C hot water for 30min, centrifuging at 10000r/min for 1min
5. Taking 1ul of supernatant, 1ul of each of universal primers L14724 GACTTGAAAAACCACCGTTG and H15915 CTCCGATCTCCGGATTACAAG AC and ddH2O7 ul, MIX 10ul, for amplification
And (3) amplification procedure: 96 ℃ for 5min
96°30S
55°30S
72 deg. 1min 30 cycles
72°5min
6. Sample application, agarose electrophoresis, recording amplification results and recovering PCR products.
Example 7
The embodiment provides a fish egg PCR amplification sequencing method, which comprises the following specific operations:
1. roe was put into a 1.5ML centrifuge tube, 50ul of the cell lysate of example 3 was added, and the cell wall was disrupted by crushing with a tip.
2. 50ul of lysis solution was added to wash the pipette tip and the wash solution was centrifuged.
3.3000r/min, and depositing roe sample by centrifuging for 1 min.
4. Soaking in 80 deg.C hot water for 30min, centrifuging at 10000r/min for 1min
5. Taking 1ul of supernatant, 1ul of universal primers L14724 GACTTGAAAAACCACCGTTG, H15915 CTCCGATCTCCGGATTACAAG AC, ddH2O 7 and MIX 10ul for amplification
And (3) amplification procedure: 96 ℃ for 5min
96°30S
55°30S
72 deg. 1min 30 cycles
72°5min
6. Sample application, agarose electrophoresis, recording amplification results and recovering PCR products.
Example 8
The embodiment provides a fish egg PCR amplification sequencing method, which comprises the following specific operations:
1. roe was put into a 1.5ML centrifuge tube, 50ul of the cell lysate of example 1 was added, and the cell wall was disrupted by crushing with a tip.
2. 50ul of lysis solution was added to wash the pipette tip and the wash solution was centrifuged.
3.3000r/min, and depositing roe sample by centrifuging for 1 min.
4. Soaking in 75 deg.C hot water for 30min, centrifuging at 8000r/min for 1min
5. Taking 1ul of supernatant, 1ul of each of universal primers L14724 GACTTGAAAAACCACCGTTG and H15915 CTCCGATCTCCGGATTACAAG AC and ddH2O7 ul, MIX 10ul, for amplification
And (3) amplification procedure: 96 ℃ for 5min
96°30S
55°30S
72 deg. 1min 30 cycles
72°5min
6. Sample application, agarose electrophoresis, recording amplification results and recovering PCR products.
It should be noted that the above examples are only for further illustration and description of the technical solution of the present invention, and are not intended to further limit the technical solution of the present invention, and the method of the present invention is only a preferred embodiment, and is not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.