CN108504726B - Method for carrying out PCR pretreatment by adopting AMF single spore trace DNA - Google Patents

Abstract

The invention adopts AMF single spore trace DNA to carry out PCR pretreatment, which is to decant AMF spores through a wet sieve; cutting small square pieces and placing on sterile filter paper; pricking small pits on the small square sheets; picking uncracked AMF spores and placing the AMF spores in the pits; crushing spores with a micro-syringe needle and injecting sterile glycerol to wash off individual spore contents remaining on the needle; and (3) placing the small square piece with the AMF single spore crushed material and the glycerol into the bottom of a PCR tube, adding positive and negative primers, Taq enzyme and PCR-grade sterile water required by PCR, and quickly centrifuging to perform PCR. Sterile glycerin quickly cleans a needle possibly stained with AMF single spore inclusion, and the glycerin can improve the PCR amplification efficiency of trace DNA, expose AMF single spore DNA and other inclusions on a sterile cube, can be directly placed at the bottom of a PCR tube, and is contacted with other reaction liquid of PCR to perform PCR reaction. The related device is easy to obtain, can be repeatedly used, and is simple to operate, high in feasibility and low in cost.

Description

Method for carrying out PCR pretreatment by adopting AMF single spore trace DNA

Technical Field

The invention relates to a PCR pretreatment method for single spores of Arbuscular Mycorrhizal Fungi (AMF), in particular to a PCR pretreatment method adopting AMF single spore trace DNA, belonging to the field of strain detection.

Background

Arbuscular Mycorrhizal Fungi (AMF) are a group of fungi that widely symbiotic with plants in the soil, are of a wide variety and have now been individually designated as gloeocystis. Because of their mutualistic symbiotic relationship with plants, efforts have been made to identify and discover strains or new species with different characteristics. However, in the process of identifying and discovering new species, molecular identification has to be resorted to because morphological characteristics of different species are difficult to distinguish. The molecular identification of AMF is to amplify the gene segment in specific area by Polymerase Chain Reaction (PCR) using the DNA of single spore of AMF as template, and identify the strain by comparing the difference between the gene segment and the homologous gene segment of known strain. Although the molecular identification of AMF has been long, due to the spore wall thickness of AMF spores, the method for PCR pretreatment by using AMF single spore micro-DNA is too cumbersome or expensive, for example, the method of repeatedly alternating cold and hot is adopted to break the single spores, and then the micro-DNA in the single spores is carefully transferred to carry out PCR; also, certain companies use expensive lysates for the extraction of single spore DNA and perform technical blockages. These tedious or costly pretreatment methods increase the resistance to AMF molecular identification. At present, a simple, cheap and highly operable pretreatment method is not available, which can not only simply break the wall of single AMF spore, but also is beneficial to transferring trace DNA in the single spore into a PCR system.

Disclosure of Invention

The invention aims to overcome the defects of the traditional AMF single spore PCR pretreatment method and discloses a method for carrying out PCR pretreatment by adopting AMF single spore trace DNA.

The technical scheme adopted by the invention for solving the technical problems is as follows:

a. obtaining a plurality of AMF spores by decantation through a wet sieve, ultrasonically cleaning a certain amount of AMF spores in sterile water, performing suction filtration on sterile filter paper, and placing the AMF spores in a super-clean workbench, wherein the following steps are all performed in the super-clean workbench;

b. shearing a flat square piece from the sterile PCR tube cover by using sterilized scissors, and placing the flat square piece on sterile filter paper;

c. placing sterile filter paper on an anatomical lens objective table, and pricking a small pit on a small square sheet by using a flame-sterilized micro-injector needle;

d. picking a full and uncracked AMF spore with a flame sterilized dissecting needle and placing the spore in a pit;

e. sucking sterile glycerin by using a micro-syringe, crushing AMF single spores in a small pit by using a needle of the micro-syringe under a dissecting mirror, and then pushing out the sterile glycerin to wash out single spore contents remained on the needle;

f. and (3) putting the small square slice with the AMF single spore crushed material and the glycerol into the bottom of a PCR tube by using forceps sterilized by flame to finish the pretreatment process, adding positive and negative primers, Taq enzyme and sterile water of PCR grade required by PCR, and quickly centrifuging to perform PCR.

The invention utilizes the micro-syringe needle to manufacture the small pit on the small square plate, the single AMF spore is put into the small pit, the needle-shaped micro-syringe needle can crush the single AMF spore at the bottom of the pit to lead the DNA and other inclusions to come out, and then the needle possibly stained with the single AMF spore inclusion is quickly cleaned by utilizing the sterile glycerol in the micro-syringe, and the glycerol can also improve the PCR amplification efficiency of the micro DNA. The AMF single spore DNA and other inclusions are exposed on the sterile small square piece and can be directly placed at the bottom of the PCR tube to be in contact with other reaction liquid of PCR for PCR reaction.

Detailed Description

The present invention will be described in further detail with reference to specific examples, which are provided for the purpose of illustration and are not intended to be limiting:

the invention adopts the following specific technical scheme for solving the technical problems:

a. decanting by wet sieve to collect mycorrhizal plant root system and AMF spore in rhizosphere soil, picking 100 AMF spores under a body type microscope, ultrasonically cleaning in 1.5mL ion tube containing 0.5mL sterile water for 10min, suction-filtering on sterile filter paper, and placing in a superclean workbench, wherein the following steps are all carried out in the superclean workbench;

b. shearing a flat 2X 2mm square piece of the sterile PCR tube cover by using sterilized scissors, and placing the flat square piece on sterile filter paper;

c. placing sterile filter paper on an anatomical lens objective table, and pricking a small pit with the depth of 0.5mm on a small square sheet by using a flame sterilized micro-syringe needle;

d. picking a full and uncracked AMF spore by using a flame sterilized dissecting needle and placing the AMF spore in a pit;

e. sucking 1 μ L of sterile glycerol with a micro-syringe, crushing AMF single spores in a pit under a dissecting scope using a needle of the micro-syringe, and then pushing out 1 μ L of sterile glycerol to wash off single spore contents remaining on the needle;

f. placing the small square slice with AMF single spore broken material and glycerol into the bottom of PCR tube by using forceps sterilized by flame to complete pretreatment process, adding 0.5. mu.L of forward and reverse primers with concentration of 10mM required by PCR, 10. mu.L of 2 XTaq enzyme mix and 8. mu.L of PCR-grade sterile water respectively, and quickly centrifuging to perform PCR.

Claims (1)

1. The method for performing PCR pretreatment by adopting AMF single spore trace DNA is characterized by adopting the technical scheme that:

a. decanting by wet sieve to collect mycorrhizal plant root system and AMF spore in rhizosphere soil, selecting 100 AMF spores under a body type microscope, ultrasonically cleaning in 1.5mL ion tube containing 0.5mL sterile water for 10min, suction-filtering on sterile filter paper, and placing in an ultra-clean workbench;

b. shearing a flat 2X 2mm square piece of the sterile PCR tube cover by using sterilized scissors, and placing the flat square piece on sterile filter paper;

c. placing sterile filter paper on an anatomical lens objective table, and pricking a small pit with the depth of 0.5mm on a small square sheet by using a flame sterilized micro-syringe needle;

d. picking a full and uncracked AMF spore by using a flame sterilized dissecting needle and placing the AMF spore in a pit;

e. sucking 1 μ L of sterile glycerol with a micro-syringe, crushing AMF single spores in a pit under a dissecting scope using a needle of the micro-syringe, and then pushing out 1 μ L of sterile glycerol to wash off the single spore contents remaining on the needle;

f. placing the small square slice with AMF single spore broken material and glycerol into the bottom of PCR tube by using forceps sterilized by flame to complete pretreatment process, adding 0.5. mu.L of forward and reverse primers with concentration of 10mM required by PCR, 10. mu.L of 2 XTaq enzyme mix and 8. mu.L of PCR-grade sterile water respectively, and quickly centrifuging to perform PCR.