Composition, method and kit for detecting bacteria by means of sequencing
OwnerJesus Mingorance Cruz, Pablo Castan Garcia +1inventors
InventorsJesus Mingorance Cruz, Pablo Castan Garcia, Pedro Manuel Franco De Sarabia Rosado
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Abstract
The present invention describes a method for detecting the presence and type of a microorganism present in a sample by means of stabilization and sequencing techniques and subsequent analysis of microsequences in genes encoding the ribosomal RNA most conserved, and on specific areas of the 16-S region with taxonomic value.
Claims10
A method for taxon-specific detection, differentiation and identification of eubacteria in a biological sample by means of sequencing analysis techniques, specifically pyrosequencing of three regions of the 16S bacterial ribosomal RNA gene, characterized in that it comprises the following steps:
- a. Stabilizing the reaction mixture by gelling by means of adding a stabilizing aqueous solution to the medium and subsequently drying to a degree of humidity between 10% and 30%.
- b. PCR amplification reaction of the total DNA extracted from the sample using the primer pairs specified in Table 2 (SEQ. ID. No. 1 to 6).
- c. Sequencing by synthesis, specifically pyrosequencing the PCR products obtained in step a. using the same non-biotinylated primers indicated in Table 2 and used for amplifying the total DNA extracted from the sample (SEQ. ID. No. 2, 4 and 6).
Dependencies are inferred from the claim text.