Shang Shi-qiang, Dong Guan-ping, Fu Jun-fen, Hong Wen-lan, Du Li-zhong, Yu Xi-lin
Department of Neonatology, Children's Hospital of Zhejiang University, Hangzhou 310003 China.
Zhonghua Er Ke Za Zhi. 2003 Sep;41(9):692-6.
To establish the specific 16S-23S rRNA gene spacer regions pattern in different bacteria using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), DNA cloning and sequences analysis.
A pair of primers were selected from highly conserved sequences adjacent to the 16S-23S rRNA spacer region. Bacterial DNA of sixty-one strains of standard bacteria and corresponding clinical isolates representative of 20 genera and 27 species was amplified by PCR, and further studied by RFLP, DNA cloning and sequences analysis. Meanwhile, all specimens were examined by bacterial culturing and PCR-RFLP analysis.
The 27 different standard strains showed one, two, three or more than three bands. The sensitivity of PCR reached 2.5 colony-forming unit (CFU), and there was no cross reaction to the human, fungal or viral genomic DNAs. Fifteen species could be distinguished immediately by PCR, while another 10 species were further identified by Hinf I or Alu I digestion. Klebsiella pneumoniae (Kp) and Enterococcus durans (Ed) could not be differentiated from each other by Alu I or Hinf I digestion. The spacer sequences of the Kp and Ed were 908 bp and 909 bp, respectively, and they differed only at the site of the 779th nucleotide. The former was G, and the latter was A. The 760 - 790 bp sequence of Kp was as follows: CGACTGCACCGCCTCCTAC / GGCCGCGTATTC. The 760 - 790 bp sequence of Ed was as follows: CGACTGCAC CGCCTCCTAC / AGCCGCGTATTC. Only one enzyme XmaIII, could discriminate the two. The cleaving site of XmaIII is C downward arrow GGCCG. Kp DNA was cleaved into 778 bp and 130 bp fragments, while E. durans was not. Of 42 specimens with suspected septicemia, 15 were positive (35.7%) on blood culture, and 27 on PCR (64.29%). The positive rate of PCR was significantly higher than that of blood culture (P < 0.01). Of the six CSF specimens, one was positive for Staphylococcus epidermidis (Se) on culture as well as by PCR, while two specimens which were negative on cultures were positive by PCR and were diagnosed as Se according to its DNA pattern. One specimen was culture-positive for Cryptococcus neoformans (Cn) but was negative by PCR. The other two specimens were negative by both PCR and culture. Fifteen blood samples from healthy children were negative by both blood culture and PCR.
The method of detecting bacterial 16S-23S rRNA spacer regions using PCR-RFLP techniques was specific, sensitive, rapid and accurate in detecting pathogens in clinical bacterial infections.
运用聚合酶链反应（PCR）、限制性片段长度多态性分析（RFLP）、DNA克隆及序列分析，建立不同细菌的特异性16S - 23S rRNA基因间隔区图谱。
从与16S - 23S rRNA间隔区相邻的高度保守序列中选取一对引物。对代表20个属27个种的61株标准菌株及相应临床分离株的细菌DNA进行PCR扩增，并进一步通过RFLP、DNA克隆及序列分析进行研究。同时，对所有标本进行细菌培养及PCR - RFLP分析。
27株不同的标准菌株呈现出一条、两条、三条或三条以上条带。PCR的灵敏度达到2.5个菌落形成单位（CFU），对人、真菌或病毒基因组DNA无交叉反应。15个菌种可通过PCR立即区分，另外10个菌种通过Hinf I或Alu I酶切进一步鉴定。肺炎克雷伯菌（Kp）和耐久肠球菌（Ed）经Alu I或Hinf I酶切无法区分。Kp和Ed的间隔序列分别为908 bp和909 bp，仅在第779个核苷酸位点不同。前者为G，后者为A。Kp的760 - 790 bp序列如下：CGACTGCACCGCCTCCTAC / GGCCGCGTATTC。Ed的760 - 790 bp序列如下：CGACTGCAC CGCCTCCTAC / AGCCGCGTATTC。只有一种酶XmaIII能区分二者。XmaIII的切割位点是C向下箭头GGCCG。Kp DNA被切割成778 bp和130 bp的片段，而耐久肠球菌则未被切割。42例疑似败血症标本中，血培养15例阳性（35.7%），PCR 27例阳性（64.29%）。PCR阳性率显著高于血培养（P < 0.01）。6例脑脊液标本中，1例表皮葡萄球菌（Se）培养及PCR均阳性，2例培养阴性标本PCR阳性，根据其DNA图谱诊断为Se。1例新型隐球菌（Cn）培养阳性但PCR阴性。另外2例标本PCR和培养均阴性。15例健康儿童血标本血培养和PCR均阴性。
运用PCR - RFLP技术检测细菌16S - 23S rRNA间隔区的方法在临床细菌感染病原体检测中具有特异性、灵敏性、快速性和准确性。
