Generation times data in Blastocystis by qPCR and by microscopy

Generation time (Tg) data of Blastocystis cultures in Barret’s and in Pavlova’s media during 48 h., from IBS patients and from asymptomatic carriers, measured by microscopy and by quantitative PCR are shown in present table. The generation times of Blastocystis isolates, were calculated according to Zhang et al. [17], with the following equation: Tg=(T2-T1)/(log2(n2/n1)), where Tg denotes the generation time, n1 represents the number of cultured parasitic organisms at the initial time (T1), and n2 represents the number of parasitic cells at subsequent time (T2). Thus, (T2-T1) = 48 hours of in vitro culture. Besides, for to absolute quantification by qPCR, it was necessary to consider i) the size of the Blastocystis genome ~ 18.8Mbp[36]; ii) 1pg of DNA ~ 978Mbp[37] and iii) the concentration of DNA control was 160ng/µL; thus, by cross multiplications, the number of copies of the genetic marker amplified were estimated.

本表格呈现了48小时体外培养周期内，分别于Barret培养基（Barret’s medium）与Pavlova培养基（Pavlova’s medium）中培养的芽囊原虫（Blastocystis）代时（Generation time, Tg）数据。该数据集的样本取自肠易激综合征（Irritable Bowel Syndrome, IBS）患者与无症状携带者，检测手段涵盖显微镜镜检与定量聚合酶链式反应（quantitative PCR, qPCR）。
本研究中芽囊原虫分离株的代时参照Zhang等人[17]的方法进行计算，计算公式如下：
Tg=(T2-T1)/(log₂(n2/n1))，其中Tg代表代时，n1为初始时刻T1的培养寄生生物数量，n2为后续时刻T2的寄生细胞数量。本实验的体外培养时长(T2-T1)为48小时。
此外，若通过qPCR实现绝对定量，需考虑以下三点：① 芽囊原虫的基因组大小约为18.8 Mbp[36]；② 1皮克（pg）DNA约对应978 Mbp[37]；③ DNA对照品的浓度为160 ng/µL。通过交叉乘法即可估算出扩增的遗传标记的拷贝数。