{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,12]],"date-time":"2025-10-12T02:46:57Z","timestamp":1760237217065,"version":"build-2065373602"},"reference-count":26,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2020,3,11]],"date-time":"2020-03-11T00:00:00Z","timestamp":1583884800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Medical Sciences"],"abstract":"<jats:p>Allogenic stem-cell therapies benefit patients in the treatment of multiple diseases; however, the side effects of stem-cell therapies (SCT) derived from the concomitant use of immune suppression agents often include triggering infection diseases. Thus, analysis is required to improve the detection of pathogen infections in SCT. We develop a polymerase chain reaction (PCR)-based methodology for the qualitative real-time DNA detection of cytomegalovirus (CMV), with reference to herpes simplex virus types 1 (HSVI), Epstein\u2013Barr virus (EBV), and varicella-zoster virus (VZV) in blood, urine, solid tissues, and cerebrospinal fluid. This real-time PCR of 96-well plate format provides a rapid framework as required by the Food and Drug Administration (FDA) for clinical settings, including the processing of specimens, reagent handling, special safety precautions, quality control criteria and analytical accuracy, precisely reportable range (analyst measurement range), reference range, limit of detection (LOD), analytical specificity established by interference study, and analyte stability. Specifically, we determined the reportable range (analyst measurement range) with the following criteria: CMV copies \u2265200 copies\/mL; report copy\/mL value; CMV copies \u2264199 copies\/mL; report detected but below quantitative range; CMV copies = 0 with report &lt;200 copies\/mL. That is, with reference range, copy numbers (CN) per milliliter (mL) of the LOD were determined by standard curves that correlated Ct value and calibrated standard DNA panels. The three repeats determined that the measuring range was 1E2~1E6 copies\/mL. The standard curves show the slopes were within the range \u22122.99 to \u22123.65 with R2 \u2265 0.98. High copy (HC) controls were within 0.17\u20130.18 log differences of DNA copy numbers; (2) low copy (LC) controls were within 0.17\u20130.18 log differences; (3) LOD was within 0.14\u20130.15 log differences. As such, we set up a fast, simple, inexpensive, sensitive, and reliable molecular approach for the qualitative detection of CMV pathogens. Conclusion: This real-time PCR of the 96-well plate format provides a rapid framework as required by the FDA for clinical settings.<\/jats:p>","DOI":"10.3390\/medsci8010014","type":"journal-article","created":{"date-parts":[[2020,3,12]],"date-time":"2020-03-12T04:13:57Z","timestamp":1583986437000},"page":"14","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Implementation and Validation of the Roche Light Cycler 480 96-Well Plate Platform as a Real-Time PCR Assay for the Quantitative Detection of Cytomegalovirus (CMV) in Clinical Specimens Using the Luminex MultiCode ASRs System"],"prefix":"10.3390","volume":"8","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9699-9204","authenticated-orcid":false,"given":"Shengwen Calvin","family":"Li","sequence":"first","affiliation":[{"name":"Neuro-Oncology and Stem Cell Research Laboratory (NSCL), CHOC Children\u2019s Research Institute (CCRI), Children\u2019s Hospital of Orange County (CHOC), 1201 W. La Veta Ave., Orange, CA 92868-3874, USA"},{"name":"Department of Neurology, University of California-Irvine School of Medicine, Orange, CA 92868, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kara J.","family":"Sparks","sequence":"additional","affiliation":[{"name":"Molecular Pathology Laboratory, Bone Marrow Transplantation, Children\u2019s Hospital of Orange County, Orange, CA 92868, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Leonard S.","family":"Sender","sequence":"additional","affiliation":[{"name":"Hyundai Cancer Institute, CHOC Children\u2019s Hospital, Orange, The Chao Family Comprehensive Cancer Center, University of California-Irvine School of Medicine, Orange, CA 92868, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2020,3,11]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1034\/j.1399-3062.1999.010305.x","article-title":"Current antiviral strategies for controlling cytomegalovirus in hematopoietic stem cell transplant recipients: Prevention and therapy","volume":"1","author":"Boeckh","year":"1999","journal-title":"Transpl. Infect. Dis."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"305","DOI":"10.1182\/asheducation-2011.1.305","article-title":"Complications, Diagnosis, Management, and Prevention of CMV Infections: Current and Future","volume":"2011","author":"Boeckh","year":"2011","journal-title":"Hematology"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"24","DOI":"10.1016\/j.bbmt.2014.11.002","article-title":"Recent advances in cytomegalovirus: An update on pharmacologic and cellular therapies","volume":"21","author":"Boeckh","year":"2015","journal-title":"Biol. Blood Marrow Transplant"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"533","DOI":"10.1128\/CMR.11.3.533","article-title":"Quantitation of Cytomegalovirus: Methodologic Aspects and Clinical Applications","volume":"11","author":"Boeckh","year":"1998","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"273","DOI":"10.4149\/av_2017_305","article-title":"PCR-based approach for qualitative molecular analysis of six neurotropic pathogens","volume":"61","author":"Ferese","year":"2017","journal-title":"Acta Virol."},{"key":"ref_6","first-page":"81","article-title":"Polymerase chain reaction for detection of mucosal cytomegalovirus infection in patients with acute ulcerative colitis","volume":"32","author":"Tun","year":"2018","journal-title":"Ann. Gastroenterol."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Basso, M., Andreis, S., Scaggiante, R., Franchin, E., Zago, D., Biasolo, M.A., Del Vecchio, C., Mengoli, C., Sarmati, L., and Andreoni, M. (2018). Cytomegalovirus, Epstein-Barr virus and human herpesvirus 8 salivary shedding in HIV positive men who have sex with men with controlled and uncontrolled plasma HIV viremia: A 24-month longitudinal study. BMC Infect. Dis., 18.","DOI":"10.1186\/s12879-018-3591-x"},{"key":"ref_8","first-page":"51","article-title":"The clinical laboratory characteristic of primary acute cytomegalovirus infection in non-immunocompromised adult patients","volume":"63","author":"Zhukova","year":"2018","journal-title":"Klin. Lab. Diagn."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1514","DOI":"10.1093\/infdis\/jix048","article-title":"Cytomegalovirus (CMV) DNA Quantitation in Bronchoalveolar Lavage Fluid From Hematopoietic Stem Cell Transplant Recipients With CMV Pneumonia","volume":"215","author":"Boeckh","year":"2017","journal-title":"J. Infect. Dis."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"527","DOI":"10.1016\/j.jmoldx.2016.02.002","article-title":"Comparative Evaluation of Four Real-Time PCR Methods for the Quantitative Detection of Epstein-Barr Virus from Whole Blood Specimens","volume":"18","author":"Buelow","year":"2016","journal-title":"J. Mol. Diagn."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"440","DOI":"10.3343\/alm.2018.38.5.440","article-title":"Evaluation of the Luminex ARIES HSV 1&2 Assay and Comparison with the FTD Neuro 9 and In-house Real-Time PCR Assays for Detecting Herpes Simplex Viruses","volume":"38","author":"Lee","year":"2018","journal-title":"Ann. Lab. Med."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"2635","DOI":"10.1128\/JCM.02170-08","article-title":"Detection and Quantification of Hepatitis C Virus (HCV) by MultiCode-RTx Real-Time PCR Targeting the HCV 3\u2032 Untranslated Region","volume":"47","author":"Mulligan","year":"2009","journal-title":"J. Clin. Microbiol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1334","DOI":"10.1128\/JCM.39.4.1334-1338.2001","article-title":"Comparison of quantitative and qualitative PCR assays for cytomegalovirus DNA in plasma","volume":"39","author":"Caliendo","year":"2001","journal-title":"J. Clin. Microbiol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1909","DOI":"10.1128\/JCM.42.5.1909-1914.2004","article-title":"Comparison of a Duplex Quantitative Real-Time PCR Assay and the COBAS Amplicor CMV Monitor Test for Detection of Cytomegalovirus","volume":"42","author":"Herrmann","year":"2004","journal-title":"J. Clin. Microbiol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1498","DOI":"10.1128\/JCM.42.4.1498-1504.2004","article-title":"Validation of clinical application of cytomegalovirus plasma DNA load measurement and definition of treatment criteria by analysis of correlation to antigen detection","volume":"42","author":"Kalpoe","year":"2004","journal-title":"J. Clin. Microbiol."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Li, S., Kirov, I., Klassen, H.K., and Schwartz, P.H. (2007). Characterization of stem cells using reverse transcriptase-polymerase chain reaction. Human Stem Cell Manual: A Laboratory Guide, Academic Press.","DOI":"10.1016\/B978-012370465-8\/50015-6"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"59","DOI":"10.21775\/cimb.024.059","article-title":"Comparing Viral Metagenomic Extraction Methods","volume":"24","author":"Klenner","year":"2017","journal-title":"Curr. Issues Mol. Boil."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"550","DOI":"10.1128\/CMR.00074-09","article-title":"Validation of Laboratory-Developed Molecular Assays for Infectious Diseases","volume":"23","author":"Burd","year":"2010","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"583","DOI":"10.1016\/j.jmoldx.2011.05.011","article-title":"Molecular methods and platforms for infectious diseases testing a review of FDA-approved and cleared assays","volume":"13","author":"Emmadi","year":"2011","journal-title":"J. Mol. Diagn."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Kuang, J., Yan, X., Genders, A., Granata, C., and Bishop, D. (2018). An overview of technical considerations when using quantitative real-time PCR analysis of gene expression in human exercise research. PLoS ONE, 13.","DOI":"10.1371\/journal.pone.0196438"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"87","DOI":"10.2353\/jmoldx.2009.080097","article-title":"Multi-Site PCR-based CMV viral load assessment-assays demonstrate linearity and precision but lack numeric standardization: A report of the association for molecular pathology","volume":"11","author":"Wolff","year":"2009","journal-title":"J. Mol. Diagn."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"303","DOI":"10.1016\/j.jmoldx.2016.10.005","article-title":"The Effect of Nucleic Acid Extraction Platforms and Sample Storage on the Integrity of Viral RNA for Use in Whole Genome Sequencing","volume":"19","author":"Lewandowski","year":"2017","journal-title":"J. Mol. Diagn."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1016\/j.diagmicrobio.2017.12.021","article-title":"Comparison of two commercial quantitative PCR assays and correlation with the first WHO International Standard for human CMV","volume":"91","author":"Engelmann","year":"2018","journal-title":"Diagn. Microbiol. Infect. Dis."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1016\/S0166-0934(01)00307-X","article-title":"Development and evaluation of a real-time quantitative PCR for the detection of human cytomegalovirus","volume":"95","author":"Kearns","year":"2001","journal-title":"J. Virol. Methods"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1016\/S1525-1578(10)60446-1","article-title":"Development of real-time PCR assays for the quantitative detection of Epstein-Barr virus and cytomegalovirus, comparison of TaqMan probes, and molecular beacons","volume":"5","author":"Jebbink","year":"2003","journal-title":"J. Mol. Diagn."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"5189","DOI":"10.1128\/JCM.42.11.5189-5198.2004","article-title":"Real-Time Quantitative PCR Assays for Detection and Monitoring of Pathogenic Human Viruses in Immunosuppressed Pediatric Patients","volume":"42","author":"Watzinger","year":"2004","journal-title":"J. Clin. Microbiol."}],"container-title":["Medical Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-3271\/8\/1\/14\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T09:06:01Z","timestamp":1760173561000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-3271\/8\/1\/14"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,3,11]]},"references-count":26,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2020,3]]}},"alternative-id":["medsci8010014"],"URL":"https:\/\/doi.org\/10.3390\/medsci8010014","relation":{},"ISSN":["2076-3271"],"issn-type":[{"type":"electronic","value":"2076-3271"}],"subject":[],"published":{"date-parts":[[2020,3,11]]}}}