新生儿常见感染病原菌的16SrDNA寡核苷酸阵列快速诊断

目的探讨PCR结合寡核苷酸阵列杂交快速诊断常见新生儿感染病原细菌方法。方法通过对40多种常见病原菌的16SrDNA序列比较分析,在中间找到一段两端高度保守中间相对变异的序列。依据其保守序列设计一对通用引物,用于扩增细菌16SrDNA目的基因。同时依据相对变异区域的序列设计常见的8种病原细菌的探针,阵列于带正电的尼龙膜上,与通用引物的PCR产物杂交,在一个反应体系中1次可检测出标本中可能存在的常见致病菌。结果设计的通用引物能有效扩增出常见的细菌目的序列。实验结果表明寡核苷酸阵列均能特异地与其检测细菌的通用引物PCR产物杂交,而不与其它所试细菌通用引物PCR产物杂交,说明探针具有很强的特异性。进一步采用寡核苷酸阵列对临床分离的菌株进行鉴定,以细菌自动鉴定仪做对比,结果表明寡核苷酸阵列能检出常见的8种病原菌。结论PCR结合寡核苷酸阵列杂交是快速诊断常见新生儿感染病原菌有效方法。

Rapid diagnosis of common pathogenic bacteria infection in newborn infants by 16SrDNA oligonucleotide array

OBJECTIVE: The rapid identification of pathogenic bacteria is important for earlier effective patient management and antimicrobial therapy, especially for the infant patient, whose immunological system is not fully developed. However conventional microbiogical techniques of bacterial identification, culture and isolation of pathogenic bacteria, identification by biochemistry and serological assay, are time-consuming and require intensive labor. On the basis of special gene sequence, PCR provides simple and rapid way to identify bacteria. But it is difficult to identify all of bacteria species which are suspicious of pathogenic agents. Oligonucleotide arrays provide a powerful tool for parallel detection of target genes. The objective of this study was to test a reverse oligonucleotide assay, which hybridize with the PCR product of 16SrDNA using a pair of universal primers, to rapidly identify common infant pathogenic bacteria. METHODS: By comparison and analysis of the 16SrDNA sequences of common 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 eight species of infant pathogenic bacteria, including staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus faecalis, Hemophilus influenzae, Enterobacter cloacae, Escherichia coli, and Acinetobacter baumannii,according to the variable sequences. The probes were fixed on the nylon membrane with positive electricity, and hybridized them with the products of PCR using the universal primers. RESULTS: The universal primers could amplify the target sequence from bacteria including the eight common infant pathogenic bacteria and Staphylococcus epidermidis, Enterobacter aerogenes, Streptococcus pneumoniae,beta-hemolytic streptococcus, Neisseria meningitides, Citrobacter freundii, Bacillus subtilis, and Salmonella infantis,but could not amplify rotavirus and human DNA as control. The results showed that the oligonucleotide array could specially hybridize with the eight bacteria to be examined and could not hybridize with other bacteria. The lowest concentration of DNA (product of PCR) for oligonucleotide array was about 25 ng/ml. The results proved that the probes are highly selective and the oligonucleotide arrays could parallelly detect the eight common infant pathogenic bacteria. The results suggested that the oligonucleotide array system was able to identify the eight common infant pathogenic bacteria from clinical specimens and the results were the same as identified by automated bacterial detection machine. From the further experiments, the oligonucleotide array system could directly diagnose the common infant pathogenic bacteria from the broths of samples culture. CONCLUSIONS: Despite limited number of identifiable bacteria and lack of information on antibiotic susceptibility of bacteria, the reverse oligonucleotide assay system, which contains amplification of the segment of 16rDNA from samples using the universal primers and parallel detection of PCR products using specific probes, is an effective method to rapidly identify the eight common infant pathogenic bacteria.