Hoorzook K B, Barnard T G
University of Johannesburg, South Africa.
MethodsX. 2022;9:101785. doi: 10.1016/j.mex.2022.101785. Epub 2022 Jul 13.
The COVID-19 pandemic lockdown created problems with importing of commercial kits resulting in extended turnaround times for consumable deliveries. One way to circumvent this was to use an inexpensive optimized in-house method for DNA extraction from water. • The DNA extraction methods were optimized on a 96-well plate using a semi-automated filtration system to increase the number of samples from 24 to 96 at a time in 2 h. The DNA extraction method optimizations included: (a) Guanidium thiocyanate method plus dilution series of celite to determine DNA binding capacity; (b) QIamp 96 Qiacube HT kit (Qiagen®); (c) Guanidium thiocyanate with the celite replaced with a binding buffer. • The in-house DNA extraction methods and adapted in-house DNA extraction method were compared to QIamp 96 Qiacube HT kit (Qiagen®), which is used on a 96-well semi-automated filtration system. The results showed maximum capacity of the 96-well filter plates was 400 μℓ broth (OD = 0.45 = 3.6 × 10 cells/mℓ) before the 96-well filters blocked. • When the methods were compared, there was no significant difference between the in-house DNA extraction method with 1:420 celite dilution (-value = 0.126) and the adapted in-house method with binding buffer (-value = 0.298) DNA yield or amplification of PCR products.
新冠疫情封锁导致商业试剂盒进口出现问题，使得耗材交付的周转时间延长。解决这一问题的一种方法是使用一种廉价的优化后的水DNA提取内部方法。• 使用半自动过滤系统在96孔板上对DNA提取方法进行优化，以便在2小时内将一次处理的样本数量从24个增加到96个。DNA提取方法的优化包括：(a) 硫氰酸胍法加硅藻土稀释系列以确定DNA结合能力；(b) QIamp 96 Qiacube HT试剂盒（Qiagen®）；(c) 用结合缓冲液替代硅藻土的硫氰酸胍法。• 将内部DNA提取方法和改进后的内部DNA提取方法与QIamp 96 Qiacube HT试剂盒（Qiagen®）进行比较，该试剂盒用于96孔半自动过滤系统。结果表明，在96孔滤板堵塞之前，其最大容量为400μℓ肉汤（OD = 0.45 = 3.6×10细胞/毫升）。• 当对这些方法进行比较时，使用1:420硅藻土稀释的内部DNA提取方法（P值 = 0.126）与使用结合缓冲液的改进后的内部方法（P值 = 0.298）在DNA产量或PCR产物扩增方面没有显著差异。
