[An evaluation of carrier detection for Spinal muscular atrophy using digital PCR assay].

OBJECTIVE: To assess the effectiveness and feasibility of carrier detection for Spinal muscular atrophy (SMA) by using digital PCR assay. METHODS: Peripheral blood samples were collected from 214 pregnant women who were routinely screened for SMA carriers, of which 204 were randomly selected samples and 10 were samples with known copy numbers of SMN1 exons 7 and 8. Samples with known copy numbers of SMN1 exons 7 and 8 were randomly mixed into the experiment to validate the performance of the digital PCR assay. RESULTS: The copy numbers of SMN1 exons 7 and 8 and SMN2 exons 7 and 8 in peripheral blood samples were detected by digital PCR assay. The results of SMN1 exons 7 and 8 were compared with those of the quantitative PCR method to assess the reliability and clinical performance of the digital PCR assay. Among the 204 random samples, digital PCR has detected five samples with simultaneous heterozygous deletion of SMN1 exons 7 and 8, three samples with heterozygous deletion of SMN1 exon 8 only, and 196 samples with no deletion of SMN1 exons 7 and 8. Ten samples with known SMN1 exons 7 and 8 copy numbers were detected with the expected values. The digital PCR test results were fully consistent with that of the quantitative PCR. CONCLUSION: The results of digital PCR for the detection of copy number variation of SMN1 exons 7 and 8 were consistent with qPCR. Digital PCR assay was able to clearly distinguish the copy number of the target genes, therefore can be used for SMA carrier screening. Moreover, it can also detect copy number of SMN2 exons 7 and 8, which can provide more information for genetic counseling.

Single-Tube Multiplex Digital Polymerase Chain Reaction Assay for Molecular Diagnosis and Prediction of Severity of Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease characterized by the degeneration of motor neurons and progressive muscle atrophy. Accurate detection of SMN1 and SMN2 copy numbers is essential for SMA diagnosis, carrier screening, disease severity prediction, therapy, and prognosis. However, a method for SMN1 and SMN2 copy number determination that is simultaneously accurate, simple, rapid, multitargeted, and applicable to various samples has not previously been reported. Here, we developed a single-tube multiplex digital polymerase chain reaction (dPCR) assay for simultaneous determination of the copy numbers of SMN1 exons 7 and 8 and SMN2 exons 7 and 8. A total of 317 clinical samples, including peripheral blood, amniotic fluid, chorionic villus, buccal swabs, and dried blood spots, were collected to evaluate the performance of this dPCR-based assay. The test results were accurate for all the clinical samples. Our assay is accurate, rapid, easy to handle, and applicable to many types of samples and uses a small amount of DNA; it is a powerful tool for SMA molecular diagnosis, large-scale screening, and disease severity assessment.

Analysis of viral load in different specimen types and serum antibody levels of COVID-19 patients.

BACKGROUND: COVID-19 has caused a global pandemic and the death toll is increasing. However, there is no definitive information regarding the type of clinical specimens that is the best for SARS-CoV-2 detection, the antibody levels in patients with different duration of disease, and the relationship between antibody level and viral load. METHODS: Nasopharyngeal swabs, anal swabs, saliva, blood, and urine specimens were collected from patients with a course of disease ranging from 7 to 69 days. Viral load in different specimen types was measured using droplet digital PCR (ddPCR). Meanwhile, anti-nucleocapsid protein (anti-N) IgM and IgG antibodies and anti-spike protein receptor-binding domain (anti-S-RBD) IgG antibody in all serum samples were tested using ELISA. RESULTS: The positive detection rate in nasopharyngeal swab was the highest (54.05%), followed by anal swab (24.32%), and the positive detection rate in saliva, blood, and urine was 16.22%, 10.81%, and 5.41%, respectively. However, some patients with negative nasopharyngeal swabs had other specimens tested positive. There was no significant correlation between antibody level and days after symptoms onset or viral load. CONCLUSIONS: Other specimens could be positive in patients with negative nasopharyngeal swabs, suggesting that for patients in the recovery period, specimens other than nasopharyngeal swabs should also be tested to avoid false negative results, and anal swabs are recommended. The antibody level had no correlation with days after symptoms onset or the viral load of nasopharyngeal swabs, suggesting that the antibody level may also be affected by other factors.

Quantitative Detection and Viral Load Analysis of SARS-CoV-2 in Infected Patients.

BACKGROUND: Coronavirus disease 2019 (COVID-19) has become a public health emergency. The widely used reverse transcription-polymerase chain reaction (RT-PCR) method has limitations for clinical diagnosis and treatment. METHODS: A total of 323 samples from 76 COVID-19-confirmed patients were analyzed by droplet digital PCR (ddPCR) and RT-PCR based 2 target genes (ORF1ab and N). Nasal swabs, throat swabs, sputum, blood, and urine were collected. Clinical and imaging data were obtained for clinical staging. RESULTS: In 95 samples that tested positive by both methods, the cycle threshold (Ct) of RT-PCR was highly correlated with the copy number of ddPCR (ORF1ab gene, R2 = 0.83; N gene, R2 = 0.87). Four (4/161) negative and 41 (41/67) single-gene positive samples tested by RT-PCR were positive according to ddPCR with viral loads ranging from 11.1 to 123.2 copies/test. The viral load of respiratory samples was then compared and the average viral load in sputum (17 429 ±â€…6920 copies/test) was found to be significantly higher than in throat swabs (2552 ±â€…1965 copies/test, P < .001) and nasal swabs (651 ±â€…501 copies/test, P < .001). Furthermore, the viral loads in the early and progressive stages were significantly higher than that in the recovery stage (46 800 ±â€…17 272 vs 1252 ±â€…1027, P < .001) analyzed by sputum samples. CONCLUSIONS: Quantitative monitoring of viral load in lower respiratory tract samples helps to evaluate disease progression, especially in cases of low viral load.

Influence of Different Inactivation Methods on Severe Acute Respiratory Syndrome Coronavirus 2 RNA Copy Number.

The outbreak of coronavirus disease 2019 (COVID-19) has spread across the world and was characterized as a pandemic. To protect medical laboratory personnel from infection, most laboratories inactivate the virus causing COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in clinical samples before testing. However, the effect of inactivation on the detection results remains unknown. Here, we used a digital PCR assay to determine the absolute SARS-CoV-2 RNA copy number in 63 nasopharyngeal swab samples and assess the effect of inactivation methods on viral RNA copy number. Viral inactivation was performed by three different methods: (i) incubation with the TRIzol LS reagent for 10 min at room temperature, (ii) heating in a water bath at 56°C for 30 min, and (iii) high-temperature treatment, including autoclaving at 121°C for 20 min, boiling at 100°C for 20 min, and heating at 80°C for 20 min. Compared to the amount of RNA in the original sample, TRIzol treatment destroyed 47.54% of the nucleocapsid protein (N) gene and 39.85% of open reading frame (ORF) 1ab. For samples treated at 56°C for 30 min, the copy number of the N gene and ORF 1ab was reduced by 48.55% and 56.40%, respectively. The viral RNA copy number dropped by 50 to 66% after heating at 80°C for 20 min. Nearly no viral RNA was detected after autoclaving at 121°C or boiling at 100°C for 20 min. These results indicate that inactivation reduced the quantity of detectable viral RNA and may cause false-negative results, especially in weakly positive cases. Thus, use of the TRIzol reagent rather than heat inactivation is recommended for sample inactivation, as the TRIzol reagent had the least effect on the RNA copy number among the tested methods.

A multiplex droplet digital PCR assay for non-invasive prenatal testing of fetal aneuploidies.

Higher multiplexing in droplet digital PCR (ddPCR) can simplify the detection process of ddPCR-based non-invasive prenatal testing (NIPT) and improve its reliability, making it a practical approach in clinical practice. However, a high level of multiplex ddPCR-based NIPT has rarely been reported. In this study, we developed a multiplex ddPCR assay using universal locked nucleic acid (LNA) probes to reliably identify fetal aneuploidies. We first performed statistical analysis based on the Poisson distribution to evaluate the required number of target DNA molecules and the total number of droplets for a ddPCR assay. Next, we designed two sets of primers and probes to quantify cfDNA from chromosomes 21 and 18 and then determined the disease status of a sample. Finally, we evaluated our multiplex ddPCR assay with 60 clinical plasma samples. All of the 60 clinical samples were correctly identified. The accessibility and cost-effectiveness of our multiplex ddPCR-based NIPT make it a competitive prenatal testing method in clinical use.

Applications of digital PCR in COVID-19 pandemic.

The coronavirus disease 2019 (COVID-19) pandemic has led to a public health crisis and global panic. This infectious disease is caused by a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Digital polymerase chain reaction (dPCR), which is an emerging nucleic acid amplification technology that allows absolute quantification of nucleic acids, plays an important role in the detection of SARS-CoV-2. In this review, we introduce the principle and advantages of dPCR, and review the applications of dPCR in the COVID-19 pandemic, including detection of low copy number viruses, measurement of the viral load, preparation of reference materials, monitoring of virus concentration in the environment, detection of viral mutations, and evaluation of anti-SARS-CoV-2 drugs. We also discuss the challenges of dPCR in clinical practice.

Comparison of qualitative and quantitative analyses of COVID-19 clinical samples.

BACKGROUND: Qualitative and quantitative detection of nucleic acids of SARS-CoV-2, the pathogen that causes coronavirus disease 2019 (COVID-19), plays a significant role in COVID-19 diagnosis, surveillance, prevention, and control. METHODS: A total of 117 samples from 30 patients with confirmed COVID-19 and 61 patients without COVID-19 were collected. Reverse transcriptase quantitative PCR (RT-qPCR) and droplet digital PCR (ddPCR) were used for qualitative and quantitative analyses of these samples to evaluate the diagnostic performance and applicability of the two methods. RESULTS: The positive detection rates of RT-qPCR and ddPCR were 93.3% and 100%, respectively. Among the 117 samples, 6 samples were tested single-gene positive by RT-qPCR but positive by ddPCR, and 3 samples were tested negative by RT-qPCR but positive by ddPCR. The viral load of samples with inconsistent results were relatively low (3.1-20.5 copies/test). There were 17 samples (37%) with a viral load below 20 copies/test among the 46 positive samples, and only 9 of them were successfully detected by RT-qPCR. A severe patient was dynamically monitored. All 6 samples from this patient were tested negative by RT-qPCR, but 4 samples were tested positive by ddPCR with a low viral load. CONCLUSION: Qualitative analysis of COVID-19 samples can meet the needs of clinical screening and diagnosis, while quantitative analysis provides more information to the research community. Although both ddPCR and RT-qPCR can provide qualitative and quantitative results, ddPCR showed higher sensitivity and lower limit of detection than RT-qPCR, and it does not rely on the standard curve to quantify viral load. Therefore, ddPCR offers greater advantages than RT-qPCR.

Decreased mitochondrial DNA copy number in children with cerebral palsy quantified by droplet digital PCR.

BACKGROUND: Mitochondrial DNA copy number is a potential biomarker for mitochondrial dysfunction and is involved in a variety of disease states including autism, neurodegenerative diseases and traumatic brain injury, but few studies on mitochondrial DNA copy number in cerebral palsy have been reported. Therefore, this study aims to investigate the role of mitochondrial DNA copy number in children with cerebral palsy. METHODS: A total of 104 children with cerebral palsy and 78 typically developing children were enrolled in this study. All children with cerebral palsy were diagnosed according to clinical criteria and furtherly divided into clinical subtypes. Mitochondrial DNA copy number was quantified by droplet digital PCR. RESULTS: We observed a significant reduction in mitochondrial DNA copy number from children with cerebral palsy comparing to healthy controls (216.76 ± 71.39 vs 359.66 ± 72.78, p < 0.001). An upward trend in mitochondrial DNA copy number alteration with the increase of age was found in healthy controls rather than in children with cerebral palsy. In addition, the mitochondrial DNA copy number in children with spastic hemiplegia was higher than that in children with spastic quadriplegia (152.27 ± 49.78 vs 90.64 ± 21.55, p = 0.001). CONCLUSIONS: Our results suggest that on the basis of accurate quantification by droplet digital PCR, the declined mitochondrial DNA copy number probably has certain implications for mitochondrial dysfunction in children with cerebral palsy, which provides a new clue for the investigation on the molecular mechanism and clinical characteristics of cerebral palsy.

Establishment and validation of a novel droplet digital PCR assay for ultrasensitive detection and dynamic monitoring of EGFR mutations in peripheral blood samples of non-small-cell lung cancer patients.

BACKGROUND: Droplet digital PCR (ddPCR)-based blood detection of EGFR mutations plays significant roles in the individualized therapy of non-small-cell lung cancer (NSCLC) patients. However, a standard assay that is approved by health authorities is still lacking. Additionally, the proper application of this method in clinical settings also needs further investigation. METHODS: The performance of a newly established ddPCR assay was first evaluated using reference samples and then validated by comparing this method with the amplification refractory mutation system (ARMS) using cell-free DNA (cfDNA) in patients' peripheral blood. Further, the correlation between dynamic quantification of EGFR mutation in the patients and their clinical outcome of tyrosine kinase inhibitors (TKIs) therapy was investigated. RESULTS: A total of 77 patients were included, with 50 in the test group and 27 in the validation group. According to the results of the reference samples and the blood samples in the test group, the cut-off value for patient detection was proposed as mutation rate ≥ 0.1% (total copy number of cfDNA ≥ 1000) or at least one copy of mutation DNA was detected (total copy number of cfDNA < 1000). With this criterion, superior sensitivity of our assay to that of ARMS was observed (P = 0.002 for Ex19Del & L858R and P < 0.001 for T790M). The dynamic quantification of EGFR mutations during TKI therapy indicated that an increase in mutation abundance was correlated with resistance, while a decline was associated with response. Notably, a rebound in mutation abundance during chemotherapy may indicate a desirable chance for TKI re-treatment. CONCLUSION: The novel ddPCR assay showed superior sensitivity in the detection of EGFR mutation in blood. The dynamic quantification of EGFR mutations by this assay would greatly facilitate the administration of TKI therapy, including the monitoring of resistance and response, as well as cohort screening for retreatment.

Evaluation of the clinical performance of single-, dual-, and triple-target SARS-CoV-2 RT-qPCR methods.

BACKGROUND: The coronavirus disease 2019 (COVID-19) has become a pandemic. Reverse transcription quantitative PCR (RT-qPCR) has played a vital role in the diagnosis of COVID-19, but the rates of false negatives is not ideal in dealing with this highly infectious virus. It is thus necessary to systematically evaluate the clinical performance of the single-, dual-, triple-target detection kits to guide the clinical diagnosis of this disease. METHODS: A series of reference materials calibrated by droplet digital PCR (ddPCR) and 57 clinical samples were used to evaluate the clinical performance of six single-, dual-, triple-target SARS-CoV-2 nucleic acid detection kits based on RT-qPCR. RESULTS: The dual-target kits, kit B and kit C had the highest and the lowest detection sensitivity, which was 125 copies/mL and 4000 copies/mL, respectively. Among the 57 clinical samples from patients with COVID-19, 47 were tested positive by the kit B, while 35, 29, 28, 30, and 29 were found positive by the kits A, C, D, E, and F, respectively. The number of targets in a detection kit is not a key factor affecting sensitivity, while the amount of sample loading may influence the performance of a detection kit. CONCLUSIONS: This study provides a guide when choosing or developing a nucleic acid detection kit for the diagnosis of COVID-19. Also, the absolute-quantification feature and high-sensitivity performance of ddPCR, suggesting that it can be used to review clinically suspected samples.