Bacterial nucleic acid sequencing identification method based on DNA bar code

A technology of bacterial nucleic acid and identification method, which is applied in the field of bacterial nucleic acid sequencing and identification based on DNA barcoding, can solve the problems of unclear and clear DNA barcoding nucleic acid sequence fragments, non-standard and unified basis for judging sequencing results, and little attention to the accuracy of sequence information.

Inactive Publication Date: 2017-05-24
SHANGHAI INST FOR FOOD & DRUG CONTROL
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  • Abstract
  • Description
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AI Technical Summary

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 domai

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  • Bacterial nucleic acid sequencing identification method based on DNA bar code
  • Bacterial nucleic acid sequencing identification method based on DNA bar code
  • Bacterial nucleic acid sequencing identification method based on DNA bar code

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1. Establishment of DNA barcodes and standard nucleic acid sequence databases for identification of common microbial pollutants in pharmaceutical production environments

[0052] 1. Log in to the NCBI Genebank database, search for the target bacterial genera under the Taxonomy item, such as Staphylococcus, Micrococcus, Pseudomonas, Bacillus, Escherichia coli, Salmonella, etc., and further filter "16SrRNAand refSeq and length 1000-2000bp", to obtain the qualified 16S rRNA nucleic acid sequence.

[0053] 2. Download the above sequence from the Genebank database in the Fasta file format, import it into the Bioedit sequence comparison software, perform ClustalW sequence comparison analysis with the ClustalW Multiple Alignment function, Full Multiple Alignment, Bootstrap NJTree (1000numbers) statistical methods, and select the sequence with identification and classification Significant nucleic acid sequence fragments, and design universal sequencing primers based on ...

Embodiment 2

[0067] Example 2. Establishment of DNA barcodes and databases for identifying staphylococcal pollutants in pharmaceutical production environments

[0068] 1. Test method and operation steps

[0069] 1. Log in to the NCBI Genebank database, search for Staphylococcus under the Taxonomy item, and further screen "16S rRNAand refSeq and length 1000-2000bp" through the filter function to obtain the qualified 16S rRNA nucleic acid sequence.

[0070] 2. Download the above sequence from the Genebank database in the Fasta file format, import it into the Bioedit sequence comparison software, perform the ClustalW sequence comparison with the ClustalW Multiple Alignment function, Full Multiple Alignment, Bootstrap NJTree (1000numbers) statistical methods, and use the general sequencing primer V1forward( 27F) and V3reverse (548R) are positioned at both ends, and the nucleic acid sequence fragments of the same length are intercepted;

[0071] 3. Delete the sequence that the nucleic acid seq...

Embodiment 3

[0075] Example 3. Establishment of DNA barcode and database for identification of Pseudomonas pollutants in drug production environment

[0076] 1. Test method and operation steps

[0077] 1. Log in to the NCBI Genebank database, search for Pseudomonas under Taxonomy, and further screen "16S rRNA and refSeq and length 1000-2000bp" through the filter function to obtain the qualified 16S rRNA nucleic acid sequence.

[0078]2. Download the above sequence from the Genebank database in the Fasta file format, import it into the Bioedit sequence comparison software, perform the ClustalW sequence comparison with the ClustalW Multiple Alignment function, Full Multiple Alignment, Bootstrap NJTree (1000numbers) statistical methods, and use the general sequencing primer V1forward( 27F) and V3reverse (548R) are positioned at both ends, and the nucleic acid sequence fragments of the same length are intercepted;

[0079] 3. Delete the sequence that the nucleic acid sequence is not completel...

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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...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/6869C12Q2531/113C12Q2565/125
Inventor 冯震杨美成李芳李琼琼杨燕洪小栩许华玉刘浩
Owner SHANGHAI INST FOR FOOD & DRUG CONTROL
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