A quick way to identify bacteria

the 1980s, the classification and identification of bacteria relied mainly on morphological characteristics,
culture
characteristics, physiological, biometric and
immunological
responses. These methods have played an important role in the classification and identification of bacteria, and there are also shortcomings such as poor accuracy and cumbersome and time-consuming identification. With the rapid development of
metrophic biology
and especially the invention of polymerase chain reaction technology
PCR
), the classification and identification of bacteria has undergone a major innovation and has begun to enter the molecular level, and a number of molecular identification methods have been produced. The 16S rDNA gene is analysis has become a standard method for the identification of bacterial genus and plays an important role in the classification of bacteria.
16S rDNA gene is found in primary nuclear organisms. It consists of conservative and variable zones, which are common to all bacteria, and there is no difference between bacteria; The 16S rDNA sequence of bacteria is about 1.6kb long, and its sequence changes at a rate appropriate to the rate of evolution, so it is widely used for genus identification. Combined with a well-developed database, 16S rDNA sequence analysis can quickly and accurately identify
microbial
genus and determine the location of microorganisms in evolution. The accuracy and importance of establishing bacterial system development classification based on 16SrDNA sequence esoxual analysis have been recognized and accepted by more and more bacterial classification scholars.
16S rDNA gene that amplifies bacteria needs its chromosomal DNA as a template. At present, the more commonly used bacterial chromosome DNA small preparation method is SDS alkali lysate method and boiling method, the former needs multi-step phenol extraction to remove
protein
, the extraction process needs about 8h; In this paper, a new method of extracting chromosome DNA from bacteria in quick and easy quantities is established, and the whole extraction process takes only 20min. Chromosomal DNA prepared by this method can be used as a template for 16S rDNA sequences of PCR amplification bacteria without any treatment.
, materials and methods
1. The 22 bacterial isolates of the test strain
for research were separated from soil samples in Yunnan and other places in the early stages of the center and were built in the Industrial Microbial Resources and Information Center of The Chinese University of Jiangnan University under the number C1 to C22.
2.
The medium
gravy medium, MRS medium, and high temperature bacteria medium.
3. The culture of the test strain
connects the test strain C1 to C22 from the glycelin storage tube and draws the line on the corresponding plate. With the exception of the high temperature bacteria plate cultured at 70 degrees C, the rest of the plates are cultured at 37 degrees C.
4. Rapid extraction of bacterial chromosome DNA
1.5 mL centrifuge tube for weighing. Add 100 L buffer I to the tube and scrape a number of cyclobacteria from the plate, oscillating evenly, 9,000r/min, 3min. Discard the liquid, weigh it, and calculate the mass of the bacteria. In accordance with the ratio of 10 L per milligram of bacteria, buffer II. is added to the centrifuge tube, the bacteria oscillates evenly, placed at 75 degrees C reaction 15min to obtain chromosomal DNA coarse fluid, directly used for subsequent PCR reaction.
5. PCR amplification and product sequencing
II, results and discussion
1. Establishment of a rapid extraction method for bacterial chromosome DNA
research attempts to establish a rapid extraction method of bacterial chromosomal DNA suitable for large-volume sample operation, and requires that the chromosomal DNA solution prepared by the established method can be used directly for subsequent PCR reactions without any post-treatment. To this end, the preparation and composition of two key solutions (buffer I and buffer II.) in the rapid DNA extraction method of bacterial chromosomes and their wide application are studied and analyzed. Buffer I contains EDTA (pH8.0) of 2 mmol/L and Tw-20 of 0.01. Buffer II consists of a 2mol/L NaOH solution and carefully adjusts the pH to 13.4 of buffer II. with a non-ionized concentration-related pH buffer.
2. Extracted chromosomal DNA quality analysis
(1)
agar
sugar gel electrophoresis test
take bacterial chromosomal DNA coarse pickup 10ptL for agarose gel electrophoresis, post-staining gel imaging. It was found that the spot hole was very bright and there were continuous bands outside the hole. The chromosomal DNA of 22 test strains was successfully extracted.
(2) PCR product inspection