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Establishment of a 10-Plex Quantitative Fluorescent-PCR Assay for rapid diagnosis of sex chromosome aneuploidies.

ABSTRACT: Sex chromosome aneuploidies occur commonly in the general population, with an incidence of 1 in 400 newborns. However, no tests specifically targeting sex chromosomes have been carried out in prenatal diagnosis or newborn screening, resulting in late recognition of these diseases. In this study, a rapid diagnostic method for sex chromosome aneuploidies was established using Quantitative Fluorescent-PCR (QF-PCR). Ten markers were included in one multiplex QF-PCR assay, including two sex determination genes (AMXY and SRY), five X-linked short tandem repeats (STRs; DXS1053, DXS981, DXS6809, DXS1187, and DXS8377), one X/Y-common STR (X22), and two autosomal STRs (D13S305 and D21S11). Retrospective tests of 70 cases with known cytogenetic results indicated that the 10-plex QF-PCR assay could well determine sex chromosome copy numbers by both allelic peak numbers and a sex chromosome dosage calculation with the autosomal STRs as internal controls. Prospective comparison with cytogenetic karyotyping on 534 cases confirmed that the 10-plex QF-PCR assay could be well employed for sex chromosome aneuploidy diagnosis in at least the Chinese Han population. This is the first QF-PCR test for the diagnosis of sex chromosome aneuploidies in the Chinese population. This test is superior to previous designs by including up to 8 sex-linked markers covering different parts of sex chromosomes as well as employing internal controls for copy number dosage calculation in a single PCR reaction. Due to simple technique and data analysis, as well as easy implementation within routine clinical services, this method is of great clinical application value and could be widely applied.

SUBMITTER: Xie X

PROVIDER: S-EPMC4160182 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Establishment of a 10-Plex Quantitative Fluorescent-PCR Assay for rapid diagnosis of sex chromosome aneuploidies.

Xie Xingmei X Liang Qiaoyi Q

PloS one 20140910 9

Sex chromosome aneuploidies occur commonly in the general population, with an incidence of 1 in 400 newborns. However, no tests specifically targeting sex chromosomes have been carried out in prenatal diagnosis or newborn screening, resulting in late recognition of these diseases. In this study, a rapid diagnostic method for sex chromosome aneuploidies was established using Quantitative Fluorescent-PCR (QF-PCR). Ten markers were included in one multiplex QF-PCR assay, including two sex determina ...[more]

PMID: 25207978

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Project description:Background & aimsCurrently available HDV PCR assays are characterized by considerable run-to-run and inter-laboratory variability. Hence, we established a quantitative reverse transcription real-time PCR (RT-qPCR) assay on the open channel of a fully automated PCR platform (cobas6800, Roche) offering improved consistency and reliability.MethodsA primer/probe-set targeting a highly conserved region upstream of the HDV antigen was adapted for use on the cobas6800. The lower limit of detection (LLOD) was determined using a dilution panel of the HDV WHO standard (n = 21/dilution). Linearity and inclusivity were tested by preparing 10-fold dilution series of cell culture-derived virus (genotype [GT]1-8; n = 5/dilution). Patient samples containing a variety of bloodborne viral pathogens were tested to confirm exclusivity (n = 60).ResultsThe LLOD of the HDV utility-channel (HDV_UTC) assay was determined as 3.86 IU/ml (95% CI 2.95-5.05 IU/ml) with a linear range from 10-10ˆ8 IU/ml (GT1). Linear relationships were observed for all HDV GTs with slopes ranging from -3.481 to -4.134 cycles/log and R2 from 0.918 to 0.994. Inter-run and intra-run variability were 0.3 and 0.6 Ct (3xLLOD), respectively. No false-positive results were observed. To evaluate clinical performance, 110 serum samples of anti-HDV-Ab+ patients were analyzed using the HDV_UTC and CE-IVD RoboGene assays. 58/110 and 49/110 samples were concordant positive or negative, respectively (overall agreement 97.3%). Quantitative comparison demonstrated a strong correlation (R2 0.8733; 95% CI 0.8914-0.9609; p value <0.0001).ConclusionThe use of highly automated, sample-to-result solutions for molecular diagnostics holds many inherent benefits over manual workflows, including improved reliability, reproducibility and dynamic scaling of testing capacity. The assay we established showed excellent analytical and clinical performance, with inclusivity for all HDV GTs and a limit of quantification of 10 IU/ml, making it a sensitive new tool for HDV screening and viral load monitoring.Lay summaryThe hepatitis delta virus (HDV) causes a severe form of inflammation in the liver. We developed a tool for molecular diagnostics, a polymerase chain reaction HDV assay that showed great performance. It can be used to improve diagnosis of HDV, as well as for monitoring treatment responses. The assay allows for quantification of the virus in the tested samples and is performed on a fully automated platform (cobas6800), which provides various benefits including less hands-on time and excellent comparability of test results.

Dataset Information

Establishment of a 10-Plex Quantitative Fluorescent-PCR Assay for rapid diagnosis of sex chromosome aneuploidies.

Publications

Establishment of a 10-Plex Quantitative Fluorescent-PCR Assay for rapid diagnosis of sex chromosome aneuploidies.

Similar Datasets

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