A Low-Contamination and High-Sensitivity Low-Cost Universal Molecular Identification Method for Bacteria

Abstract

The invention discloses a low-cost general strain molecular identification method with low pollution and high sensitivity, belonging to the technical field of biology. The method comprises the following steps: 1) pretreating a strain; 2) preparing a column with high adsorption capacity; 3) performing strain splitting and nucleic acid adsorption; 4) cleaning; 5) eluting; 6) performing first ground of PCR (polymerase chain reaction) amplification; 7) performing second ground of PCR amplification; 8) performing electrophoresis detection and purification; 9) sequencing and comparing by using a primer M13F, and determining genus and species. According to the low-cost general strain molecular identification method with low pollution and high sensitivity, the strain can be relatively accurately identified in an initial stage of microorganism colony formation by using a simple and efficient wall-breaking and extracting technique and combining with an efficient nucleic acid adsorption technique as well as two times of PCR amplification effects; furthermore, the primer for identification adopted by the invention gives consideration to the uniformity of fungus sites and bacterium sites, so that the bacterium can be identified at the level of species, the fungus can also reach the level of genus or even species, thereby the simultaneous identification of certain bacterium and fungus is facilitated, the efficiency is greatly improved, and the experiment cost is reduced.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a low-pollution and high-sensitivity low-cost universal strain molecular identification method. Background technique [0002] The identification of bacterial species by means of molecular biology is an important means for the evolution and taxonomic identification of microbial systems. When determining the bacterial species, most of them first carry out DNA extraction, then use the DNA sample for PCR amplification, and finally determine the taxonomic status of the bacteria through sequencing and sequence comparison of the PCR products. Existing detection methods usually require the isolation and cultivation of bacteria when extracting sample DNA, which takes a long time and is relatively low in efficiency. In addition, some bacteria can only be cultivated under specific conditions, and routine cultivation is not easy, resulting in the loss of sample collection. difficult...

AI Technical Summary

Problems solved by technology

Existing detection methods usually require the isolation and cultivation of bacteria when extracting sample DNA, which takes a long time and is relatively low in efficiency. In addition, some bacteria can only be cultivated under specific conditions, and routine cultivation is not easy, resulting in sample collection. difficulty

If a small amount of sample is extracted, the sensitivity of the identification method is often not enough to meet the requirements

If simply using the PCR amplification mechanism to improve the detection sensitivity, it is feasible in theory to directly perform PCR after lysing the sample, but it is often not easy to succeed in the actual operation process, especially for most Gram-positive bacteria. , actinomycetes and fungi, because the cell wall is relatively thick, it is difficult to collect the target DNA, and some very small amounts of contamination that are easy to amplify are easily mixed into the reaction, so that the final amplification result is often a large number of false positives

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