| Objective Pathogenic bacteria is a important factor involved in disease and death of infant of Neonatal intensive care unit (NICU). The rapid identification of pathogenic bacteria is important for earlier effective patient management and antimicrobial therapy, especially for the infant patients, which immunological system is not fully developed. However conventional microbiogical techniques of bacteria identification, culture and isolation of pathogenic bacteria, then identified by biochemistry and serological assay, are time-comsued as well as intensive labor. It causes abuse of antibiotics and accumulation of anti-antibiotics of pathogenic bacteria. Furthermore, these methods do not operate for difficult cultured or non-cultured agents. It is urgent problem to find an effective method to rapidly, simply, sensitively diagnose pathogenic agents at earlier phase. The objective of this study was to design and test a reverse oligonucleotide assays, which hybrid the PCR product of 16SrDNA using a pair of universal primers, to rapidly identify common NICU pathogenic bacteria.Methods By comparison and analysis of the 16SrDNA sequences of 83 species of pathogenic bacteria, a region, which has numerous sequence variations and flanked by highly conserved sequences, was found. A pair of universal primers was designed according to its flanking conservative sequence, and a set of probes specially targeting to twelve species of infant pathogenic bacteria according to the variable sequences. The probes were fixed on the nylon membranes with positive electricity, and hybridized them with the production of PCR using the universal primers.To optimize speciality, sensitivity and effect of 16SrDNA oligonucleotide array, different methods of probes fixation, condition of PCR, condition of hybrid, and improvement of hybrid liquid were tested in this study.The 16SrDNA oligonucleotide arrays were valuated by mathematic methods using 45 standard trains and 123 wild trains. The sensitive value, special value, Youden index, likelihood ratio, predictive value, and true value were tested in this study.At the last, this method was directly used to detect pathogenic bacteria from 568 clinical samples, as which the results were compared by translational cultured method.Results The universal primers can amplify the target sequence from the common infant pathogenic bacteria, but Rotavirus and human DNA as control. The results showed that the oligonucleotide array can specially hybridize with the twelve bacteria to be examined and can't hybridize with other bacteria. It proved that the probes are of relative highly selective and the oligonucleotide arrays can parallelly detect the twelve common infant pathogenic bacteria. The result showed that the sensitive of oligonucleotide arrays was relative high. The limit concentration of DNA sample for positive detection was lpg/ul.At optimization of oligonucleotide arrays, it was found that the effect of fixation probes to nylon membranes though amino side modification and treatment of membranes with DEC were better than that though addition tail to probes and conjunction by UV. The optimized temperature for hybrid is 42C-45C, and the process of hybrid was shortened from 6 hours to 30 minutes, when the optimized liquid was used.The obvious results were obtained when the concentration of probes for fixation membranes was 2pmol-20pmol, and the concentrations of 4pmol-10pmol were favored according to integrated request. Obvious results were obtained when the membranes were pre-hybridizated or not. However,the later takes the advance of saving about an hour.It proved that oligonucleotide arrays have high values. The sensitive value of assay was 96.1%, the special value was 95.0%, Youden index was 0.91, positive likelihood ratio was 19.22, negative likelihood ratio was 0.04, the predictive positive value was 98.4%, and the predictive negative value was 88.4%, when 168 trains were test. There was no obvious difference (P=0.453) , when the results were compared with cultured method, and th...
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