摘要
目的 建立连接酶链反应-酶联免疫吸附试验(LCR-ELISA)检测沙眼衣原体DNA的方法,并初步应用于检测新生儿沙眼衣原体感染。方法 针对沙眼衣原体外膜蛋白基因设计4条引物,并分别在其两端标记地高辛和生物素。用此4条引物进行LCR扩增沙眼衣原体DNA。带有生物素和地高辛双标记的扩增产物以ELISA方法显色判断结果。采集328份肺炎新生儿鼻咽标本,分别对其进行培养和LCR扩增,应用ELISA检测有无沙眼衣原体感染。结果 LCR-ELISA方法可以检测出6种沙眼衣原体标准株,而对2种肺炎衣原体和其他细菌DNA无反应,显示出较强的特异性;可检测出10fg的DNA,较传统电泳法敏感10倍,且避免了溴化乙啶的污染。328份标本中,LCR-ELISA检测出68份阳性,培养仅检测出60份,两者相比差异有显著性。以扩大的金标准判断,LCR-ELISA的特异性、敏感性分别为100％和98.6％;而培养的特异性、敏感性分别为100％和、86.9％。结论LCR-ELISA是一种特异而敏感的基因扩增方法,避免了溴化乙啶污染,判断结果客观,检测方便。经临床初步应用,取得良好结果,值得进一步研究和完善。
Objective To establish a LCR-ELISA to detect Chlamydia trachomatis infection in neonates with pneumonia. Methods Two pairs of probes of chlamydial outer membrane protein gene (omp1) were labeled with biotin and digoxin, respectively. Ligase chain reaction was used to amplify C. trachomatis DNA with these probes in six chlamydial strains. The amplified products were captured by microplates coated with streptavidin. After revelation with an anti-digoxi-POD-combined antibody, the amount was determined by optical reading. Nasopharyngeal swabs taken from 328 neonates with pneumonia were analyzed by LCR-ELISA and cell culture. Results A method of LCR-ELISA for detection C. trachomatis infection was established. It could detect six species of C. trachomatis and was no cross-reaction with C. psittaci and other bacteria. The detection limit of LCR-ELISA was 10fg template DNA, which was 10-folds sensitive than routine LCR using polyacrylamide gel electrophoresis for the detection of amplified products. It could be operated easily without radioactive and ethidium bromide contamination. The prevalence of C. trachomatis in 328 cases of neonatal pneumonia, tested by an expanded gold standard of a positive cell culture or two confirmed positive non-culture tests, was 21% (69/328). After analysis of discrepant results, the sensitivity, specificity, positive and negative predictive values for the LCR-ELISA were 98 .6% , 100% , 100% and 99.6% ,respectively, whereas those for culture were 86.9% , 100% , 100% and 96. 6% , respectively. Conclusion This study demonstrated that the LCR-ELISA was a highly sensitive and special non-culture technique and good alternative test for the detection of chlamydial infections. Initial application indicated the ELISA plate achieved good results and worth further studying and expanding detectable DNA of CT patients.
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2004年第5期295-298,共4页
Chinese Journal of Laboratory Medicine
基金
国家自然科学基金资助项目(30200300)
关键词
连接酶链反应
酶联免疫吸附试验
诊断
沙眼衣原体感染
Polymerase chain reaction
Chlamydia trachomatis
Enzyme-linked immunosorbent assay
Neonatal diseases
