关键词: 芽囊原虫, 形态学, 荧光定量PCR, 体外增殖, 降解

Abstract:

The morphology, proliferation and stability of in vitro cultured Blastocystis were studied. Blastocystis were isolated from Blastocystis positive feces of patients with diarrhea and cultured in IMDM medium to observe the parasite morphology and proliferation. The density of Blastocystis was quantified by microscopic counting and the copy number of Blastocystis 18S small subunit ribosomal DNA (SSU rDNA) was measured by fluorescence quantitative PCR (qPCR) to analyze the proliferation of Blastocystis in the in vitro culture, and the proliferation curve was plotted to analyze the correlation between the two methods. Blastocystis were stored at 4, -20, and -80 ℃ for 1 to 7 days and 1 to 5 weeks respectively, and the Blastocystis SSU rDNA was detected by qPCR to analyze the stability. Morphological studies showed that the four common morphological forms were observed in the IMDM medium, including vacuolar form, granular form, ameboid form and cyst form; and the three reproductive modes including fission, gemmation and endodyogeny were observed. The rapid growth period of Blastocystis was observed from day 3 to 7 from the in vitro culture, and the Blastocystis density peaked on day 7. The microscopic counting and qPCR results were (2.55 ± 0.22) × 10⁶/ml and (1.06 ± 0.10) × 10⁶ copies/μl, respectively. Correlation analysis showed that the Pearson correlation coefficient of proliferation curve generated by microscopic counting and qPCR was 0.95, which was highly correlated. The degradation of Blastocystis began on the second day after storage at 4, -20 and -80 ℃, and the copy numbers of SSU rDNA on the seventh day were (2.75 ± 0.20) × 10⁴, (6.84 ± 1.33) × 10⁴ and (1.39 ± 0.06) × 10⁵ copies/μl, respectively, which accounted for 11.65%, 28.67% and 62.42% of the copy volume (2.36 ± 0.06) × 10⁵, (2.39 ± 0.06) × 10⁵, (2.23 ± 0.21) × 10⁵ copies/μl on the first day. The difference was statistically significant (F = 130.67, P < 0.05). At the fifth week, the copy numbers of SSU rDNA were (3.77 ± 0.23) × 10³, (4.37 ± 0.59) × 10³ and (3.86 ± 0.26) × 10⁴ copies/μl, accounted for 2.98%, 3.41% and 28.74% of the copy volume (2.23 ± 0.21) × 10⁵, (2.23 ± 0.21) × 10⁵, (2.23 ± 0.21) × 10⁵ copies/μl in the first week. The difference was statistically significant (F = 500.51, P < 0.05). The morphology of in vitro cultured Blastocystis showed diversive forms at the rapid proliferation stage. Both microscopic counting and qPCR can be used for the quantification of Blastocystis and the stability of Blastocystis stored at -80 ℃ is less than that stored at 4 and -20 ℃.

Key words: Blastocystis, Morphology, Fluorescent quantitative PCR, Proliferation, Degradation