METHODS, SYSTEMS AND COMPOSITIONS FOR MICROBIAL DNA DETECTION BY PCR

摘要

FIELD: pharmacy.;SUBSTANCE: method includes: hybridization of multiple hybridization probes selected from SEQ ID NO: 1 or SEQ ID NO: 6, with the said target bacterial DNA, wherein each of the said hybridization probes contains a 5'-terminal portion with a non-bacterial DNA sequence selected from a viral sequence or an artificially designed sequence and 3'-terminal portion with a sequence complementary to the portion of the bacterial 16S rRNA gene sequence; removal of unhybridized hybridization probes and any unbound 3'-terminal portion of the target bacterial DNA; extension of 3'-terminal hybridization probes and the bacterial target DNA to form a double-stranded matricas elongated by means of primer. PCR is then carried out for selective amplification of the primer-elongated matrices using a set of primers, which comprises at least one primer with a non-bacterial sequence, complementary to the non-bacterial sequence of hybridization probes. A method for bacterial infection detection in a subject is provided, comprising: addition of multiple hybridization probes selected from SEQ ID NO: 1 or SEQ ID NO: 6 in the subject sample, wherein each of the said hybridization probes contains a 5'-terminal portion having a non-bacterial DNA sequence selected from viral a sequence or an artificially constructed sequence, 3'-terminal portion having a sequence complementary to the portion of the bacterial 16S rRNA gene sequence; hybridizing of hybridization probes with bacterial DNA in the sample; removal of unhybridized hybridization probes and any unbound 3'-terminal portion of the bacterial DNA; extension of the 3'-terminal hybridization probes and bacterial DNA form a double-stranded matricas elongated by means of primer; amplification of the primer-elongated matrices by the PCR method using a set of primers, which comprises at least one primer with a non-bacterial sequence, complementary to the non-bacterial sequence of the hybridization probe; and analysis of the amplified PCR-products to determine bacteria presence or absence. Also, a hybridization probe to create primer-elongated DNA matrix from the target bacterial DNA in the sample for selective amplification by the PCR method is described. The said hybridization probe comprises: a 5'-terminal portion having a non-bacterial sequence selected from a viral sequence and an artificially constructed sequence; and 3'-terminal portion having sequence complementary to portion of the bacterial 16S rRNA gene sequence portion, wherein the said hybridization probe is selected from SEQ ID NO: 1 or SEQ ID NO: 6. A kit comprising a plurality of such probes is described. Methods of this invention are based on a new approach of marking of the 5' terminals of DNA matrices by a non-bacterial marked sequence in order to distinguish the matrices from endogene contaminants present in PCR agents.;EFFECT: invention provides a method for selective amplification of one or more target bacterial DNA in the sample.;13 cl, 6 dwg, 2 tbl, 1 ex