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 ± 6920 copies/test) was found to be significantly higher than in throat swabs (2552 ± 1965 copies/test, P < .001) and nasal swabs (651 ± 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 ± 17 272 vs 1252 ± 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.
The outbreak of COVID-19 has spread across the world and was characterized as a pandemic. To protect medical laboratory personnel from infection, most laboratories inactivate the 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 samples and assess the effect of inactivation methods on viral RNA copy number. Viral inactivation was performed with three different methods: (1) incubation with TRIzol® LS Reagent for 10 min at room temperature, (2) heating in a waterbath at 56°C for 30 min, and (3) high-temperature treatment, including 121°C autoclaving for 20 min, 100°C boiling for 20 min, and 80°C heating for 20 min. Compared to the amount of RNA in the original sample, TRIzol treatment destroyed 47.54% of N gene and 39.85% of ORF 1ab. For samples treated at 56°C for 30 min, the copy number of N gene and ORF 1ab was reduced by 48.55% and 56.40%, respectively. Viral RNA copy number dropped by 50–66% after 80°C heating for 20 min. Nearly no viral RNA was detected after autoclaving at 121°C or boiling at 100°C for 20 min. These results indicated that inactivation reduced the quantity of detectable viral RNA and may cause false negative results especially in weakly positive cases. Thus, TRIzol is recommended for sample inactivation in comparison to heat inactivation as Trizol has the least effect on RNA copy number among the tested methods.
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.
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.
Using universal locked nucleic acid probes, a high multiplexing ddPCR-based NIPT was developed to reliably identify fetal aneuploidies.
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