The application value and research progress of urine free DNA in tumor molecular diagnosis / 中华检验医学杂志

Urine cell-free DNA (ucfDNA) contains DNA fragments released from the lysis of cells in the urinary system, and circulating cell-free DNA (ccfDNA) can also enter the urine after glomerular filtration, and the mutation information carried by tumor DNA contained in ucfDNA and ccfDNA is consistent. Therefore, as a biomarker for molecular diagnosis of urological and non-urinary tumors, ucfDNA, has become a research hotspot in the field of liquid biopsy in recent years. UcfDNA has potential application value in individualized treatment, early diagnosis, dynamic monitoring of therapeutic efficacy, and prognosis assessment, etc. However, in order to realize the clinical application of urine ucfDNA, the extraction and accurate detection of ucfDNA still need to be solved.

An acceptable method to evaluate the analytical performance of real-time fluorescent RT-PCR targeting SARS-CoV-2

It was necessary to carry out methodologies evaluations of real-time fluorescent reverse-transcription PCR (RT-PCR) targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Considering biosafety issues and lack of positive specimens in some special locations in China, the routine specimens from healthy individuals were used to perform methodologies evaluations, in which the indexes were the differences of quantification cycle values ({Delta} Cq) between human derived internal reference control (IRC) genes of a specimen and quality control (QC). Serial experiments were carried out to evaluate various factors that might affect aforementioned methodologies, such as types of virus transport mediums, methods of specimen pretreatment and template preparation, specimen vortex strength, specimen storage temperature and duration. The results showed that using {Delta}Cq values as indexes, among various factors that might affect analytical performance, it was better to store specimens in the normal saline transport mediums, inactivate pathogens using water or metal bath, release more virus particles from swabs by vortex mixing, extract nucleic acids with centrifuge methods, and perform amplification assays timely. Aforementioned opinions and optimum conditions were further confirmed by SRAS-CoV-2 pseudovirus and clinical positive specimens. Altogether, the results of this study indicated that the routine specimens from healthy individuals could be used to evaluate the analytical performance of real-time fluorescent RT-PCR targeting SRAS-CoV-2, of which the indexes were the {Delta}Cq values between IRC genes of a specimen and QC. This acceptable method was extremely valuable in both theoretical and practical significance under current pandemic of coronavirus disease 2019 (COVID-19).