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.本表格展示了48小时培养周期内，在Barret培养基与Pavlova培养基中培养的芽囊原虫（Blastocystis）代时（Tg）数据，样本来源于肠易激综合征（IBS）患者与无症状携带者，检测方式涵盖显微镜计数与定量PCR（qPCR）。芽囊原虫分离株的代时按照Zhang等人[17]提出的公式计算得出：Tg=(T2-T1)/(log₂(n2/n1))，其中Tg代表代时，n1为初始时刻（T1）体外培养的寄生生物数量，n2为后续时刻（T2）的寄生细胞数量。本次实验中，(T2-T1)即体外培养时长为48小时。此外，为通过qPCR实现绝对定量，需参考以下参数：i）芽囊原虫基因组大小约为18.8 Mbp[36]；ii）1 pg DNA对应约978 Mbp碱基对[37]；iii）DNA对照品浓度为160 ng/µL；通过交叉相乘运算，即可估算扩增得到的遗传标记的拷贝数。