PCR in diagnosis of pneumocystosis of rats

, Hong, S T

doi:1996.34.3.191

Korean J Parasito > Volume 34(3):1996 > Article

Original Article


Korean J Parasitol. 1996 Sep;34(3):191-196. English. Published online Sep 20, 1996. http://dx.doi.org/10.3347/kjp.1996.34.3.191
Copyright © 1996 by The Korean Society for Parasitology


PCR in diagnosis of pneumocystosis of rats
Sung-Tae Hong^*
Department of Parasitology and Institute of Endemic Diseases, Seoul National University College of Medicine, Seoul 110-799, Korea.

Received August 17, 1996; Accepted August 24, 1996.

Abstract
Polymerase chain reaction (PCR) is a powerful technique to detect scanty amount of DNA from living organisms. The present study intended to develop specific primers for PCR diagnosis of pneumocystosis and to evaluate diagnostic efficacy by preparation of template DNAs from invasive BAL fluid and also to screen serum or blood as a non-invasive specimen. Albino rats of Wistar or Fischer strains were experimentally infected by Pneumocystis carinii. Extracted DNAs or cell lysates of their blood, bronchoalveolar lavage fluid, and lung homogenate were used as the template DNA. Primers were synthetic oligonucleotides among 16s rDNA sequences. All of the primer combinations gave PCR products, but the primer pair of #24 and #27 gave best quality product of 666 bp. The sensitivity of PCR with lysates of BAL fluid was 57.7% but it increased to 84.6% with extracted DNAs. None of BAL lysate or DNA was positive among 13 microscopically negatives. The serum DNAs were positive only in 2 cases out of 20 morphologically positive rats. DNAs of human, rat, other parasites, yeast, and microorganisms were negative. The findings suggest that the present primers are specific but simple lysate of BAL fluid is not sensitive. PCR may be used as a routine diagnostic method of pneumocystosis if simple and rapid preparation of non-invasive clinical specimens are available.

Figures

Fig. 1
PCR products of 666 bp by various amounts of template DNA in a reaction tube. The Template DNA was extracted DNA of purified P. carinii (W 13-20). Reaction volume was 50 µl and the reaction parameters were 94℃ 30 sec., 61℃ 30 sec., 72℃ 40 sec. in a thermal cycler 9600. 1, size marker; 2, 10 ng of DNA; 3, 1 ng og DNA; 4, 0.1 ng of DNA; 5, 10 fg of DAN; 6, 1 fg of DNA; 7, 0.1 fg of DNA; 8, 0.01 fg of DNA. The lane 3 showed best reaction, and the lane 5 was marginally positive.

Fig. 2
PCR product with DNA of other organisms. The reaction parameters were same as those of Fig. 1. 1, size marker; 2, purified P. carinii DNA (W13-20); 3, Escherichia coli; 4, Saccharomyces cerevisiae; 5, Entamoeba dispar, 6, Trichomonas vaginalis; 7, Leishmania major, 8, size marker; 9, sparganum; 10, Clonorchis sinensis; 11, Ascaris lumbricoides; 12, Gastroconger myriaster, 13, rat; 14, human; 15, size marker; 16, Streptococcus pneumoniae; 17, Neisseria sicca; 18, Staphylococcus aureus; 19, Candida albicans; 20, sputum of a patient; 21, rat P. carinii (F19). Only P. carinii DNA from rats were positive and all with others were negative.


	Fig. 3 PCR product separated in 0,7% agarose gel. Primers were #24 and #27. and PCR parameters were 94℃ 2 min., 62℃ 2 min., 74℃ 2 min. in a thermal cycler. 1, lysate of an infected lung homogenate; 2-4, BAL fluid lysates from infected rats; 5-7, Sera from non-infected Wistar rats; 8-9, sera from infected Wistar rats; 10-15, blood from infected Sprague-Dawley rats.

Tables


	Table 1 Primers of 16s rDNA for PCR


	Table 2 Sensitivity and specificity of PCR amplification of rDNA with extracted DNAs and lysates of BAL fluid or serum

References


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