Bacterial nucleic acid sequencing identification method and bacterial identification kit based on DNA characteristic sequence

Abstract

The invention relates to a bacterial nucleic acid sequencing identification method and kit based on DNA characteristic sequence. The method comprises the steps: selecting a bacterial strain to be identified, and extracting and purifying a genomic DNA; amplifying a characteristic sequence fragment of the purified genomic DNA by PCR; extracting and purifying the PCR amplified product; determining asequence of the purified PCR amplified product, and removing a primer region by universal sequence splicing software, to obtain a DNA sequence of the corresponding fragment; importing the DNA sequenceof the corresponding fragment into a standard nucleic acid sequence database, carrying out sequence comparison, and carrying out bacterial strain identification, wherein the extraction and purification of the genomic DNA of the bacterial strain adopts lysozyme, sodium dodecyl sulfate and cetyltriethylammnonium bromide for bacterial crushing; nucleic acid is precipitated by phenol-chloroform-isoamyl alcohol and ethanol to obtain the genomic DNA which meets the quality control requirements. An objective and accurate judgment basis can be provided for the bacterial nucleic acid sequencing identification method, and rapid and integrated identification of bacterial pollutants is achieved.

Description

technical field [0001] The invention relates to a nucleic acid sequencing identification method, in particular to a DNA characteristic sequence-based bacterial nucleic acid sequencing identification method and a bacterial identification kit. Background technique [0002] Using molecular biology technology to identify, classify and trace the polluting microorganisms in pharmaceutical raw materials, excipients, pharmaceutical water, intermediates, final products and the environment is an important way to strengthen the quality control of the pharmaceutical production process, improve product quality and safety, and reduce medication risks. effective means. Compared with traditional morphological observation, microscopy, physical and chemical identification and biochemical analysis, molecular biology technology uses biological nucleic acid as the target detection object, which can explain the species and source of the strain from the fundamental level of genetic material , the...

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Problems solved by technology

[0004] The full length of the 16S rRNA gene covers the most comprehensive information on the genetic evolution of strains, but is limited by the operability of nucleic acid sequencing methods, sequencing quality, sequencing cost, length of nucleic acid sequencing reactions, and especially the read length of high-throughput nucleic acid sequencing reactions. Still use partial nucleic acid sequence (Partial Sequence) for research, and the core region with identification and classification significance remains to be discussed, such as: USP40 believes that the 5' end of the 16S rRNA gene is more meaningful for the identification of bacterial "species" level; Chan et al. reported that in the population identification of clinically isolated microorganisms, the V1-V2 region has more classification significance; Kataoka et al. used the V1-V3 region as the identification target in the classification and identification of streptomycin; Becker et al. used in the classification and identification of staphylococcus The comparison method of V1~V3 region and V1~V8 region; Engelbrektson et al. used the Roche454 nucleic acid sequencing platform to find that V1, V2 and V8 regions have more specific sites; Kim et al. used the theory of OTUs (Operational taxonomic units) analysis to think V1-V3 regions and V1-V4 regions are more meaningful in the identification of microbial flora; Chakravorty et al. found that the tandem nucleotide sequences of V2, V3, and V6 regions in 16S rRNA can realize the identification and classification of most bacterial "species" levels; Lu used PCR and RFLP methods to classify and identify the microbial flora isolated from clinical cerebrospinal fluid, and believed that the resolution of the V4-V9 region in 16S rRNA was low, etc.

[0005] At present, microbial identification methods based on 16S rRNA nucleic acid sequencing technology have been established and applied to the study of clinically isolated strains, but there are few reports on their use in drug quality control and the identification of common pollutants in drug production environments; In the literature on microbial identification by rRNA nucleic acid sequencing method, the selected sequencing targets and sequencing primers are different, and the characteristic DNA sequence fragments that meet the requirements of drug quality control and have identification and classification significance are not clear; Most of the sequencing results are compared with the nucleic acid sequences in the Genebank database through the Blast analysis method. There is little attention paid to the quality verification of the sequencing results and the accuracy of the sequence information in the Genebank database, and the basis for judging the sequencing results is not yet standardized.

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