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Simple preparation method of rapid PCR template of filamentous fungi

A filamentous fungus and template technology, applied in the field of molecular biology, can solve the problems of multiple instruments or steps, shorten the preparation time of PCR templates, and difficulty in adapting to large-scale rapid detection, etc.

Pending Publication Date: 2021-05-18
贵州中医药大学
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the Chelex-100 extraction method, alkali lysis method, cell wall lysate method, mechanical wall breaking combined with microwave method, etc. have been developed in the field of plant research, these methods shorten the preparation time of PCR templates to a certain extent, but the required reagents and instruments Or there are still many steps, the experimental cost is high, and it is difficult to adapt to the needs of large-scale rapid detection

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  • Simple preparation method of rapid PCR template of filamentous fungi
  • Simple preparation method of rapid PCR template of filamentous fungi
  • Simple preparation method of rapid PCR template of filamentous fungi

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preparation example Construction

[0023] A simple preparation method for rapid PCR template of filamentous fungi, comprising the following steps:

[0024] (1) Cultivate the filamentous fungus to be detected with a conventional medium to obtain mycelium; the filamentous fungus to be detected can be cultured on a solid medium or in a liquid medium. Filamentous fungi include, but are not limited to, Monascus purpureus and Isaria cicadae.

[0025] (2) Pick about 20 mg of the mycelium block of rice grain-sized filamentous fungi with an inoculation needle, put into a glass test tube filled with 1 ml of distilled water to obtain the mycelial mixture;

[0026] (3) The glass test tube containing the mycelia mixture obtained in step (2) is subjected to ultrasonic treatment for 5-10 min under 40 Hz ultrasonic conditions.

[0027] (4) The mycelia mixture liquid treated with sonication in step (3) is directly used as a DNA template for PCR amplification.

[0028] The supernatant of the mycelium mixture is directly used a...

Embodiment 1

[0031] Use solid Sabouraud medium to purify and cultivate Monascus purpura, use an inoculation needle to pick a little hyphae from the purified culture of Monascus purpura on solid Sabouraud medium, the size of a rice grain is about 20 mg, and put it into a glass test tube filled with 1 mL of distilled water. Then put the test tube into an ultrasonic cleaning machine for ultrasonic treatment, the ultrasonic treatment conditions are 40Hz, 8min. Take 1 μL of the sonicated bacterial solution directly as a DNA template for PCR amplification. Reaction system 20 μL, universal primers ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) 1 μL each, 2×Taq PCR Mix 10 μL, finally use ddH 2 Make up 20 μL of O, pre-denature at 94°C for 5 min in a PCR machine, then denature at 94°C for 30 s, anneal at 55°C for 30 s, extend at 72°C for 30 s, a total of 30 cycles, and finally extend at 72°C for 10 min. After the PCR reaction, 5ul of the amplification product was taken and d...

Embodiment 2

[0033] Use solid Sabouraud medium to purify and cultivate Monascus purpura, use an inoculation needle to pick out the mycelium of Monascus purpura and put it into a test tube filled with 1ml of distilled water to disperse the mycelium in the water. Then put the test tube into an ultrasonic cleaning machine for ultrasonic treatment, the ultrasonic treatment conditions are 40Hz, 8min. Then put the test tube in a water bath at 60°C for 15 minutes, then put it into a centrifuge at 6000 rpm for 1 minute, and take 2 μL of the supernatant for PCR amplification. At the same time, use the DNA extraction kit to extract the purified DNA as a template, take 2 μL for PCR amplification, and compare the PCR results with the ultrasonic product as a DNA template. Use the CtnC-F / CtnC-R and CtnD-F / CtnD-R primer pairs to amplify the CtnC and CtnD genes of Monascus purpura, respectively. The reaction system is 20 μL, each primer is 1 μL, 2×Taq PCR Mix is ​​10 μL, and finally ddH 2 Add O to 20 μL,...

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Abstract

The invention discloses a simple preparation method of a rapid PCR template of filamentous fungi. The simple preparation method comprises the following steps of: (1) culturing to-be-detected filamentous fungi to obtain mycelia; (2) picking the mycelia of the filamentous fungi and putting the mycelia into a glass test tube filled with distilled water to obtain a mycelia mixed solution; (3) carrying out ultrasonic treatment on the glass test tube containing the mycelia mixed solution; and (4) directly taking the mycelia mixed solution subjected to ultrasonic treatment as a DNA template to carry out PCR amplification. The method disclosed by the invention does not need liquid nitrogen grinding, does not need to add any extraction reagent, is simple and rapid, and saves time and labor. The defects that existing filamentous fungi are long in DNA extraction time, more in added reagent and more in required instrument are overcome. The DNA template extracted by the method disclosed by the invention can obtain a stable and reliable experimental result through PCR reaction, is low in cost, good in effect, suitable for rapid analysis of large-batch samples, and also suitable for detection in a laboratory with imperfect instrument conditions.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a simple and simple preparation method for a rapid PCR template of a filamentous fungus. Background technique [0002] PCR is the polymerase chain reaction. As the most basic technology in the field of molecular biology, it is widely used in gene amplification and genetic analysis of animals, plants and microorganisms. However, the time and cost of PCR are greatly affected by the reaction DNA template. At present, most DNA extraction methods use cetyltrimethylammonium bromide (CTAB) method, sodium dodecylsulfonate (SDS) method or commercialized kits. Although these methods can obtain high-quality DNA, they need to add a lot of reagents, and the operation steps are complicated and time-consuming, which increases the time and cost of the experiment. Therefore, it is of great application value to realize rapid PCR by targetedly improving the DNA template of the reaction. Although ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C12Q1/686
CPCC12Q1/6806C12Q1/686
Inventor 赵杰宏桂艳玲韩洁唐光甫满海乔
Owner 贵州中医药大学
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