Comparability of CMV DNA Extraction Methods and Validation of Viral Load

Abstract

Human cytomegalovirus is a herpesvirus that has a worldwide seroprevalence of more than 60% of adults in developed countries and 90% in developing countries. Severe disabilities in newborns are characteristic of the human cytomegalovirus congenital infection, and this virus is implicated in graft rejection in transplant patients. To treat and follow-up the infection, the CMVPCR viral loads are required, and the DNA extraction step remains very important; however, the quantity, quality, and purity of extracted DNA from different biological fluids influence the results of PCR amplification, that is why for reliable results, the choice of nucleic acid extraction methods requires careful attention. Materials and methods: In this study, we compare 4 protocols, I (EZ1 DSP Virus kit), II (EZ1 Virus mini kit), III (QIAamp DSP virus kit), and IV (heating); the extractions are made from plasma collected on EDTA tubes, and the concentration of extracted DNA was measured on NanoDrop Lite followed by real-time CMVPCR using an Artus CMV QS-RGQ kit. All protocols are performed following the manufacturer’s instructions. Results: This study is conducted on the samples of 135 transplant patients whose follow-up medical tests related to human cytomegalovirus infection; since most of the CMVPCR results are negative, we have chosen the 10 CMVPCR positive samples and 2 negative samples as controls to conduct this comparison study. By using NanoDrop Lite to evaluate the DNA concentration, the yield of extracted DNA is higher in our heating protocol than other protocols, the EZ1 DSP virus kit and EZ1 Virus mini kit show homogeneous quantities, and the QIAamp DSP virus kit shows very low DNA yields. Comparing cycle threshold and viral loads by real-time PCR, all these protocols identified negative samples (100%), and the previously positive samples used were as follows: protocol IV (90%), protocol II (60%), and protocol I (40%). QIAamp DSP virus kit results were not real-time PCR applicable and were non-conclusive because of the low DNA yields. Conclusion: Our developed heating method (protocol IV) is very effective, reliable, simple, fast, and cheap compared to the other protocols in our study.

Comparative analyses of DNA extraction methods for whole blood quantification of HCMV DNAemia in patients with hematological diseases: false negative cases in manual method

“…Based on this rationale, we evaluated the hypothesis that is a significant difference in the efficiency of two different DNA extraction methods from WB of patients with suspected HCMV disease for a subsequent HCMV DNA quantitation by qPCR [ 33 , 34 ].…”

The impact of preanalytical variables on the analysis of cell-free DNA from blood and urine samples

“…These methods vary in terms of recovery efficiency, fragment discrimination, and reproducibility ( Ungerer et al, 2020 ), making it challenging to select the optimal method for cfDNA isolation. Factors such as the efficiency of extracting low-content DNA ( Lee et al, 2018 ), purity ( Uwiringiyeyezu et al, 2022 ), repeatability ( Letendre and Goggs, 2017 ), and cost ( Diefenbach et al, 2018 ) are usually considered when applying extraction protocols. Commercial specialized kits based on current optimized technologies seem to offer clear advantages for cfDNA extraction ( Janovicova et al, 2020 ) and are routinely used in many studies.…”