阅读说明：本技术 一种丝状真菌快速pcr模板的简易制备方法 (Simple preparation method of filamentous fungus rapid PCR template ) 是由 赵杰宏 桂艳玲 韩洁 唐光甫 满海乔 于 2021-01-11 设计创作，主要内容包括：本发明公开了一种丝状真菌快速PCR模板的简易制备方法,包括以下步骤：(1)培养待检测的丝状真菌,得到菌丝体；(2)挑取丝状真菌的菌丝放入盛有蒸馏水的玻璃试管中得到菌丝混合液；(3)将含有菌丝混合液的玻璃试管进行超声处理；(4)将超声处理过的菌丝混合液直接作为DNA模板进行PCR扩增。本发明的方法不需要液氮研磨,不需要添加任何提取试剂,简单快捷、省时省力。克服现有丝状真菌DNA提取时间长、添加试剂多、需要仪器多的缺点。本发明方法提取的DNA模板经PCR反应能够获得稳定、可靠的实验结果,成本低廉,效果良好,适合用于大批量样品快速分析,也适合于仪器条件不完善的实验室开展检测。(The invention discloses a simple preparation method of a filamentous fungus rapid PCR template, which comprises the following steps: (1) culturing the filamentous fungi to be detected to obtain mycelium; (2) picking hyphae of filamentous fungi and putting the hyphae into a glass test tube filled with distilled water to obtain hyphae mixed liquor; (3) carrying out ultrasonic treatment on the glass test tube containing the hypha mixed solution; (4) and directly using the hypha mixed solution after ultrasonic treatment as a DNA template for PCR amplification. The method disclosed by the invention does not need liquid nitrogen grinding, does not need to add any extraction reagent, and is simple, quick, time-saving and labor-saving. Overcomes the defects of long extraction time, more added reagents and more required instruments of the existing filamentous fungi DNA. The DNA template extracted by the method can obtain stable and reliable experimental results through PCR reaction, has low cost and good effect, is suitable for rapid analysis of mass samples and is also suitable for laboratory development detection with incomplete instrument conditions.)

1. A simple preparation method of a filamentous fungus rapid PCR template is characterized by comprising the following steps:

(1) culturing filamentous fungi to be detected by using a conventional culture medium to obtain a mycelium;

(2) picking hyphae of filamentous fungi, and putting the hyphae into a glass test tube filled with distilled water to obtain hyphae mixed liquor;

(3) carrying out ultrasonic treatment on the glass test tube containing the hypha mixed solution obtained in the step (2);

(4) and (4) directly taking the hypha mixed liquor subjected to ultrasonic treatment in the step (3) as a DNA template for PCR amplification.

2. The method for preparing a rapid PCR template of filamentous fungi according to claim 1, wherein the filamentous fungi to be detected in step (1) is cultured on a solid medium or in a liquid medium.

3. The method for preparing a filamentous fungus rapid PCR template according to claim 1, wherein the step (2) further comprises: a20 mg piece of rice-sized filamentous fungus mycelia was picked and placed in a glass test tube containing 1ml of water.

4. The method for preparing a filamentous fungus rapid PCR template according to claim 1, wherein the step (3) further comprises: treating the glass test tube containing the mycelium mixture under 40Hz ultrasonic condition for 5-10 min.

5. The simple preparation method of the filamentous fungus rapid PCR template as claimed in claim 1, wherein the mycelium mixture solution after the ultrasonic treatment in the step (4) is directly used as a DNA template and added into a PCR reaction system for PCR amplification.

6. The sonicated hyphal mixture according to claim 5 directly as a DNA template, wherein the supernatant of the hyphal mixture directly serves as a DNA template; or carrying out water bath on the mycelium mixed solution at the high temperature of 50-65 ℃ for 10-15min, and taking the supernatant as a DNA template; or centrifuging the mycelium mixed solution at 1000-6000 rpm, and taking supernatant as a DNA template; or carrying out high-temperature water bath on the hypha mixed solution at 50-65 ℃ for 10-15min, and centrifuging at 1000-6000 rpm to obtain supernatant serving as a DNA template.

7. The method for PCR amplification by adding proper amount of the compound to a PCR reaction system according to claim 6, wherein the step (4) further comprises: and (3) sucking 1-3 mu l of supernatant of the hypha mixed solution, and adding the hypha mixed solution into a PCR reaction system for PCR amplification.

8. The method for preparing a rapid PCR template of filamentous fungus according to claim 1, wherein the filamentous fungus comprises Aspergillus kawachii and Isaria cicadae.

Technical Field

The invention relates to the field of molecular biology, in particular to a simple preparation method of a filamentous fungus rapid PCR template.

Background

PCR, i.e., polymerase chain reaction, is widely used for gene amplification and genetic analysis of animals, plants and microorganisms as the most basic technique in the field of molecular biology, but the time and cost of PCR are greatly affected by the reaction DNA template. Currently, most of the methods for extracting DNA employ the Cetyl Trimethyl Ammonium Bromide (CTAB) method, the Sodium Dodecyl Sulfate (SDS) method, or a commercially available kit. These methods, although capable of obtaining high quality DNA, require the addition of many reagents, and are complicated and time-consuming in the procedure, increasing the time and cost of the experiment. Therefore, the method has important application value in realizing rapid PCR by using the DNA template of the targeted improvement reaction. Although the Chelex-100 extraction method, the alkaline lysis method, the cell wall lysate method, the mechanical wall breaking combined microwave method and the like have been developed in the field of plant research, the preparation time of the PCR template is shortened to a certain extent by the methods, the required reagents, instruments or steps are still many, the experiment cost is high, and the method is difficult to adapt to the requirement of large-scale rapid detection.

Disclosure of Invention

In view of the above, the present invention aims to provide a simple, fast, safe and economical method for preparing a filamentous fungus rapid PCR template.

According to one aspect of the present invention, there is provided a simple method for preparing a filamentous fungus rapid PCR template, comprising the steps of:

(1) culturing filamentous fungi to be detected by using a conventional culture medium to obtain a mycelium;

(2) picking hyphae of filamentous fungi, and putting the hyphae into a glass test tube filled with distilled water to obtain hyphae mixed liquor;

(3) carrying out ultrasonic treatment on the glass test tube containing the hypha mixed solution obtained in the step (2);

(4) and (4) directly taking the hypha mixed liquor subjected to ultrasonic treatment in the step (3) as a DNA template for PCR amplification.

In some embodiments, the filamentous fungus to be detected in step (1) is cultured on a solid medium or in a liquid medium.

In some embodiments, step (2) further comprises: a20 mg piece of rice-sized filamentous fungus mycelia was picked and placed in a glass test tube containing 1ml of water.

In some embodiments, step (3) further comprises: treating the glass test tube containing the mycelium mixture under 40Hz ultrasonic condition for 5-10 min.

In some embodiments, step (4) further comprises: and directly taking the hypha mixed solution after ultrasonic treatment as a DNA template, and adding the DNA template into a PCR reaction system for PCR amplification.

In some embodiments, the supernatant of the hyphal mixture is used directly as a DNA template; or carrying out water bath on the mycelium mixed solution at the high temperature of 50-65 ℃ for 10-15min, and taking the supernatant as a DNA template; or centrifuging the mycelium mixed solution at 1000-6000 rpm, and taking supernatant as a DNA template; or carrying out high-temperature water bath on the hypha mixed solution at 50-65 ℃ for 10-15min, and centrifuging at 1000-6000 rpm to obtain supernatant serving as a DNA template.

In some embodiments, the step (4) further comprises: and (3) sucking 1-3 mu l of supernatant of the hypha mixed solution, and adding the hypha mixed solution into a PCR reaction system for PCR amplification.

In some embodiments, the filamentous fungi include aspergillus purpureus (Monascus purpureus) and Isaria cicadae (Isaria cicadae).

The invention has the beneficial effects that: compared with other methods, the method disclosed by the invention does not need liquid nitrogen grinding, does not need any extraction reagent, and is simple, quick, time-saving and labor-saving. The defects of the existing rapid detection technology are overcome: long extraction time of filamentous fungi DNA, more added reagents, more required instruments and the like. The DNA template extracted by the method can obtain stable and reliable experimental results through PCR reaction, has low cost and good effect, is very suitable for rapid analysis of mass samples, and is also suitable for laboratory development detection with incomplete instrument conditions.

Drawings

FIG. 1 is an electrophoresis chart of PCR products of DNA templates directly obtained by taking supernatant after red yeast rice hypha is subjected to ultrasonic treatment;

FIG. 2 is a PCR comparison graph of the supernatant of the monascus mycelia treated by ultrasound and DNA extracted by the kit respectively as templates;

FIG. 3 is an electrophoresis chart of the supernatant liquid after the monascus mycelia are treated by ultrasonic treatment and then treated by high temperature and centrifugation as a DNA template PCR product;

FIG. 4 is an electrophoretogram of PCR product obtained by directly using supernatant after ultrasonication of Isaria cicadae mycelia as a DNA template.

Detailed Description

The present invention will be described in further detail with reference to specific examples.

FIGS. 1 to 4 are schematic diagrams showing electrophoresis of PCR products of filamentous fungi as DNA templates according to a simple method for preparing a rapid PCR template of filamentous fungi of the present invention.

A simple preparation method of a filamentous fungus rapid PCR template comprises the following steps:

(1) culturing filamentous fungi to be detected by using a conventional culture medium to obtain a mycelium; the filamentous fungus to be detected may be cultured on a solid medium or in a liquid medium. Filamentous fungi include, but are not limited to, Monascus purpureus (Monascus purpureus) and Isaria cicadae (Isaria cicadae).

(2) Picking 20mg of rice-grain-sized hypha blocks of filamentous fungi by using an inoculating needle, and putting the hypha blocks into a glass test tube filled with 1ml of distilled water to obtain hypha mixed liquor;

(3) and (3) carrying out ultrasonic treatment on the glass test tube containing the hypha mixed solution obtained in the step (2) for 5-10min under the ultrasonic wave condition of 40 Hz.

(4) And (4) directly taking the hypha mixed liquor subjected to ultrasonic treatment in the step (3) as a DNA template for PCR amplification.

Directly taking the supernatant of the hypha mixed solution as a DNA template; or carrying out water bath on the mycelium mixed solution at the high temperature of 50-65 ℃ for 10-15min, and taking the supernatant as a DNA template; or centrifuging the mycelium mixed solution at 1000-6000 rpm, and taking supernatant as a DNA template; or carrying out high-temperature water bath on the mycelium mixed solution at 50-65 ℃ for 10-15min, and centrifuging the mycelium mixed solution at 1000-6000 rpm by using a centrifugal machine to obtain supernatant serving as a DNA template.

The addition amount of the supernatant of the hypha mixed solution added into a PCR reaction system for PCR amplification is 1-3 mul.

Example 1

Purifying and culturing Monascus purpureus with solid Sabouraud's medium, and inoculating with inoculating needleA small amount of hyphae with a grain size of about 20mg were picked from the purified and cultured monascus purpureus and placed in a glass test tube containing 1mL of distilled water. And then putting the test tube into an ultrasonic cleaning machine for ultrasonic treatment under the ultrasonic treatment conditions of 40Hz and 8 min. Taking 1 mu L of the bacteria liquid after ultrasonic treatment as a DNA template for PCR amplification. mu.L of the reaction system, 1. mu.L of each of the universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'), 10. mu.L of 2 XTaq PCR Mix, and finally, ddH₂O20. mu.L, pre-denatured in a PCR instrument at 94 ℃ for 5min, then denatured at 94 ℃ for 30s, annealed at 55 ℃ for 30s, extended at 72 ℃ for 30s for 30 cycles, and finally extended at 72 ℃ for 10 min. After the PCR reaction was completed, 5ul of the amplified product was detected by 1% agarose gel electrophoresis to obtain a clear and correct PCR product, see FIG. 1. FIG. 1 is an electrophoresis chart of PCR products obtained by taking supernatant directly as DNA templates after red yeast rice hypha is subjected to ultrasonic treatment. Wherein, the PCR amplification product is ITS fragment (579 bp). M: DL1000 Marker; in the figure, 1-4 are PCR results of 4 red yeast rice samples.

Example 2

Purifying and culturing Monascus purpureus by using solid Sabouraud's medium, picking Monascus purpureus hypha with inoculating needle, placing into test tube containing 1ml distilled water, and dispersing the hypha in water. And then putting the test tube into an ultrasonic cleaning machine for ultrasonic treatment under the ultrasonic treatment conditions of 40Hz and 8 min. Then placing the test tube into a water bath with the temperature of 60 ℃ for heat preservation for 15min, then placing the test tube into a centrifuge for centrifugal treatment at 6000rpm for 1min, and taking 2 mu L of supernatant for PCR amplification. Meanwhile, DNA extracted and purified by the DNA extraction kit is used as a template, 2 mu L of DNA is taken for PCR amplification, and the PCR result is compared with that of an ultrasonic product used as a DNA template. CtnC-F/CtnC-R and CtnD-F/CtnD-R primer pairs are selected to respectively amplify the CtnC and CtnD genes of the monascus purpureus, the reaction system is 20 mu L, the primers are respectively 1 mu L, 2 XTaq PCR Mix is 10 mu L, and finally ddH is used₂O is supplemented to 20 μ L, pre-denatured at 94 ℃ for 5min in a PCR instrument, then denatured at 94 ℃ for 30s, annealed at 55 ℃ for 30s, extended at 72 ℃ for 30s, and finally extended at 72 ℃ for 10 min. After the PCR reaction, 5ul of the amplified product was subjected to agarose gel electrophoresis. Both DNA templates gave clear and stable PCR products, see FIG. 2. FIG. 2 shows the ultrasonic treatment of the supernatant of red yeast mycelia and the extraction with the kitThe DNA was used as a template for PCR comparison. Wherein, the PCR amplification products are CtnC (316bp) and CtnD (346bp) sequences. M: DL1000 Marker; in the figure, 1: amplifying CtnC; 2: amplifying CtnD; s: performing ultrasonic treatment on hypha supernatant to serve as a DNA template; k: the kit extracts DNA as a template.

Example 3

Purifying and culturing Monascus purpureus by using liquid Sabouraud's medium, and selecting Monascus purpureus hyphae from the liquid Sabouraud's medium by using an inoculating needle, placing into a test tube filled with 1ml of distilled water, and dispersing the hyphae in the water. And then putting the test tube into an ultrasonic cleaning machine for ultrasonic treatment under the ultrasonic treatment conditions of 40Hz and 8 min. Then placing the test tube into a water bath with the temperature of 60 ℃ for heat preservation for 15min, then placing the test tube into a centrifugal machine for centrifugal treatment for 30s at 6000rpm, and adopting 4 treatment conditions to respectively carry out experiments to obtain treated samples: ultrasonic, ultrasonic + centrifugation, ultrasonic + high temperature + centrifugation, respectively sucking 2 μ L of supernatant as DNA template for PCR. Clear bands can be amplified by four treated samples, wherein the sample effect of ultrasonic treatment, high temperature treatment and centrifugation is the best, and the reference figure 3 shows that the sample has the best effect. FIG. 3 is an electrophoresis chart of PCR products using the supernatant after the monascus mycelia are treated by ultrasonic treatment and then treated by high temperature and centrifugation as a DNA template. Wherein, the PCR amplification product is an ITS (579bp) fragment. M: DL1000 Marker; in the figure, 1: carrying out ultrasonic contrast; 2: performing ultrasonic and centrifugation; 3: ultrasonic plus high temperature; 4: ultrasound + high temperature + centrifugation.

Example 4

A small amount of hyphae were picked from 3 Isaria cicadae strains which had been purified and cultured in a solid Shake's medium using an inoculating needle, and placed in a glass test tube containing 1mL of distilled water to disperse the hyphae in the water. And then putting the test tube into an ultrasonic cleaning machine for ultrasonic treatment under the ultrasonic treatment conditions of 40Hz and 8 min. Taking 2 mu L of the ultrasonic bacterial liquid as a DNA template to carry out PCR amplification. Universal primers ITS1 and ITS4 each 1. mu.L, 2 XTaq PCR Mix 10. mu.L, finally ddH₂O make up to 20. mu.L. Pre-denaturation at 94 ℃ for 5min in a PCR instrument, followed by denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles, and finally extension at 72 ℃ for 10 min. After the PCR reaction was completed, 5ul of the amplified products were electrophoretically detected to obtain clear and correct PCR products, as shown in FIG. 4. FIG. 4 shows an ultrasonic-treated cicada rollTaking supernatant after mycelia of the mycelia as an electrophoresis picture of a PCR product of a DNA template directly. Wherein, the PCR amplification product is an ITS fragment (about 560 bp). M: DL1000 Marker; in the figure, 1-3 are the PCR results of 3 Isaria cicadae strains.

What has been described above are merely some embodiments of the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the inventive concept herein, and it is intended to cover all such modifications and variations as fall within the scope of the invention.