Reverse Transcription Droplet Digital Polymerase Research Articles

Development of Human Rhinovirus RNA Reference Material Using Digital PCR.

The human rhinovirus (RV) is a positive-stranded RNA virus that causes respiratory tract diseases affecting both the upper and lower halves of the respiratory system. RV enhances its replication by concentrating RNA synthesis within a modified host membrane in an intracellular compartment. RV infections often occur alongside infections caused by other respiratory viruses, and the RV virus may remain asymptomatic for extended periods. Alongside qualitative detection, it is essential to accurately quantify RV RNA from clinical samples to explore the relationships between RV viral load, infections caused by the virus, and the resulting symptoms observed in patients. A reference material (RM) is required for quality evaluation, the performance evaluation of molecular diagnostic products, and evaluation of antiviral agents in the laboratory. The preparation process for the RM involves creating an RV RNA mixture by combining RV viral RNA with RNA storage solution and matrix. The resulting RV RNA mixture is scaled up to a volume of 25 mL, then dispensed at 100 µL per vial and stored at -80 °C. The process of measuring the stability and homogeneity of RV RMs was conducted by employing reverse transcription droplet digital polymerase chain reaction (RT-ddPCR). Digital PCR is useful for the analysis of standards and can help to improve measurement compatibility: it represents the equivalence of a series of outcomes for reference materials and samples being analyzed when a few measurement procedures are employed, enabling objective comparisons between quantitative findings obtained through various experiments. The number of copies value represents a measured result of approximately 1.6 × 105 copies/μL. The RM has about an 11% bottle-to-bottle homogeneity and shows stable results for 1 week at temperatures of 4 °C and -20 °C and for 12 months at a temperature of -80 °C. The developed RM can enhance the dependability of RV molecular tests by providing a precise reference value for the absolute copy number of a viral target gene. Additionally, it can serve as a reference for diverse studies.

Detecting the Neuraminidase R294K Mutation in Avian Influenza A (H7N9) Virus Using Reverse Transcription Droplet Digital PCR Method.

The R294K mutation in neuraminidase (NA) causes resistance to oseltamivir in the avian influenza virus H7N9. Reverse transcription droplet digital polymerase chain reaction (RT-dd PCR) is a novel technique for detecting single-nucleotide polymorphisms. This study aimed to develop an RT-dd PCR method for detecting the R294K mutation in H7N9. Primers and dual probes were designed using the H7N9 NA gene and the annealing temperature was optimized at 58.0 °C. The sensitivity of our RT-dd PCR method was not significantly different from that of RT-qPCR (p = 0.625), but it could specifically detect R294 and 294K in H7N9. Among 89 clinical samples, 2 showed the R294K mutation. These two strains were evaluated using a neuraminidase inhibition test, which revealed that their sensitivity to oseltamivir was greatly reduced. The sensitivity and specificity of RT-dd PCR were similar to those of RT-qPCR and its accuracy was comparable to that of NGS. The RT-dd PCR method had the advantages of absolute quantitation, eliminating the need for a calibration standard curve, and being simpler in both experimental operation and result interpretation than NGS. Therefore, this RT-dd PCR method can be used to quantitatively detect the R294K mutation in H7N9.

A multistate assessment of population normalization factors for wastewater-based epidemiology of COVID-19.

Wastewater-based epidemiology (WBE) has become a valuable tool for monitoring SARS-CoV-2 infection trends throughout the COVID-19 pandemic. Population biomarkers that measure the relative human fecal contribution to normalize SARS-CoV-2 wastewater concentrations are needed for improved analysis and interpretation of community infection trends. The Centers for Disease Control and Prevention National Wastewater Surveillance System (CDC NWSS) recommends using the wastewater flow rate or human fecal indicators as population normalization factors. However, there is no consensus on which normalization factor performs best. In this study, we provided the first multistate assessment of the effects of flow rate and human fecal indicators (crAssphage, F+ Coliphage, and PMMoV) on the correlation of SARS-CoV-2 wastewater concentrations and COVID-19 cases using the CDC NWSS dataset of 182 communities across six U.S. states. Flow normalized SARS-CoV-2 wastewater concentrations produced the strongest correlation with COVID-19 cases. The correlation from the three human fecal indicators were significantly lower than flow rate. Additionally, using reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) significantly improved correlation values over samples that were analyzed with real-time reverse transcription quantitative polymerase chain reaction (rRT-qPCR). Our assessment shows that utilizing flow normalization with RT-ddPCR generate the strongest correlation between SARS-CoV-2 wastewater concentrations and COVID-19 cases.

Development and application of a reverse transcription droplet digital PCR assay for detection and quantification of Plantago asiatica mosaic virus

Plantago asiatica mosaic virus (PlAMV), which infects lilies worldwide, constitutes a potential risk factor for lily production. Development of effective and sensitive methods for the detection and specific quantification of PlAMV is required to prevent the spread of PlAMV in lilies. In this study, we established a sensitive and accurate quantification method for PlAMV infection in lily samples using a reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) assay. The specificity and sensitivity of the proposed RT-ddPCR assay was evaluated for precise detection and quantification of PlAMV. The assay exhibited high specificity for PlAMV and showed no cross-reactivity with other lily viruses. It was about 10-fold more sensitive than reverse-transcription followed by quantitative polymerase chain reaction (RT-qPCR). RT-ddPCR (R2 = 0.9985) and RT-qPCR (R2 = 0.9869) showed high degrees of linearity. In addition, field validation tests on 92 lily samples confirmed that RT-ddPCR (45/92) has higher sensitivity than RT-qPCR (8/92). In summary, RT-ddPCR outperforms RT-qPCR in the analysis of low-viral-load samples and can be utilized for PlAMV infection diagnosis, especially in plant quarantine inspection and PlAMV-free certification programs.

Detection of SARS-CoV-2 RNA in wastewater and comparison to COVID-19 cases in two sewersheds, North Carolina, USA

Wastewater surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may be useful for monitoring population-wide coronavirus disease 2019 (COVID-19) infections, especially given asymptomatic infections and limitations in diagnostic testing. We aimed to detect SARS-CoV-2 RNA in wastewater and compare viral concentrations to COVID-19 case numbers in the respective counties and sewersheds. Influent 24-hour composite wastewater samples were collected from July to December 2020 from two municipal wastewater treatment plants serving different population sizes in Orange and Chatham Counties in North Carolina. After a concentration step via HA filtration, SARS-CoV-2 RNA was detected and quantified by reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) and quantitative PCR (RT-qPCR), targeting the N1 and N2 nucleocapsid genes. SARS-CoV-2 RNA was detected by RT-ddPCR in 100 % (24/24) and 79 % (19/24) of influent wastewater samples from the larger and smaller plants, respectively. In comparison, viral RNA was detected by RT-qPCR in 41.7 % (10/24) and 8.3 % (2/24) of samples from the larger and smaller plants, respectively. Positivity rates and method agreement further increased for the RT-qPCR assay when samples with positive signals below the limit of detection were counted as positive. The wastewater data from the larger plant generally correlated (⍴ ~0.5, p < 0.05) with, and even anticipated, the trends in reported COVID-19 cases, with a notable spike in measured viral RNA preceding a spike in cases when students returned to a college campus in the Orange County sewershed. Correlations were generally higher when using estimates of sewershed-level case data rather than county-level data. This work supports use of wastewater surveillance for tracking COVID-19 disease trends, especially in identifying spikes in cases. Wastewater-based epidemiology can be a valuable resource for tracking disease trends, allocating resources, and evaluating policy in the fight against current and future pandemics.

Detection and Quantification of Apple Stem Grooving Virus in Micropropagated Apple Plantlets Using Reverse-Transcription Droplet Digital PCR.

Apple stem grooving virus (ASGV) is a destructive viral pathogen of pome fruit trees that causes significant losses to fruit production worldwide. Obtaining ASGV-free propagation materials is essential to reduce economic losses, and accurate and sensitive detection methods to screen ASGV-free plantlets during in vitro propagation are urgently necessary. In this study, ASGV was sensitively and accurately quantified from in vitro propagated apple plantlets using a reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) assay. The optimized RT-ddPCR assay was specific to other apple viruses, and was at least 10-times more sensitive than RT-real-time quantitative PCR assay. Furthermore, the optimized RT-ddPCR assay was validated for the detection and quantification of ASGV using micropropagated apple plantlet samples. This RT-ddPCR assay can be utilized for the accurate quantitative detection of ASGV infection in ASGV-free certification programs, and can thus contribute to the production of ASGV-free apple trees.

Late Complications of COVID-19.

Studies of lungs in patients with COVID-19 have focused on early findings. To systematically study histopathologic and imaging features and presence of SARS-CoV-2 RNA in lung tissue from patients in later stages of COVID-19. Autopsies, explants, surgical lung biopsies, transbronchial biopsies, cryobiopsies, and needle biopsies from patients with COVID-19 whose onset of symptoms/confirmed diagnosis was more than 28 days before the procedure were studied. Available images were reviewed. Reverse transcription droplet digital polymerase chain reaction for SARS-CoV-2 RNA was performed on lung tissue. Of 44 specimens (43 patients; median age, 59.3 years; 26 [60.5%] male) features of acute lung injury (ALI) were seen in 39 (88.6%), predominantly organizing pneumonia and diffuse alveolar damage, up to 298 days after onset of COVID-19. Fibrotic changes were found in 33 specimens (75%), most commonly fibrotic diffuse alveolar damage (n = 22) and cicatricial organizing pneumonia (n = 12). Time between acquiring COVID-19 and specimen was shorter in patients with diffuse ALI (median, 61.5 days) compared with patients with focal (140 days) or no ALI (130 days) (P = .009). Sixteen (of 20; 80%) SARS-CoV-2 reverse transcription droplet digital polymerase chain reaction tests were positive, up to 174 days after COVID-19 onset. Time between COVID-19 onset and most recent computed tomography in patients with consolidation on imaging was shorter (median, 43.0 days) versus in patients without consolidation (87.5 days; P = .02). Reticulations were associated with longer time to computed tomography after COVID-19 onset (median, 82 versus 23.5 days; P = .006). ALI and SARS-CoV-2 RNA can be detected in patients with COVID-19 for many months. ALI may evolve into fibrotic interstitial lung disease.

Development and application of reverse transcription droplet digital PCR assay for sensitive detection of apple scar skin viroid during in vitro propagation of apple plantlets

Apple scar skin viroid (ASSVd), of the genus Apscaviroid, causes serious pome fruit diseases, such as apple scar skin, dapple apple, pear rusty skin, pear fruit crinkle, and pear dimple fruit. This study aimed at establishing a sensitive and accurate method for quantification of ASSVd in apple leaves and plantlets using a reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) assay. The specificity was analyzed using other apple viruses, and the negative amplification of the cross-reaction assay demonstrated the high specificity of RT-ddPCR. The detection limit of ASSVd by RT-ddPCR was 1.75 × 102 copies/μL (0.14 concentration), and the sensitivity was ten-fold higher than that of RT-qPCR. Similarly, positive detection in apple plantlet samples by RT-ddPCR was higher than that by RT-qPCR. The RT-ddPCR assay represents a promising alternative for accurate quantitative detection and diagnosis of ASSVd infection in ASSVd-free certification programs.

Application of Reverse Transcription Droplet Digital PCR for Detection and Quantification of Tomato Spotted Wilt Virus

Plant viruses cause significant yield losses, continuously compromising crop production and thus representing a serious threat to global food security. Tomato spotted wilt virus (TSWV) is the most harmful plant virus that mainly infects horticultural crops and has a wide host range. Reverse-transcription quantitative real-time PCR (RT-qPCR) has been widely used for detecting TSWV with high sensitivity, but its application is limited owing to the lack of standardization. Therefore, in this study, a sensitive and accurate reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) method was established for TSWV detection. Additionally, we compared the sensitivities of RT-qPCR and RT-ddPCR for TSWV detection. Specificity analysis of RT-ddPCR for TSWV showed no amplification for main pepper viruses and negative control. TSWV transcripts levels measured by RT-ddPCR and RT-qPCR showed a high degree of linearity; however, the former yielded results that were at least 10-fold more sensitive and detected lower TSWV copy numbers than the latter. Collectively, our findings show that RT-ddPCR provides improved analytical sensitivity and specificity for TSWV detection, making it suitable for identifying low TSWV concentrations in field samples.

O-179 Safety of ovarian tissue cryopreservation and transplantation in patients with central nervous system cancers

Abstract Study question Is cryopreserved ovarian tissue transplantation safe in patients with central nervous system (CNS) tumors? Summary answer Cancer cell contamination was not detected in any ovarian samples from patients with CNS tumors by histological analysis, immunohistochemistry, molecular biology or long-term xenotransplantation. What is known already Frequently encountered CNS cancers in childhood include astrocytoma, medulloblastoma, ependymoma, glioblastoma and germinoma. CNS tumors have the capacity for extraneural metastases in 0.5-18% of cases. There are two publications reporting metastases to patients’ ovaries from medulloblastoma. Study design, size, duration Prospective experimental study conducted in an academic gynecology research laboratory using frozen-thawed ovarian tissue from 20 patients suffering from 6 types of CNS tumors, including the most common forms mentioned above and primitive neuroectodermal tumors (PNET). Five-month xenotransplantation was performed to severe combined immunodeficient (SCID) mice. Participants/materials, setting, methods Cryopreserved ovarian tissue from 20 patients with CNS cancers was thawed and analyzed for minimal disseminated disease and long-term xenografting to immunodeficient mice. The presence of malignant cells was assessed in both cryopreserved and xenografted ovarian tissue using histological analysis, immunohistochemistry for disease-­specific markers (neuron-specific enolase [NSE] and glial fibrillary acidic protein [GFAP]) and reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) for quantification of GFAP gene amplification. Main results and the role of chance No malignant cells were detected in frozen-thawed ovarian tissue from any of the patients by histology, immunolabeling for NSE and GFAP, RT-ddPCR for detection of GFAP gene amplification or xenotransplantation to SCID mice. One patient successfully underwent frozen-thawed ovarian tissue transplantation, resulting in the birth of 3 healthy children, but suffered a recurrence of her PNET 6 years after reimplantation and sadly died. Scrupulous analysis of her remaining frozen tissue showed no infiltration by malignant cells, neither after thawing nor long-term xenotransplantation. No relationship was ever established between the patient’s relapsed cancer and reintroduction of her cryopreserved ovarian tissue. The risk of reseeding cancer cells when transplanting ovarian tissue in patients with CNS cancers can therefore be considered low. Limitations, reasons for caution The risk of ovarian metastases cannot be completely ruled out for any type of tumor because we cannot analyze the actual fragments that will be reimplanted. Wider implications of the findings Our results indicate that the risk of disseminated disease in ovarian tissue from CNS patients is minimal. This is useful information for doctors when counseling women looking to undergo ovarian tissue transplantation. Trial registration number Not applicable

Development of a reverse transcription droplet digital PCR assay for sensitive detection of peach latent mosaic viroid

Peach latent mosaic viroid (PLMVd) represents a continuing threat to peach tree production worldwide. In this study, a sensitive and accurate quantification of PLMVd in peach leaves was established using a reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) assay. The quantitative linearity, accuracy, and sensitivity of RT-ddPCR for the detection of PLMVd were comparatively assessed to those of reverse-transcription real-time quantitative polymerase chain reaction (RT-qPCR) assay. The specificity assay shows no amplification in major peach viruses, apple chlorotic leaf spot virus and prunus necrotic ring spot virus and negative control. Furthermore, the levels of PLMVd transcripts determined using RT-ddPCR and RT-qPCR showed a high degree of linearity and quantitative correlation. Our results also indicated that the RT-ddPCR assay is at least two-fold more sensitive than qPCR and could therefore, be used to detect PLMVd in field samples. Moreover, optimization of RT-ddPCR was found to enhance the sensitivity of PLMVd detection in the peach leaf samples with low viral loads. In summary, the established RT-ddPCR assay represents a promising alternative method for the precise quantitative detection of PLMVd; it would be particularly applicable for diagnosing PLMVd infections in plant quarantine inspection and PLMVd-free certification program.

Ovarian tissue cryopreservation and transplantation in patients with central nervous system tumours.

Is there a possibility of reseeding cancer cells potentially present in frozen ovarian tissue from patients with central nervous system (CNS) tumours? Malignancy reseeding in cryopreserved ovarian tissue from 20 patients with CNS tumours was not detected by histology, immunohistochemistry (IHC), molecular biology or xenotransplantation. Ovarian metastasis potential has been documented in patients with leukaemia, borderline ovarian tumours, advanced breast cancer and Ewing sarcoma. However, data on the safety of transplanting frozen-thawed ovarian tissue from cancer patients with CNS tumours are still lacking. This prospective experimental study was conducted in an academic gynaecology research laboratory using cryopreserved ovarian cortex from 20 patients suffering from CNS tumours. Long-term (5 months) xenografting was performed in immunodeficient mice. Subjects enrolled in the study were suffering from one of six types of CNS tumours including medulloblastoma, ependymoma, primitive neuroectodermal tumours, astrocytoma, glioblastoma and germinoma. The presence of malignant cells was investigated with disease-specific markers for each patient in cryopreserved and xenografted ovarian tissue by histology, IHC via expression of neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP), and reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) for quantification of GFAP and ENO2 gene amplification. Serial sections of cryopreserved and xenografted ovarian tissue from 20 patients showed no malignant cells by histology. All samples were negative for NSE and GFAP, although these neural markers were expressed extensively in the patients' primary tumours. Analysis by RT-ddPCR revealed no cancer cells detected in cryopreserved and xenografted ovarian fragments from subjects with astrocytoma, ependymoma, glioblastoma or medulloblastoma. Taken together, the study found no evidence of malignancy seeding in frozen-thawed and xenotransplanted ovarian tissue from patients affected by CNS cancers. This analysis cannot guarantee complete elimination of disseminated disease from all cryopreserved ovarian cortex, since we are unable to examine the fragments used for transplantation. This is the first study to be conducted in patients with CNS cancers undergoing ovarian tissue cryopreservation and transplantation, and clearly demonstrates no tumour seeding in their frozen-thawed and xenografted tissue. This information is vital for doctors to provide patients with meaningful and accurate advice on the possibilities and risks of ovarian tissue reimplantation. This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique-the Excellence of Science (FNRS-EOS), number 30443682 awarded to M.-M.D. and T.Y.T.N., FNRS grant number 5/4/150/5 and FNRS-PDR Convention grant number T.0077.14 awarded to M.-M.D., grant 2018-042 from the Foundation Against Cancer awarded to A.C., and private donations (Ferrero, de Spoelberch). The authors declare no competing financial interests. N/A.

Transcriptome analysis of a ring chromosome 20 patient cohort.

Ring chromosomes occur when the ends of normally rod-shaped chromosomes fuse. In ring chromosome 20 (ring 20), intellectual disability and epilepsy are usually present, even if there is no deleted coding material; the mechanism by which individuals with complete ring chromosomes develop seizures and other phenotypic abnormalities is not understood. We investigated altered gene transcription as a contributing factor by performing RNA-sequencing (RNA-seq) analysis on blood from seven patients with ring 20, and 11 first-degree relatives (all parents). Geographic analysis did not identify altered expression in peritelomeric or other specific chromosome 20 regions. RNA-seq analysis revealed 97 genes potentially differentially expressed in ring 20 patients. These included one epilepsy gene, NPRL3, but this finding was not confirmed on reverse transcription Droplet Digital polymerase chain reaction analysis. Molecular studies of structural chromosomal anomalies such as ring chromosome are challenging and often difficult to interpret because many patients are mosaic, and there may be genome-wide chromosomal instability affecting gene expression. Our findings nevertheless suggest that peritelomeric altered transcription is not the likely pathogenic mechanism in ring 20. Underlying genetic mechanisms are likely complex and may involve differential expression of many genes, the majority of which may not be located on chromosome 20.

Relationships between UBE3A and SNORD116 expression and features of autism in chromosome 15 imprinting disorders

Chromosome 15 (C15) imprinting disorders including Prader–Willi (PWS), Angelman (AS) and chromosome 15 duplication (Dup15q) syndromes are severe neurodevelopmental disorders caused by abnormal expression of genes from the 15q11–q13 region, associated with abnormal DNA methylation and/or copy number changes. This study compared changes in mRNA levels of UBE3A and SNORD116 located within the 15q11–q13 region between these disorders and their subtypes and related these to the clinical phenotypes. The study cohort included 58 participants affected with a C15 imprinting disorder (PWS = 27, AS = 21, Dup15q = 10) and 20 typically developing controls. Semi-quantitative analysis of mRNA from peripheral blood mononuclear cells (PBMCs) was performed using reverse transcription droplet digital polymerase chain reaction (PCR) for UBE3A and SNORD116 normalised to a panel of internal control genes determined using the geNorm approach. Participants completed an intellectual/developmental functioning assessment and the Autism Diagnostic Observation Schedule-2nd Edition. The Dup15q group was the only condition with significantly increased UBE3A mRNA levels when compared to the control group (p < 0.001). Both the AS and Dup15q groups also had significantly elevated SNORD116 mRNA levels compared to controls (AS: p < 0.0001; Dup15q: p = 0.002). Both UBE3A and SNORD116 mRNA levels were positively correlated with all developmental functioning scores in the deletion AS group (p < 0.001), and autism features (p < 0.001) in the non-deletion PWS group. The findings suggest presence of novel interactions between expression of UBE3A and SNORD116 in PBMCs and brain specific processes underlying motor and language impairments and autism features in these disorders.

One-step duplex RT-droplet digital PCR assay for the detection of norovirus GI and GII in lettuce and strawberry

The study was designed to develop a sensitive one-step duplex reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) to detect norovirus genogroup I and II (NoV GI and GII) in lettuce and strawberry. The specificity, sensitivity, repeatability and robustness of the assay was compared with RT-qPCR. The lowest concentration detected by RT-ddPCR for NoV GI and NoV GII were 4.68 and 8.47 copies/μL respectively, much lower than that of RT-qPCR with a number of 46.8 and 84.7 copies/μL, respectively. Lettuce and strawberry samples were artificially contaminated with NoV GI and GII suspensions, with inoculum size of 3.00 × 106 to 1.70 × 108 copies and 4.80 × 105 to 2.50 × 107 copies, respectively. Strawberry spiked with low inoculum size revealed positive results by RT-ddPCR, while recorded negative by RT-qPCR. Meanwhile, RT-ddPCR also showed a higher average recovery rate for NoV in lettuce and strawberry than RT-qPCR.The limit of detection (LoDs) of RT-ddPCR for NoVs in lettuce was 2.32 × 104 copies/25g (NoV GI) and 2.36 × 104 ciopies/25g (NoV GII), and that in strawberry was 2.56 × 104 copies/25g (NoV GI) and 2.64 × 104 ciopies/25g (NoV GII), which were 10 folds lower than that of RT-qPCR. The developed duplex RT-ddPCR assay exhibited stability and increased capacity to resist inhibitors in food samples with low concentration of NoV, making it a reliable method to avoid false negative result as opposed to RT-qPCR. In conclusion, one-step RT-ddPCR method developed in this study is pertinent in detecting foodborne virus such as NoV.

Reverse transcriptase droplet digital PCR vs reverse transcriptase quantitative real-time PCR for serum HBV RNA quantification.

Serum hepatitis B virus (HBV) RNA is a novel marker reflecting the activity of covalently closed circular DNA. However, the methodology for detecting HBV RNA has been a technical challenge. In this study, the performance of reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) for quantifying HBV RNA was compared with that of reverse transcription quantitative real-time PCR (RT-qPCR) in serum samples collected from treatment-naïve patients with different phases of chronic hepatitis B (CHB). A total of 417 serum samples, including 136 HBeAg-positive CHB and 281 HBeAg-negative CHB were examined. HBV RNA levels measured by RT-ddPCR and RT-qPCR showed a high degree of linearity and quantitative correlation. The limit of detections of RT-ddPCR and RT-qPCR assays were 102 and 103 copies/mL, respectively. Our results also demonstrated that RT-ddPCR was superior to RT-qPCR in terms of its consistency for quantifying HBV RNA across all concentrations. In the HBeAg-positive group, serum HBV RNA levels based on RT-ddPCR were moderately correlated with HBV DNA (r = 0.591, P < .001) and HBsAg (r = 0.502, P < .001). Among patients with HBeAg-negative CHB, serum HBV RNA levels were moderately correlated with HBV DNA (r = 0.603, P < .001) but had weak correlation with HBsAg (r = 0.203, P = .001). In summary, RT-ddPCR could enhance the sensitivity of serum HBV RNA detection, particularly among the HBeAg-negative group with low viral loads. Thus, RT-ddPCR could serve as an optimal method for HBV RNA quantification in clinical practice.

Covalent chemistry on nanostructured substrates enables noninvasive quantification of gene rearrangements in circulating tumor cells.

Well-preserved mRNA in circulating tumor cells (CTCs) offers an ideal material for conducting molecular profiling of tumors, thereby providing a noninvasive diagnostic solution for guiding treatment intervention and monitoring disease progression. However, it is technically challenging to purify CTCs while retaining high-quality mRNA.Here, we demonstrate a covalent chemistry-based nanostructured silicon substrate ("Click Chip") for CTC purification that leverages bioorthogonal ligation-mediated CTC capture and disulfide cleavage-driven CTC release. This platform is ideal for CTC mRNA assays because of its efficient, specific, and rapid purification of pooled CTCs, enabling downstream molecular quantification using reverse transcription Droplet Digital polymerase chain reaction. Rearrangements of ALK/ROS1 were quantified using CTC mRNA and matched with those identified in biopsy specimens from 12 patients with late-stage non-small cell lung cancer. Moreover, CTC counts and copy numbers of ALK/ROS1 rearrangements could be used together for evaluating treatment responses and disease progression.