Non-invasive prenatal screening & diagnosis of ?-thalassaemia in an affected foetus

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.

Detection of BCR-ABL Fusion Gene in Chronic Myeloid Leukemia by Novel Digital PCR / 中国实验血液学杂志

OBJECTIVE@#To establish a new digital polymerase chain reaction (dPCR) system for the detection of BCR-ABL fusion gene in patients with chronic myeloid leukemia (CML), and explore its analytical performance and clinical applicability in the detection of BCR-ABLp190/210/230.@*METHODS@#A new dPCR system for detecting BCR-ABLp190/210/230 was successfully developed, and its sensitivity difference with qPCR and improvement of drug side effects in patients with CML during drug reduction or withdrawal were compared.@*RESULTS@#Among 176 samples, qPCR and dPCR showed high consistency in the sensitivity of detecting BCR-ABL (82.39%), and the positive rate of dPCR was about 5 times higher that of qPCR (20.45% vs 3.98%). During follow-up, blood routine (25% vs 10%), kidney/liver/stomach (25% vs 20%) and cardiac function (10% vs 0) were significantly improved after drug reduction or withdrawal in patients with initial dPCR negative compared with before drug reduction or withdrawal.@*CONCLUSIONS@#This new dPCR detection system can be applied to the detection of BCR-ABLp190/210/230. It has better consistency and higher positive detection rate than qPCR. Drug withdrawal or dose reduction guided by dPCR has a certain effect on improving drug side effects.

Comparison of BCR::ABL (P210) mRNA levels detected by dPCR and qPCR methods in patients with chronic myeloid leukemia / 中华血液学杂志

Objective: To compare digital polymerase chain reaction (dPCR) and real-time quantitative PCR (qPCR) measurements of BCR::ABL (P210) mRNA expression in patients with chronic myeloid leukemia (CML) . Methods: In this non-interventional, cross-sectional study, BCR::ABL (P210) mRNA was simultaneously measured by dPCR and qPCR in peripheral blood samples collected from patients with CML who underwent tyrosine kinase inhibitor therapy and who achieved at least a complete cytogenetic response from September 2021 to February 2023 at Peking University People's Hospital. The difference, correlation, and agreement between the two methods were evaluated using the Wilcoxon signed-rank test, Spearman's correlation, and Bland-Altman analysis, respectively. Results: In total, 459 data pairs for BCR::ABL mRNA expression measured by dPCR and qPCR from 356 patients with CML were analyzed. There was a significant difference in BCR::ABL mRNA expression between the two methods (P<0.001). When analyzed by the depth of the molecular response (MR), a significant difference only existed for patients with ≥MR4.5 (P<0.001). No significant difference was observed for those who did not achieve a major MR (no MMR; P=0.922) or for those who achieved a major MR (MMR; P=0.723) or MR4 (P=0.099). There was a moderate correlation between the BCR::ABL mRNA expression between the two methods (r=0.761, P<0.001). However, the correlation gradually weakened or disappeared as the depth of the MR increased (no MMR: r=0.929, P<0.001; MMR: r=0.815, P<0.001; MR4: r=0.408, P<0.001; MR4.5: r=0.176, P=0.176). In addition, the agreement in BCR::ABL mRNA expression between the two methods in those with MR4.5 was weaker than other groups (no MMR: ▉= 0.042, P=0.846; MMR:▉=0.054, P=0.229; MR4:▉=-0.020, P=0.399; MR4.5:▉=-0.219, P<0.001) . Conclusions: dPCR is more accurate than qPCR for measuring BCR::ABL (P210) mRNA expression in patients with CML who achieve a stable deep MR.

Application and evaluation of droplet digital polymerase chain reaction in detection of severe acute respiratory syndrome corona virus-2 / 中国热带医学

@#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.

Application of digital PCR technology in the precise molecular diagnosis: opportunities and challenges / 中华检验医学杂志

Digital polymerase chain reaction (dPCR) is an absolute quantitative technique that has been rapidly developed in recent years. This technique assigns the reaction system containing DNA template to a large number of independent reaction units for PCR, and calculates the DNA copy number according to the Poisson distribution and statistical positive signals. In contrast to conventional qPCR, dPCR does not depend on amplification curves, is not affected by amplification efficiency, thus has high accuracy and repeatability, and can achieve the absolute quantification. This article reviews the development history of dPCR and its application in molecular diagnosis, tumor liquid biopsy and prenatal diagnosis of infectious diseases, and looks forward to the application prospect of this technology.

Application of Duplex Droplet Digital PCR Detection of miR-888 and miR-891a in Semen Identification / 法医学杂志

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.

Clinical importance of screening differential gene set of monocytes based on single-cell sequencing and digital polymerase chain reaction technology for early diagnosis of sepsis / 中华危重病急救医学

Objective:To verify the specific differentiated subsets of monocytes in sepsis, and to screen and construct the differential gene set of monocytes used for early diagnosis of sepsis.Methods:Patients with sepsis admitted to Guangdong Provincial People's Hospital from June 2020 to March 2021 were enrolled, and peripheral blood mononuclear cells (PBMC) were extracted. Single-cell sequencing technology and pseudo-time analysis were used to verify the differential subsets of monocytes. Bioinformatics methods were used to analyze the expression of genes in differential subsets of monocytes and screen out differential genes for the preliminary construction of a candidate differential gene set. The digital polymerase chain reaction (PCR) technology was used to verify the candidate differential genes in PBMC of sepsis patients and sepsis human myeloid leukemia mononuclear cells (THP-1) models, and the Venn diagram was used to construct the final differential gene set of monocytes. Gene Expression Omnibus (GEO) database was used to validate the differential gene set of monocytes.Results:① The results of cell annotation and pseudo-time analysis showed that the differentiation of NEAT1 +CD163 + monocyte occurred in the early stage of sepsis was significantly different from other subsets, which validated that NEAT1 +CD163 + monocyte was the characteristic subset in the pathological process of sepsis. ② Twenty-two differential genes related to sepsis were screened out from the gene expression of NEAT1 +CD163 + monocyte. After further verification by digital PCR, basic leucine zipper ATF-like transcription factor (BATF), JUNB proto-oncogene, carcinoembryonic antigen-related cell adhesion molecule 4 (CEACAM4), chromosome 9 open reading frame 95 (C9orf95), G protein subunit alpha 15 (GNA15), complement C3a receptor 1 (C3AR1), transforming growth factor beta 1 (TGFB1) and mitochondrial carrier homolog 1 (MTCH1) were screened out to construct the final differential gene set of monocytes. ③ The external validation results showed that C9orf95 gene had no data in GSE154918 and GSE133822 from GEO, it was excluded during validation. In GSE154918, the expressions of BATF, JUNB, CEACAM4, GNA15, C3AR1, TGFB1, and MTCH1 in the sepsis group were significantly higher than those in the healthy control group (log 2expression level: BATF was 12.78±0.08 vs. 11.39±0.35, JUNB was 16.88±0.07 vs. 16.04±0.03, CEACAM4 was 14.73±0.08 vs. 13.77±0.05, GNA15 was 13.16±0.06 vs. 12.30±0.04, C3AR1 was 14.62±0.13 vs. 12.87±0.05, TGFB1 was 16.95±0.05 vs. 16.57±0.36, MTCH1 was 14.80±0.02 vs. 14.61±0.15, all P < 0.05). In GSE133822, the expressions of BATF, CEACAM4, GNA15, and C3AR1 in the sepsis group were significantly higher than those in the health control group (log 2expression level: BATF was 8.66±0.16 vs. 7.92±0.14, CEACAM4 was 9.20±0.16 vs. 8.36±0.20, GNA15 was 10.66±0.18 vs. 10.13±0.16, C3AR1 was 11.49±0.27 vs. 10.48±0.16, all P < 0.05), while the expressions of JUNB, TGFB1, and MTCH1 were not statistically different between two groups. The results of gene set variation analysis (GSVA) showed that the enrichment scores of monocytes differential gene set of sepsis group were significantly higher than those of the healthy control group in both GSE154918 (0.38±0.04 vs. -0.44±0.02) and GSE133822 (0.56±0.02 vs. 0.20±0.05, both P < 0.01). Receiver operator characteristic curve (ROC curve) analysis showed that the differential gene set of monocytes had a reliable diagnostic value for early sepsis with the area under ROC curve (AUC) of 0.993 [95% confidence interval (95% CI) was 0.980-1.000] in GSE154918 and 0.944 (95% CI was 0.873-1.000) in GSE133822. Conclusion:A differential gene set of monocytes (BATF, JUNB, CEACAM4, GNA15, C3AR1, TGFB1, and MTCH1) screened out by single-cell sequencing and digital PCR technology has a reliable diagnostic value for the early sepsis, and may provide a new idea for the early diagnosis of sepsis.

Clinical Value of Droplet Digital PCR and Super-ARMS Detection of Epidermal Growth Factor Receptor Gene Mutation in Plasma Circulating Tumor DNA of Patients with Advanced Lung Adenocarcinoma / 中国肺癌杂志

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.

Diagnosis and treatment progress of chronic myeloid leukemia / 白血病·淋巴瘤

Chronic myeloid leukemia (CML) has made a milestone progress due to the development of the first generation tyrosine kinase inhibitor(TKI). Nowadays, most clinical trials in CML focus on discontinuation, even the second discontinuation, and the third generation TKI against T315I mutation. The conventional treatments are more focused on decreasing BCR-ABL transcripts rapidly. At the same time, the treatment management of some special patients has been valued.

Sensitivity and detection of chikungunya viral genetic material using several PCR-based approaches

Abstract INTRODUCTION: Chikungunya fever is a condition resulting from infection by chikungunya virus (CHIKV), an Aedes sp.-transmitted virus. This disease has been diagnosed in thousands of cases in the Americas, particularly in Brazil, in recent years, and there is an ongoing epidemic of chikungunya fever in Brazil that began in 2014. Clinical diagnosis is difficult; only a few cases have been confirmed by laboratory tests due to the low number of specific, efficient tests available for virus or antibody detection. Here, we aimed to evaluate different polymerase chain reaction (PCR) approaches for detection of CHIKV genetic material. METHODS: Specific primers and probes within the viral capsid gene region were designed for this work. To evaluate the analytic sensitivity of detection, human sera were spiked with serial dilutions of the viral stock. Several PCR protocols were performed to investigate the sensitivity of CHIKV RNA detection in serum dilutions ranging from 106 to 1 PFU equivalents. RESULTS: The technique showing the greatest sensitivity was a real-time PCR assay using specific probes that could detect the genetic material of the virus at all dilutions, followed by conventional PCR. Digital PCR showed low sensitivity and was much more expensive than other technologies. Digital PCR should be used for specific purposes other than clinical diagnosis. CONCLUSIONS: Although quantitative PCR using probes was more expensive than the use of intercalating dyes or conventional PCR, it had the highest sensitivity out of all tested PCR approaches.

Recent Development of Droplet Microfluidics in Digital Polymerase Chain Reaction / 分析化学

Digital polymerase chain reaction ( PCR) has been experiencing a rapid development during the past few years. Comparing with the traditional real-time quantitative PCR ( RT-qPCR) , using the same primer and probe, the accuracy for the absolute quantification of target gene is significantly improved. The development of digital PCR is directly related to the development of microfluidics. The integrated fluid circuit is an early combination of the microfluidics and digital PCR, which has a complicated fabrication process with high cost. Recently, researchers are trying to apply the droplet microfluidics in digital PCR, and the droplet microfluidic chip is able to generate millions of droplets within a short time. Each of these droplets containing no more than one target gene is a reaction chamber during the amplification process. After amplification, each droplet is tested to achieve the absolute quantification of the target gene. This paper reviews the recent progresses of droplet digital PCR, and the applications of droplet digital PCR in biological, medical and environmental fields.

Development of Droplet Digital Polymerase Chain Reaction for Quantifying Escherichia Coli O157:H7 / 分析化学

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.