Bacterial nucleic acid sequencing identification method based on DNA bar code

Abstract

The invention discloses a bacterial nucleic acid sequencing identification method based on a DNA bar code. Common bacterial contaminants in a medicine production environment can be effectively and accurately identified. The bacterial nucleic acid sequencing identification method comprises the steps that 1, a monoclonal bacterial strain is obtained through culture; 2, a genome DNA of the monoclonal bacterial strain obtained in the step 1 is extracted; 3, PCR is performed by using a general nucleic acid sequencing primer to amplify a specific sequence of nucleic acids; 4, a PCR product is extracted and purified; 5, a nucleic acid sequence of the PCR product obtained in the step 4 is determined; 6, a sequence analysis software is applied to perform sequence splicing, positioning is performed by using a general nucleic acid sequencing primer pair, a primer region is removed, and a DNA sequence of a corresponding fragment is obtained; 7, the DNA sequence of the corresponding fragment in the step is imported into a DNA bar code standard sequence core database or a GenBank database for sequence comparison, and bacteria are identified.

Description

technical field [0001] The invention relates to a nucleic acid sequencing identification method, in particular to a DNA barcode-based bacterial nucleic acid sequencing identification method. background technology [0002] 16S ribosomal RNA (16S ribosomal RNA, 16S rRNA) is the most commonly used molecular marker in the study of bacterial taxonomy, with a total length of about 1500bp, including 9 variable regions (Variable Region, V region) and 10 constant regions ( Constant Region, C region), is highly conserved in structure and function, and is suitable for the study of the phylogenetic relationship of various bacteria with different evolutionary distances. With the improvement of the drug production quality standard (GMP2010) and the national drug quality standard, the requirements for microbial identification in drug production and quality control are getting higher and higher, such as the requirements in the Parenteral Drug Association Technical Report (PDA TR) All class...

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Problems solved by technology

At present, a microbial identification method based on 16S rRNA nucleic acid sequencing technology has been established and applied to the research of clinical isolates (16S rRNA gene sequence analysis for bacterial identification in the clinical laboratory. RinshoByori. 2013Dec; 61(12): 1107-15 ; The use of molecular techniques based onribosomal RNA and DNA for rumen microbial ecosystem studies: a review.Mol BiolRep.2008Jun; 35(2):265-74.), but it is used for drug quality control and common contaminating bacteria in drug production environment The identification of 16S rRNA nucleic acid sequencing method is rarely reported; in the literature of microbial identification by 16S rRNA nucleic acid sequencing method, the selected sequencing targets and sequencing primers are different (Review and re-analysis of domain-specific 16S primers.JMicrobiol Methods.2003Dec; 55 (3):541-55; Identification of bacteria through 16SrRNA sequencing: principles, methods and applications in clinical microbiology.Enferm Infecc Microbiol Clin.2004Apr; 22(4):238-45.), which meet the requirements of drug quality control with identification and The nucleic acid sequence fragments of DNA barcodes with taxonomic significance are not yet clear; most of the nucleic acid sequencing results in existing studies are compared with the nucleic acid sequences in the Genebank database by Blast analysis, and the quality check of the sequencing results and the sequence information in the Genebank database There is less attention paid to the accuracy of the sequencing results, and the basis for judging the sequencing results is not yet standardized and unified

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