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Background & objectives: Non-invasive prenatal testing (NIPT) of maternally inherited alleles of ?-thalassaemia (MIB) remains to be a challenge. Furthermore, current techniques are not available for use as routine tests. NIPT for ?-thalassaemia disease was developed by using a specific droplet digital polymerase chain reaction (ddPCR) assay to analyze the cell-free foetal DNA (cffDNA) derived from maternal plasma. Methods: Pregnant women and their spouses who are at risk of bearing an offspring with ?-thalassaemia disease from common MIB mutations (CD 41/42-TCTT, CD17A>T, IVS1-1G>T and CD26G>A) were enrolled. The ddPCR assay sets were constructed for each of the four mutations. All cell-free DNA samples were first screened for the paternally inherited ?-thalassaemia (PIB) mutation. The PIB-negative samples were considered as non-disease and were not further analyzed. For PIB-positive samples, DNA fragments of 50-300 base pairs in size were isolated and purified, and further analyzed for MIB mutation. The allelic ratio between the mutant and the wild-type was used to determine the presence of MIB in cffDNA. All cases underwent a prenatal diagnosis by amniocentesis for a definite diagnosis. Results: Forty two couples at risk were enrolled. Twenty two samples were positive for PIBs. Among these 22 samples, there were 10 cases with allelic ratio >1.0 (MIB positive). All foetuses with over-represented mutant alleles were further diagnosed with ?-thalassaemia disease; eight with compound heterozygous and two with homozygous mutations. The 20 PIB-negative and 12 MIB-negative foetuses were non-affected. Interpretation & conclusions: The results of this study suggest that NIPT utilizing the ddPCR assay can be effectively used for the screening and diagnosis of foetal ?-thalassaemia in at risk pregnancies.
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@#Abstract: Objective To analyze the nucleic acid detection results of severe acute respiratory syndrome corona virus-2 (SARS-CoV-2) by droplet digital PCR (ddPCR) and compare with the detection results of real-time fluorescence quantitative RT-PCR (qRT-PCR), so as to evaluate the advantages and disadvantages of detection, and to provide data support for optimizing the nucleic acid detection scheme of SARS-CoV-2. Methods According to the SARS-CoV-2 specific primer probe published by the China Center for Disease Control and Prevention, a ddPCR detection method for SARS-CoV-2 was designed. One sample was selected for sensitivity test after gradient dilution; six respiratory virus nucleic acid positive samples including seasonal H3N2 influenza virus and SARS-CoV-2 positive samples were selected for specificity test; five SARS-CoV-2 positive samples were selected for repeatability test; in addition, 30 positive and 20 negative SARS-CoV-2 samples were selected for multiple clinical samples testing, and the results were analyzed and compared with those of qRT-PCR. Results The ddPCR method can specifically detect SARS-CoV-2, and directly obtain the original copy number of the sample target gene to achieve accurate quantification; the sensitivity test of gradient dilution positive samples showed that qRT-PCR detected target genes in part of the 10-5 dilution of samples, and no target genes were detected in 10-6 dilution, while ddPCR detected all target genes in both 10-5 and 10-6 dilution of samples. The detection limit of ddPCR was two orders of magnitude higher than that of qRT-PCR, and the sensitivity was higher than that of qRT-PCR; in the comparison of the repeatability test results of the two methods, the coefficient of variation of ddPCR was 1.266%-11.814%, lower than 1.729%-26.174% of qRT PCR, and the repeatability was higher than qRT-PCR; among 50 clinical samples, 30 positive samples of confirmed cases of Coronavirus Disease 2019 (COVID-19) were detected by both methods, SARS-CoV-2 was successfully detected by both methods, and 20 negative samples of COVID-19 were detected by both methods, and the results were negative, with a coincidence rate of 100.00% (50/50). Conclusion The ddPCR method can accurately quantify SARS-CoV-2 with strong specificity, and its sensitivity and repeatability are higher than those of qRT-PCR, but it also has certain detection limitations and is more suitable for the detection of low load samples. In the actual detection, the two methods can be reasonably combined to improve the detection accuracy.
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OBJECTIVES@#To establish a system for simultaneous detection of miR-888 and miR-891a by droplet digital PCR (ddPCR), and to evaluate its application value in semen identification.@*METHODS@#The hydrolysis probes with different fluorescence modified reporter groups were designed to realize the detection of miR-888 and miR-891a by duplex ddPCR. A total of 75 samples of 5 body fluids (including peripheral blood, menstrual blood, semen, saliva and vaginal secretion) were detected. The difference analysis was conducted by Mann-Whitney U test. The semen differentiation ability of miR-888 and miR-891a was evaluated by ROC curve analysis and the optimal cut-off value was obtained.@*RESULTS@#There was no significant difference between the dual-plex assay and the single assay in this system. The detection sensitivity was up to 0.1 ng total RNA, and the intra- and inter-batch coefficients of variation were less than 15%. The expression levels of miR-888 and miR-891a detected by duplex ddPCR in semen were both higher than those in other body fluids. ROC curve analysis showed that the AUC of miR-888 was 0.976, the optimal cut-off value was 2.250 copies/μL, and the discrimination accuracy was 97.33%; the AUC of miR-891a was 1.000, the optimal cut-off value was 1.100 copies/μL, and the discrimination accuracy was 100%.@*CONCLUSIONS@#In this study, a method for detection of miR-888 and miR-891a by duplex ddPCR was successfully established. The system has good stability and repeatability and can be used for semen identification. Both miR-888 and miR-891a have high ability to identify semen, and the discrimination accuracy of miR-891a is higher.
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Female , Humans , Male , Body Fluids/chemistry , MicroRNAs/analysis , Real-Time Polymerase Chain Reaction/methods , Saliva/chemistry , Semen/chemistryABSTRACT
BACKGROUND@#The patients with advanced lung adenocarcinoma should select targeted drugs based on the type of tumor epidermal growth factor receptor (EGFR) gene mutation. However, it is difficult to collect tumor tissue of advanced lung adenocarcinoma, and some experts agree that peripheral blood can be used as a substitute for tumor tissue as a test specimen. This paper aimed to investigate the clinical value of ddPCR and super-amplification refractory mutation system (ARMS) in detecting EGFR gene mutation in peripheral blood of patients with advanced lung adenocarcinoma.@*METHODS@#A total of 119 patients diagnosed in Beijing Chest Hospital Affiliated to Capital Medical University from February 2016 to February 2019 were collected, and the sensitivity and specificity of plasma ctDNA EGFR gene mutation detected by ddPCR and super-arms were compared. Some patients with positive EGFR gene mutations received oral treatment with first-line EGFR tyrosine kinase inhibitors (EGFR-TKI). The patients were divided into subgroups according to the test results. In group 1, both ddPCR and super-arms showed positive EGFR gene mutation results, with 21 cases. In group 2, ddPCR and super-arms detection of EGFR gene mutation were all negative, with 16 cases. In group 3, the ddPCR test was positive and the super-arms test was negative, with 5 cases. In group 4, the ddPCR test result was negative while the super-arms test result was positive. Since the number of patients in group 4 was 0, no statistics were included. Objective response rate (ORR) and disease control rate (DCR) were used to evaluate the short-term outcome, and progression-free survival (PFS) was compared with survival analysis to evaluate the long-term outcome.@*RESULTS@#EGFR mutations were detected in 58 (48.7%) of 119 patients with advanced lung adenocarcinoma. The coincidence rate between ddPCR and EGFR gene mutation in tumor tissues was 82.4% (Kappa=0.647, P0.05). Survival analysis showed that the PFS of the three groups was compared. The difference was not statistically significant (χ²=2.221, P=0.329).@*CONCLUSIONS@#ddPCR, as a high sensitivity and specificity liquid gene detection method, can be used as a reliable method to detect the mutation of plasma ctDNA EGFR gene in patients with advanced lung adenocarcinoma. The results of plasma genetic testing can also be used as the basis for predicting the efficacy of EGFR-TKIs in patients.
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A droplet digital polymerase chain reaction ( ddPCR) method for quantifying E. coli O157:H7 by targeting rfbE gene was developed. The probe concentration in ddPCR was optimized and the linearity range, precision, limit of detection ( LOD) and limit of quantification ( LOQ) were also evaluated. The optimized probe concentration was 300 nmol/L. The ddPCR response was linear over the E. coli O157:H7 genome DNA concentration range from 4 to 1. 25×105 copies in 20 μL ddPCR system and the linear correlation coefficient (R2) was 0. 999. The ddPCR precision (RSD) was less than 5% over the DNA concentration range from 760 to 88400 copies/20 μL. The LOD and LOQ was 3 copies in 20 μL and 4 copies in 20 μL, respectively. Specificity test showed that the ddPCR was specific for detecting E. coli O157:H7. Both ddPCR and standard real time quantitative PCR showed the same results for 16 real samples of chicken meat, pork and beef, which indicated that ddPCR method was suitable for detection of E. coli O157:H7 in food.