Comparability of CMV DNA Extraction Methods and Validation of Viral Load

Théophile Uwiringiyeyezu; Bouchra El Khalfi; Rachid Saile; Jamal Belhachmi; Abdelaziz Soukri

doi:10.3390/mps5010006

Comparability of CMV DNA Extraction Methods and Validation of Viral Load

Methods Protoc. 2022 Jan 4;5(1):6. doi: 10.3390/mps5010006.

Authors

Théophile Uwiringiyeyezu^{1

2}, Bouchra El Khalfi¹, Rachid Saile³, Jamal Belhachmi², Abdelaziz Soukri¹

Affiliations

¹ Research Center of Biotechnology and Health, Laboratory of Physiopathology, Molecular Genetics and Biotechnology, Faculty of Sciences Aïn Chock, Hassan II University of Casablanca, B.P 5366 Maarif, Casablanca 20000, Morocco.
² Laboratory Al Kindy of Medicals Analysis, Casablanca 20100, Morocco.
³ Research Center of Biotechnology and Health, Laboratory of Biology and Health, Faculty of Sciences, Ben M'sik, Hassan II University of Casablanca, B.P 5366 Maarif, Casablanca 20000, Morocco.

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.

Keywords: cytomegalovirus; extraction techniques; graft rejection; medical analysis; molecular biology; nucleic acids; polymerase chain reaction; threshold cycle (Ct); transplanting.