Specific and universal probes and amplification primers to rapidly detect and identify common bacterial pathogens and antibiotic resistance genes from clinical specimens for routine diagnosis in microbiology laboratories
a technology of amplification primers and probes, which is applied in the direction of bacteria peptides, biochemistry apparatus and processes, peptide sources, etc., can solve the problems of low sensitivities and specificities, system error margin, rapid test results, etc., to reduce rapid and sensitive bacterial identification, the effect of reducing the time currently required for identification
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example 1
[0037]Isolation and cloning of fragments. Genomic DNAs from Escherichia coli strain ATCC 25922, Klebsiella pneumoniae strain CK2, Pseudomonas aeruginosa strain ATCC 27853, Proteus mirabilis strain ATCC 35657, Streptococcus pneumoniae strain ATCC 27336, Staphylococcus aureus strain ATCC 25923, Staphylococcus epidermidis strain ATCC 12228, Staphylococcus saprophyticus strain ATCC 15305, Haemophilus influenzae reference strain Rd and Moraxella catarrhalis strain ATCC 53879 were prepared using standard procedures. It is understood that the bacterial genomic DNA may have been isolated from strains other than the ones mentioned above. (For Enterococcus faecalis and Streptococcus pyogenes oligonucleotide sequences were derived exclusively from data banks). Each DNA was digested with a restriction enzyme which frequently cuts DNA such as Sau3AI. The resulting DNA fragments were ligated into a plasmid vector (pGEM3Zf) to create recombinant plasmids and transformed into competent E. coli cell...
example 2
[0040]Same as example 1 except that testing of the strains is by colony hybridization. The bacterial strains were inoculated onto a nylon membrane placed on nutrient agar. The membranes were incubated at 37° C. for two hours and then bacterial lysis and DNA denaturation were carried out according to standard procedures. DNA hybridization was performed as described earlier.
example 3
[0041]Same as example 1 except that bacteria were detected directly from clinical samples. Any biological samples were loaded directly onto a dot blot apparatus and cells were lysed in situ for bacterial detection. Blood samples should be heparizined in order to avoid coagulation interfering with their convenient loading on a dot blot apparatus.
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