Detection of Pneumocystis carinii DNA by loop-mediated isothermal amplification / 中华微生物学和免疫学杂志

ABSTRACT

Objective To detect Pneumocystis carinii (Pc) DNA by loop-mediated isothermal amplification (LAMP). Methods After injected with hydrocortisone acetate for 8 weeks, the bronchoalveolar lavage fluid (BALF) of Wistar rats were collected and a portion of BALF were examined for identifying Pc organisms using microscope. Then Pc DNA was extracted by phenol-chloroform extraction. Four primers which recognized 6 distinct regions on the mtrRNA gene of Pc were designed and used for LAMP assay. To evaluate the specificity of the assay, M. tuberculosis, M. pneumoniae, C. pneumoniae, P. gondii and rat leucocyte were used as negative controls. To compare the sensitivity of the LAMP to that of conventional PCR, Pc DNA were 10-fold serially diluted and was amplified by LAMP and PCR. LAMP results were judged by naked eye, electrophoretic analysis and restriction digestion. Results Pc organisms were detected from BALF of rats injected with hydrocortisone acetate. After LAMP reaction, positive signal was observef rats injected with hydrocortisone acetate. By contrast, no positive signal was observed for the negative controls in the study. The amplified product digested by restriction enzyme demonstrated 3 bands (82, 135, 189 lip) upon agarose gel electrophoresis, in good agreement with the predicted sizes. The detection limit of LAMP assay was 1 pg/μl of Pc DNA per reaction and that of PCR was 10 pg/μ1 of Pc DNA per reaction. Conclusion LAMP assay has usefulness for rapid detection of Pc.