阅读说明：本技术 一种芽孢杆菌的pcr模板及其制备方法和应用 (PCR template of bacillus and preparation method and application thereof ) 是由 曹树威 廖玉英 邱磊 王自豪 卢慧林 甘露 陈政瑜 刘克俊 黄焕昌 卢文宜 向雷 于 2021-01-20 设计创作，主要内容包括：本发明适用于生物技术领域,提供了一种芽孢杆菌的PCR模板及其制备方法和应用,该PCR模板的制备方法包括以下步骤：将芽孢杆菌菌体与DNA提取液进行混合后,再加入石英砂进行混合,同时进行沸水浴处理,得到混合液；将混合液进行离心处理,取上清液,得到所述PCR模板；其中,DNA提取液包括三羟甲基氨基甲烷盐酸盐、乙二胺四乙酸、聚乙烯吡咯烷酮、蛋白酶K和溶菌酶。本发明实施例提供的制备方法,结合物理和生物等方法,利用石英砂、溶菌酶、蛋白酶K等物质,充分裂解菌体,可在短时间内制备出满足PCR需求的DNA模板,其具有安全、高效、环保等特点。(The invention is suitable for the technical field of biology, and provides a PCR template of bacillus, a preparation method and application thereof, wherein the preparation method of the PCR template comprises the following steps: mixing bacillus thallus with the DNA extracting solution, adding quartz sand for mixing, and simultaneously carrying out boiling water bath treatment to obtain a mixed solution; centrifuging the mixed solution, and taking supernatant to obtain the PCR template; wherein, the DNA extracting solution comprises trihydroxymethyl aminomethane hydrochloride, ethylene diamine tetraacetic acid, polyvinylpyrrolidone, proteinase K and lysozyme. The preparation method provided by the embodiment of the invention combines physical and biological methods, utilizes substances such as quartz sand, lysozyme, proteinase K and the like to fully crack thalli, can prepare the DNA template meeting the PCR requirement in a short time, and has the characteristics of safety, high efficiency, environmental protection and the like.)

1. A preparation method of a PCR template of bacillus is characterized by comprising the following steps:

mixing bacillus thallus with the DNA extracting solution, adding quartz sand for mixing, and simultaneously carrying out boiling water bath treatment to obtain a mixed solution; the DNA extracting solution comprises trihydroxymethyl aminomethane hydrochloride, ethylene diamine tetraacetic acid, polyvinylpyrrolidone, proteinase K and lysozyme;

and centrifuging the mixed solution, and taking supernatant to obtain the PCR template.

2. The method for preparing PCR template of Bacillus according to claim 1, wherein the Bacillus is any one of Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus megaterium, and Bacillus cereus.

3. The method for preparing a PCR template of bacillus according to claim 1, wherein the mass-to-volume ratio of the quartz sand to the DNA extracting solution is (0.3-0.7): 1 in mg/μ L.

4. The method of claim 1, wherein the pH of the DNA extract is 7.5-8.5, and the concentration of tris hydrochloride is 15-25 mmol/L.

5. The method for preparing PCR template of Bacillus according to claim 1 or 3, wherein the concentration of EDTA in the DNA extract is 1.5-2.5 mmol/L.

6. The method for preparing a PCR template of Bacillus according to claim 1 or 3, wherein the concentration of polyvinylpyrrolidone in the DNA extract is 8-12 g/L.

7. The method for preparing PCR template of Bacillus according to claim 1 or 3, wherein the concentration of proteinase K in the DNA extract is 80-120 μ g/L.

8. The method for preparing PCR template of Bacillus according to claim 1 or 3, wherein the concentration of lysozyme in the DNA extract is 0.8-1.2 mg/mL.

9. A PCR template prepared by the preparation method of any one of claims 1 to 8.

10. Use of the PCR template of claim 9 for bacillus identification and/or classification.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a PCR template of bacillus, and a preparation method and application thereof.

Background

In recent years, the bacillus is widely applied in the field of ecological breeding, has various varieties and different functions, and is an important method for realizing the rapid classification of strains by combining a PCR (polymerase chain reaction) method to carry out molecular identification on the bacillus. When a genome template of a PCR reaction strain is prepared, the traditional genome extraction method is time-consuming and labor-consuming, cannot meet the requirements of light weight and less quantification, and needs to be contacted with highly toxic chemical substances such as phenol, chloroform and the like.

In addition, although the existing kit can improve the extraction efficiency of the genomic DNA under the low-toxicity condition, the cost is high, and the requirement of mass strain identification is difficult to meet under the condition of limited experimental expenditure budget.

Disclosure of Invention

The embodiment of the invention aims to provide a method for preparing a PCR template of bacillus, and aims to solve the problems in the background art.

The embodiment of the invention is realized in such a way that the preparation method of the PCR template of bacillus comprises the following steps:

mixing bacillus thallus with the DNA extracting solution, adding quartz sand for mixing, and simultaneously carrying out boiling water bath treatment to obtain a mixed solution; the DNA extracting solution comprises Tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl), Ethylene Diamine Tetraacetic Acid (EDTA), polyvinylpyrrolidone (PVP), proteinase K and lysozyme;

and centrifuging the mixed solution, and taking supernatant to obtain the PCR template.

As another preferable mode of the embodiment of the present invention, the Bacillus is any one of Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus megaterium and Bacillus cereus.

As another preferable mode of the embodiment of the invention, the mass volume ratio of the quartz sand to the DNA extracting solution is (0.3-0.7): 1 in mg/μ L

In another preferred embodiment of the present invention, the pH of the DNA extract is 7.5 to 8.5.

In another preferred embodiment of the present invention, the concentration of tris hydrochloride in the DNA extract is 15 to 25 mmol/L.

In another preferable embodiment of the present invention, the concentration of ethylenediaminetetraacetic acid in the DNA extraction solution is 1.5 to 2.5 mmol/L.

In another preferred embodiment of the present invention, the concentration of polyvinylpyrrolidone in the DNA extraction solution is 8 to 12 g/L.

In another preferred embodiment of the present invention, the concentration of proteinase K in the DNA extract is 80 to 120. mu.g/L.

As another preferable scheme of the embodiment of the invention, the concentration of lysozyme in the DNA extracting solution is 0.8-1.2 mg/mL.

Another objective of the embodiments of the present invention is to provide a PCR template prepared by the above preparation method.

It is another object of the embodiments of the present invention to provide a use of the PCR template in bacillus identification and/or classification.

The preparation method of the PCR template of the bacillus provided by the embodiment of the invention combines physical and biological methods, utilizes quartz sand, lysozyme, proteinase K and other substances, fully cracks thalli, can prepare the DNA template meeting the PCR requirement in a short time, and has the characteristics of safety, high efficiency, environmental protection and the like. Specifically, before the thalli are subjected to enzymolysis, the thalli are abraded by quartz sand, so that the enzymolysis is more sufficient; the bacillus is gram-positive bacteria, the main component of the cell wall is peptidoglycan, and the lysozyme can efficiently hydrolyze the peptidoglycan, so that the enzymolysis method is used for the bacillus, and the cell lysis efficiency is greatly improved. After enzymolysis, the bacteria can be thoroughly cracked by polishing with quartz sand again.

In addition, aiming at substances which are easy to pollute DNA, such as polysaccharide, protein and the like released by the cracking of the thalli, the invention utilizes PVP to combine with the polysaccharide, utilizes proteinase K to digest the protein, finally utilizes boiling water bath to fully increase the cell cracking efficiency and simultaneously denature and coagulate the protein, and finally utilizes high-speed centrifugation to settle uncracked thalli, denatured organic macromolecules, cell fragments and the like and separate the cells from the DNA in a supernatant.

Tris-HCl in the DNA extracting solution provides a weak alkali buffer environment, meanwhile, EDTA can inhibit the activity of DNase, and the combination of the two can protect the stability of template DNA and prevent the template DNA from being degraded.

The invention fully cracks thalli, releases DNA, and simultaneously eliminates the pollution of polysaccharide, protein and the like to the DNA to the maximum extent, and prepares template mother liquor. After the mother solution is diluted by high times, the residual small amount of DNA pollutants can not influence the PCR reaction.

The invention only needs to prepare a small amount of DNA templates which can meet the requirement of PCR reaction, and does not need concentration and purification of genome DNA, so the operation method of the invention is greatly simplified compared with the traditional genome extraction method, not only saves a large amount of time, but also avoids highly toxic chemical substances such as phenol, chloroform and the like, and is safe and efficient.

According to the invention, a small amount of bacteria can be picked by directly using the toothpick, so that the experimental requirements can be met, complicated operations such as liquid culture and bacteria collection are not needed, a large amount of genome DNA templates of bacillus strains meeting the PCR requirements can be prepared in a short time, the cost is far lower than that of a kit, and compared with the experimental method of the kit, the operation steps are simplified; under the condition of limited scientific research expenditure, the method can meet the requirement of identifying a large batch of strains.

Drawings

FIG. 1 is a comparison of the results of electrophoresis detection of the amplification products of PCR templates obtained by different methods.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

This embodiment provides a method for preparing a PCR template for bacillus, comprising the steps of:

s1, picking mung bean-sized bacteria from the surface of the bacillus subtilis solid culture medium by using an aseptic toothpick into a 1.5mL EP tube, adding 1 mL of aseptic normal saline, repeatedly purging for 5 times by using a gun head, centrifuging for 1min at 12000 rpm/min, and removing supernatant to obtain the bacillus bacteria.

S2, mixing the bacillus thallus with 600 mu L of DNA extracting solution, repeatedly purging for 3 times by using a gun head until the thallus is uniformly distributed, then adding 300mg of quartz sand for mixing, performing vortex oscillation for 3min, then placing in a 58 ℃ water bath for 15min, then taking out from the water bath, continuing to perform vortex oscillation for 3min, and then performing boiling water bath for 5min to obtain a mixed solution;

wherein, the DNA extracting solution comprises 20mmol/L of tris (hydroxymethyl) aminomethane hydrochloride, 2mmol/L of ethylene diamine tetraacetic acid, 10g/L of polyvinylpyrrolidone, 100 mu g/L of protease and 1mg/mL of lysozyme, and the pH value is 8.

S3, centrifuging the mixed solution at 12000 rpm/min for 1min, taking the supernatant to another sterile EP tube to be used as template mother solution, freezing and preserving at-20 ℃, and diluting the mother solution by 500 times when the template is used, thus being directly used as a PCR template.

Example 2

This embodiment provides a method for preparing a PCR template for bacillus, comprising the steps of:

s1, picking mung bean particle-size thalli from the surface of the bacillus licheniformis solid culture medium by using an aseptic toothpick into a 1.5mL EP tube, adding 1 mL of aseptic normal saline, repeatedly purging for 6 times by using a gun head, centrifuging for 1min at 12000 rpm/min, and removing supernatant to obtain the bacillus thalli.

S2, mixing the bacillus thallus with 600 mu L of DNA extracting solution, repeatedly purging for 3 times by using a gun head until the thallus is uniformly distributed, then adding 180mg of quartz sand for mixing, performing vortex oscillation for 3min, then placing in a 58 ℃ water bath for 15min, then taking out from the water bath, continuing to perform vortex oscillation for 3min, and then performing boiling water bath for 5min to obtain a mixed solution;

wherein, the DNA extracting solution comprises 15mmol/L of tris (hydroxymethyl) aminomethane hydrochloride, 1.5mmol/L of ethylene diamine tetraacetic acid, 8g/L of polyvinylpyrrolidone, 80 mu g/L of protease and 0.8mg/mL of lysozyme, and the pH value is 7.5.

S3, centrifuging the mixed solution at 12000 rpm/min for 1min, taking the supernatant to another sterile EP tube to be used as template mother solution, freezing and preserving at-20 ℃, and diluting the mother solution by 500 times when the template is used, thus being directly used as a PCR template.

Example 3

This embodiment provides a method for preparing a PCR template for bacillus, comprising the steps of:

s1, selecting mung bean-sized bacteria from the surface of a bacillus amyloliquefaciens solid culture medium by using an aseptic toothpick to a 1.5mL EP tube, adding 1 mL aseptic normal saline, repeatedly purging for 6 times by using a gun head, centrifuging for 1min at 12000 rpm/min, and removing supernatant to obtain the bacillus bacteria.

S2, mixing the bacillus thallus with 600 mu L of DNA extracting solution, repeatedly purging for 3 times by using a gun head until the thallus is uniformly distributed, then adding 420mg of quartz sand for mixing, performing vortex oscillation for 3min, then placing in a 58 ℃ water bath for 15min, then taking out from the water bath, continuing to perform vortex oscillation for 3min, and then performing boiling water bath for 5min to obtain a mixed solution;

wherein, the DNA extracting solution comprises 25mmol/L of tris (hydroxymethyl) aminomethane hydrochloride, 2.5mmol/L of ethylene diamine tetraacetic acid, 12g/L of polyvinylpyrrolidone, 120 mu g/L of protease and 1.2mg/mL of lysozyme, and the pH value is 8.5.

S3, centrifuging the mixed solution at 12000 rpm/min for 1min, taking the supernatant to another sterile EP tube to be used as template mother solution, freezing and preserving at-20 ℃, and diluting the mother solution by 500 times when the template is used, thus being directly used as a PCR template.

Example 4

This embodiment provides a method for preparing a PCR template for bacillus, comprising the steps of:

s1, picking mung bean-sized bacteria from the surface of a bacillus megaterium solid culture medium by using an aseptic toothpick into a 1.5mL EP tube, adding 1 mL aseptic normal saline, repeatedly purging for 5 times by using a gun head, centrifuging for 1min at 12000 rpm/min, and removing supernatant to obtain the bacillus bacteria.

S2, mixing the bacillus thallus with 600 mu L of DNA extracting solution, repeatedly purging for 3 times by using a gun head until the thallus is uniformly distributed, then adding 240mg of quartz sand for mixing, performing vortex oscillation for 3min, then placing in a 58 ℃ water bath for 15min, then taking out from the water bath, continuing to perform vortex oscillation for 3min, and then performing boiling water bath for 5min to obtain a mixed solution;

wherein, the DNA extracting solution comprises 18mmol/L of trihydroxymethyl aminomethane hydrochloride, 1.8mmol/L of ethylene diamine tetraacetic acid, 9g/L of polyvinylpyrrolidone, 90 mu g/L of protease K and 0.9mg/mL of lysozyme, and the pH value is 7.8.

S3, centrifuging the mixed solution at 12000 rpm/min for 1min, taking the supernatant to another sterile EP tube to be used as template mother solution, freezing and preserving at-20 ℃, and diluting the mother solution by 500 times when the template is used, thus being directly used as a PCR template.

Example 5

This embodiment provides a method for preparing a PCR template for bacillus, comprising the steps of:

s1, picking mung bean particle-size bacteria from the surface of the bacillus cereus solid culture medium by using an aseptic toothpick into a 1.5mL EP tube, adding 1 mL aseptic normal saline, repeatedly purging for 6 times by using a gun head, centrifuging for 1min at 12000 rpm/min, and removing supernatant to obtain the bacillus bacteria.

S2, mixing the bacillus thallus with 600 mu L of DNA extracting solution, repeatedly purging for 3 times by using a gun head until the thallus is uniformly distributed, then adding 360mg of quartz sand for mixing, performing vortex oscillation for 3min, then placing in a 58 ℃ water bath for 15min, then taking out from the water bath, continuing to perform vortex oscillation for 3min, and then performing boiling water bath for 5min to obtain a mixed solution;

wherein, the DNA extracting solution comprises 22mmol/L of tris (hydroxymethyl) aminomethane hydrochloride, 2.2mmol/L of ethylene diamine tetraacetic acid, 11g/L of polyvinylpyrrolidone, 110 mu g/L of protease and 1.1mg/mL of lysozyme, and the pH value is 8.2.

S3, centrifuging the mixed solution at 12000 rpm/min for 1min, taking the supernatant to another sterile EP tube to be used as template mother solution, freezing and preserving at-20 ℃, and diluting the mother solution by 500 times when the template is used, thus being directly used as a PCR template.

Genomic DNAs (PCR templates) of 4 strains of Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, Bacillus amyloliquefaciens and the like were extracted by 4 different methods, such as the colony PCR method of the prior art, the method provided in example 1 of the present invention, the CTAB method of the prior art, the kit method of the prior art and the like, and PCR reactions were performed, respectively, and the electrophorograms of the amplification products obtained were as shown in FIG. 1.

In FIG. 1, L1, L2, L3 and L4 are 16srDNA bands of 4 strains of Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, Bacillus amyloliquefaciens and the like obtained by a colony PCR method; l5, L6, L7 and L8 are 16srDNA bands of 4 strains of bacillus subtilis, bacillus licheniformis, bacillus megaterium, bacillus amyloliquefaciens and the like obtained by the method provided by the embodiment 1 of the invention; l9, L10, L11 and L12 are 16srDNA bands of 4 strains of bacillus subtilis, bacillus licheniformis, bacillus megaterium, bacillus amyloliquefaciens and the like obtained by a CTAB method; l13, L14, L15 and L16 are 16srDNA bands of 4 strains of bacillus subtilis, bacillus licheniformis, bacillus megaterium, bacillus amyloliquefaciens and the like obtained by a kit method.

The 16srDNA gene size of the 4 bacteria in FIG. 1 is about 1500 bp; as is clear from FIG. 1, the colony PCR method is the simplest, time-saving and safe method among the 4 methods, but the concentration of the amplification product is low and unstable, and it is difficult to satisfy the requirement of sequencing; the gene product amplified by the method has high concentration and good specificity, the using effect can be completely comparable to or even superior to that of a CTAB method and a kit method, and the PCR product of the method meets the sequencing requirement.

In summary, the main advantages of the present invention are as follows:

1. safety: the traditional CTAB genome extraction method needs 3 types of substances such as mercaptoethanol, phenol, chloroform and the like, and the 3 types of substances are known to have strong toxicity, have volatility, are easy to enter a respiratory tract, and are difficult to be taken into a body in a timely experiment operation regular mode. The DNA extracting solution used in the invention is composed of Tris-HCl, EDTA, PVP, proteinase K, lysozyme and other components, has no known toxic chemical substances, and the combination of the substances has safety while ensuring high-efficiency experimental effect;

2. and (3) fast: the invention can quickly complete genome template extraction work, which is mainly embodied in the following 3 aspects:

(1) collection of the cells without liquid culture: according to the traditional CTAB genome extraction method and the kit genome extraction method, bacteria need to be cultured in advance, namely the bacteria are inoculated to a liquid culture set for culture for several days and then thallus is collected, but the requirements on the thallus quantity are very little, only a small amount of thallus is required to be picked from a solid flat plate or a test tube inclined plane to be placed in a 1.5ml EP tube, and the work of strain liquid shaking table culture, thallus collection and the like is avoided; and the risk of mixed bacteria pollution in the liquid culture process is also avoided. Because the strain collection work is simple, only a small amount of strains are picked into a 1.5ml EP tube by using a sterile toothpick, one person can simultaneously complete the sampling work of dozens of strains or even hundreds of strains in a short time, thereby greatly improving the working efficiency.

(2) The experimental operation steps are few, the operation is simple, and the extraction time of the single strain genome is saved: the method has simple steps, good safety and greatly simplified steps, and can finish the genome extraction work of 1 strain within 35 min.

(3) Electrophoresis detection is not needed after genome extraction is finished: the extracted genome template can be directly diluted to be used as the genome template for PCR without electrophoresis detection after extraction, thereby saving the experimental steps and time.

3. The extracted template has good PCR effect: the invention utilizes the improved DNA extracting solution, combines a physical wall breaking method, a biological enzymolysis method and a chemical method, can degrade protein while efficiently breaking walls to release the genome of the thallus, inhibit the interference of protein and polysaccharide substances on PCR, and inhibit the degradation of DNA of a template by DNA enzyme.

4. The cost is low: among 4 different methods for extracting genome DNA, such as a colony PCR method, the method of the invention, a CTAB method, a kit method and the like, the colony PCR method is the method with the lowest cost and the least time consumption, but is difficult to meet the application requirements; the cost of extracting single strain materials by the method is slightly higher than that of a CTAB method, but far lower than that of a kit method; the time cost (35 min) for extracting a single strain by the method is slightly higher than that of a kit method (30 min), but is far lower than that of a CTAB method (90 min) (the time does not comprise the culture of bacteria, the thallus collecting time and the electrophoresis gel detection time of genome extract).

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.