Amoah I D, Singh G, Troell K, Reddy P, Stenström T A, Bux F
Institute for Water and Wastewater Technology, Durban University of Technology, PO Box 1334, Durban 4000, South Africa.
Department of Microbiology, National Veterinary Institute, SE-751 89, Uppsala, Sweden.
J Helminthol. 2019 Aug 28;94:e78. doi: 10.1017/S0022149X19000683.
A central and critical step in the molecular detection of soil-transmitted helminths from environmental sources is the extraction of DNA from the eggs. In this study, we investigated the yield of DNA extracted from known quantities (500, 100, 50, 20, 10 and 5) of Ascaris suum eggs, as well as directly from wastewater and sludge samples containing Ascaris spp. eggs, using six commercial DNA extraction kits. The amount of DNA extracted was quantified with NanoDrop, Qubit and Ct values from quantitative polymerase chain reaction (qPCR) assay using CFX96 Touch™ real-time PCR equipment. The PowerLyzer Ultraclean Microbial DNA isolation kit and PowerSoil DNA isolation kit gave the highest yield of DNA based on the NanoDrop, Qubit and Ct values. However, the qPCR results indicate that in some of the kits, PCR inhibitors may have been carried over to the PCR reaction. DNA extraction kits that incorporate a bead-beating step as well as other mechanical eggshell disruption steps were superior in extracting DNA from Ascaris spp. eggs. Additionally, for the accurate quantification of extracted DNA, the use of Ct values from qPCR and Qubit readings gives better results compared to the NanoDrop readings. For efficient downstream applications, the use of DNA extraction kits with superior inhibitor removal technology is essential, in addition to a high yield of DNA.
从环境样本中分子检测土源性蠕虫的一个核心且关键的步骤是从虫卵中提取DNA。在本研究中，我们使用六种商用DNA提取试剂盒，研究了从已知数量（500、100、50、20、10和5）的猪蛔虫卵中，以及直接从含有蛔虫属虫卵的废水和污泥样本中提取的DNA产量。使用CFX96 Touch™实时PCR设备，通过NanoDrop、Qubit以及定量聚合酶链反应（qPCR）检测的Ct值对提取的DNA量进行定量。基于NanoDrop、Qubit和Ct值，PowerLyzer Ultraclean微生物DNA分离试剂盒和PowerSoil DNA分离试剂盒的DNA产量最高。然而，qPCR结果表明，在某些试剂盒中，可能有PCR抑制剂被带入到PCR反应中。包含珠磨步骤以及其他机械破坏卵壳步骤的DNA提取试剂盒在从蛔虫属虫卵中提取DNA方面表现更优。此外，为了准确量化提取的DNA，与NanoDrop读数相比，使用qPCR的Ct值和Qubit读数能得到更好的结果。为了实现高效的下游应用，除了要有高产量的DNA外，使用具有卓越抑制剂去除技术的DNA提取试剂盒至关重要。
