scholarly journals Performance of COVIDSeq and Swift normalase amplicon SARS-CoV-2 panels for SARS-CoV-2 Genomes Sequencing: Practical Guide and Combining FASTQ Strategy

2022 ◽  
Author(s):  
Hosoon Choi ◽  
Munok Hwang ◽  
Dhammika Navarathna ◽  
Jing Xu ◽  
Janell Lukey ◽  
...  
Keyword(s):  
Viral Load ◽  
Sequence Analysis ◽  
Crucial Role ◽  
Digital Pcr ◽  
Clinical Samples ◽  
Genomic Sequencing ◽  
Sequence Coverage ◽  
Sequencing Coverage ◽  
Practical Guide ◽  
Cutoff Values

The whole genomic sequencing (WGS) of SARS-CoV-2 has been performed extensively and is playing a crucial role in fighting against COVID-19 pandemic. Obtaining sufficient WGS data from clinical samples is often challenging especially from the samples with low viral load. We evaluated two SARS-CoV-2 sequencing protocols for their efficiency/accuracy and limitations. Sequence coverage of >95% was obtained by Swift normalase amplicon SARS-CoV-2 panels (SNAP) protocol for all the samples with Ct ≤ 35 and by COVIDSeq protocol for 97% of samples with Ct ≤ 30. Sample RNA quantitation obtained using digital PCR provided more precise cutoff values. The quantitative digital PCR cutoff values for obtaining 95% coverage are 10.5 copies/μL for SNAP protocol and 147 copies/μL for COVIDSeq protocol. Combining FASTQ files obtained from 2 protocols improved the outcome of sequence analysis by compensating for missing amplicon regions. This process resulted in an increase of sequencing coverage and lineage call precision.

scholarly journals ddPCR: a more sensitive and accurate tool for SARS-CoV-2 detection in low viral load specimens

2020 ◽  
Author(s):  
Tao Suo ◽  
Xinjin Liu ◽  
Jiangpeng Feng ◽  
Ming Guo ◽  
Wenjia Hu ◽  
...  
Keyword(s):  
Viral Load ◽  
Limit Of Detection ◽  
False Negative ◽  
Digital Pcr ◽  
Clinical Samples ◽  
Double Blind Study ◽  
Rt Pcr ◽  
Current Standard ◽  
Double Blind ◽  
Sensitivity Specificity

AbstractReal time fluorescent quantitative PCR (RT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. However, due to the low viral load in patient throats and the limitations of RT-PCR, significant numbers of false negative reports are inevitable, which results in failure to timely diagnose, early treat, cut off transmission, and assess discharge criteria. To improve this situation, an optimized droplet digital PCR (ddPCR) was used for detection of SARS-CoV-2, which showed that the limit of detection of ddPCR is significantly lower than that of RT-PCR. We further explored the feasibility of ddPCR to detect SARS-CoV-2 nucleic acid from 77 clinical throat swab samples, including 63 suspected outpatients with fever and 14 supposed convalescents who were about to discharge after treatment, and compared with RT-PCR in terms of the diagnostic accuracy. In this double-blind study, we tested, surveyed subsequently and statistically analyzed 77 clinical samples. According to our study, 26 samples from COVID-19 patients with RT-PCR negative were detected as positive by ddPCR. No FPRs of RT-PCR and ddPCR were observed. The sensitivity, specificity, PPV, NPV, NLR and accuracy were improved from 40% (95% CI: 27–55%), 100% (95% CI: 54–100%), 100%, 16% (95% CI: 13–19%), 0.6 (95% CI: 0.48–0.75) and 47% (95% CI: 33–60%) for RT-PCR to 94% (95% CI: 83–99%), 100% (95% CI: 48–100%), 100%, 63% (95% CI: 36–83%), 0.06 (95% CI: 0.02–0.18) and 95% (95% CI: 84–99%) for ddPCR, respectively. Moreover, 14 (42.9 %) convalescents still carry detectable SARS-CoV-2 after discharge. Overall, ddPCR shows superiority for clinical diagnosis of SARS-CoV-2 to reduce the false negative reports, which could be a powerful complement to the current standard RT-PCR. It also suggests that the current clinical practice that the convalescent after discharge continues to be quarantined for at least 2 weeks is completely necessary which can prevent potential viral transmission.

scholarly journals Essential properties and pitfalls of colorimetric Reverse Transcription Loop-mediated Isothermal Amplification as a point-of-care test for SARS-CoV-2 diagnosis

2021 ◽  
Vol 27 (1) ◽  
Author(s):  
Bruna de Oliveira Coelho ◽  
Heloisa Bruna Soligo Sanchuki ◽  
Dalila Luciola Zanette ◽  
Jeanine Marie Nardin ◽  
Hugo Manuel Paz Morales ◽  
...  
Keyword(s):  
Viral Load ◽  
Internal Control ◽  
Reverse Transcription ◽  
Color Change ◽  
Point Of Care ◽  
Colorimetric Detection ◽  
False Negative ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification

Abstract Background SARS-CoV-2 Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) colorimetric detection is a sensitive and specific point-of-care molecular biology technique used to detect the virus in only 30 min. In this manuscript we have described a few nuances of the technique still not properly described in the literature: the presence of three colors clusters; the correlation of the viral load with the color change; and the importance of using an internal control to avoid false-negative results. Methods To achieve these findings, we performed colorimetric RT-LAMP assays of 466 SARS-CoV-2 RT-qPCR validated clinical samples, with color quantification measured at 434 nm and 560 nm. Results First we determinate a sensitivity of 93.8% and specificity of 90.4%. In addition to the pink (negative) and yellow (positive) produced colors, we report for the first time the presence of an orange color cluster that may lead to wrong diagnosis. We also demonstrated using RT-qPCR and RT-LAMP that low viral loads are related to Ct values > 30, resulting in orange colors. We also demonstrated that the diagnosis of COVID-19 by colorimetric RT-LAMP is efficient until the fifth symptoms day when the viral load is still relatively high. Conclusion This study reports properties and indications for colorimetric RT-LAMP as point-of-care for SARS-CoV-2 diagnostic, reducing false results, interpretations and optimizing molecular diagnostics tests application.

Droplet digital PCR for the simultaneous analysis of minimal residual disease and hematopoietic chimerism after allogeneic cell transplantation

2019 ◽  
Vol 57 (5) ◽  
pp. 641-647 ◽  
Author(s):  
Miguel Waterhouse ◽  
Dietmar Pfeifer ◽  
Jesus Duque-Afonso ◽  
Marie Follo ◽  
Justus Duyster ◽  
...  
Keyword(s):  
Minimal Residual Disease ◽  
Cell Transplantation ◽  
Residual Disease ◽  
Digital Pcr ◽  
Mixed Chimerism ◽  
Clinical Samples ◽  
Molecular Remission ◽  
Remission Status ◽  
Hematopoietic Chimerism ◽  
Minimal Residual

Abstract Background Minimal residual disease (MRD) and hematopoietic chimerism testing influences clinical decision and therapeutic intervention in patients after allogeneic stem cell transplantation (HSCT). However, treatment approaches to induce complete donor chimerism and MRD negativity can lead to complications such as graft-versus-host disease (GvHD) and marrow aplasia. Therefore, there is a need for comprehensive characterization of the molecular remission status after transplantation. Methods We analyzed 764 samples from 70 patients after HSCT for the simultaneous measurement of chimerism and molecular targets used for MRD testing with a digital PCR (dPCR) platform. Results Mixed chimerism (MC) was detected in 219 samples from 37 patients. The mean percentage of host derived DNA in these clinical samples was 4.3%. Molecular relapse with a positive MRD marker and/or increased WT1 expression was observed in 15 patients. In addition to WT1 overexpression, other MRD positive markers were: NPM1 (Type A, B, K), DNMT3A (R882H), MLL-PTD, IDH1 (R132H) and KRAS (G12S). Increasing MC was observed in 15 patients. This group of patients showed either a positive MRD marker, increased WT1 expression or both. Next, we analyzed whether MC or the molecular target for MRD was first detected. MC and MRD marker positivity in this group was first detected in six and two patients, respectively. In the remaining seven patients MC and MRD positivity was detected simultaneously. Conclusions The combination of MRD and chimerism markers in a dPCR platform represents a practical, sensitive and accurate diagnostic tool for the comprehensive assessment of the molecular remission status of patients undergoing HSCT.

scholarly journals Quantification of SARS-CoV-2 viral copy number in saliva mouthwash samples using digital droplet PCR

2020 ◽  
Author(s):  
Lisa Oberding ◽  
Jia Hu ◽  
Byron Berenger ◽  
Abu Naser Mohon ◽  
Dylan R. Pillai
Keyword(s):  
Viral Load ◽  
Copy Number ◽  
Clinical Samples ◽  
Digital Droplet Pcr ◽  
Viral Copy Number ◽  
Droplet Pcr ◽  
Precise Quantification ◽  
Viral Copy ◽  
Mouth Wash

AbstractSaliva samples were collected through a simple mouth wash procedure and viral load quantified using a technology called digital droplet PCR. Data suggest ddPCR allows for precise quantification of viral load in clinical samples infected with SARS-CoV-2.

Detecting SARS-CoV-2 by Realtime RT-PCR in clinical samples collected in the North of Vietnam

2021 ◽  
Vol 30 (9) ◽  
pp. 11-17
Author(s):  
Hoang Vu Mai Phuong ◽  
Ung Thi Hong Trang ◽  
Nguyen Vu Son ◽  
Le Thi Thanh ◽  
Nguyen Le Khanh Hang ◽  
...  
Keyword(s):  
Viral Load ◽  
Average Rate ◽  
Clinical Samples ◽  
Viet Nam ◽  
Rt Pcr ◽  
Ct Value ◽  
The North ◽  
Viral Load Data ◽  
The Mean ◽  
High Level

From January to August 2020, Northern Viet Nam faced a COVID-19 outbreak, up to September 2020, there were 1122 confrmed cases of SARS-CoV-2, of which 465 cases were imported from Europe, America and Asia, 657 cases were identifed domestically. A total of 30,686 samples were collected during the SARS-CoV-2 outbreak in Northern Viet Nam and examined by Real-time RT-PCR using primers and probe from Charite - Berlin protocol. This study showed the initial results of SARS-CoV-2 detection and RNA quantitative in positive samples. The positive rate was 0.8%, ranging from 0.4 to 3.5% according to collection sites. Out of 251 positive samples, the mean Ct value was 28 (IQR: 22.3-32; range 14 - 38). The positive samples had a Ct value below 30 was 68.5%, there was no signifcant difference between the Ct value of the group ≤ 30 and > 30. The mean of the RNA copies/µl was 8.4.107, (IQR: 2.29.106 - 1.83.109 RNA copies/µl, range: 1.95.103 – 4.95.1011). In the group of imported COVID-19 cases, the rate of virus at low level was 29%, an average was 56% and at high level was 15%. In the community groups, the viral load data showed that the average rate at low, intermediate and high level were 20%, 63% and 17% respectively. The proportion of high-level viral load may raise an alert to start the quarantine process to reduce the transmission of SARS-CoV-2

scholarly journals Sensitivity and specificity of metatranscriptomics as an arbovirus surveillance tool

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Jana Batovska ◽  
Peter T. Mee ◽  
Stacey E. Lynch ◽  
Tim I. Sawbridge ◽  
Brendan C. Rodoni
Keyword(s):  
Viral Load ◽  
Sensitivity And Specificity ◽  
High Specificity ◽  
Digital Pcr ◽  
Infected Mosquito ◽  
Clear Understanding ◽  
Acid Extraction ◽  
Analytical Sensitivity ◽  
Illumina Hiseq ◽  
Arbovirus Surveillance

AbstractThe ability to identify all the viruses within a sample makes metatranscriptomic sequencing an attractive tool to screen mosquitoes for arboviruses. Practical application of this technique, however, requires a clear understanding of its analytical sensitivity and specificity. To assess this, five dilutions (1:1, 1:20, 1:400, 1:8,000 and 1:160,000) of Ross River virus (RRV) and Umatilla virus (UMAV) isolates were spiked into subsamples of a pool of 100 Culex australicus mosquitoes. The 1:1 dilution represented the viral load of one RRV-infected mosquito in a pool of 100 mosquitoes. The subsamples underwent nucleic acid extraction, mosquito-specific ribosomal RNA depletion, and Illumina HiSeq sequencing. The viral load of the subsamples was also measured using reverse transcription droplet digital PCR (RT-ddPCR) and quantitative PCR (RT-qPCR). Metatranscriptomic sequencing detected both RRV and UMAV in the 1:1, 1:20 and 1:400 subsamples. A high specificity was achieved, with 100% of RRV and 99.6% of UMAV assembled contigs correctly identified. Metatranscriptomic sequencing was not as sensitive as RT-qPCR or RT-ddPCR; however, it recovered whole genome information and detected 19 other viruses, including four first detections for Australia. These findings will assist arbovirus surveillance programs in utilising metatranscriptomics in routine surveillance activities to enhance arbovirus detection.

A novel duplex ddPCR assay for the diagnosis of schistosomiasis japonica: proof of concept in an experimental mouse model

Parasitology ◽  
2017 ◽  
Vol 144 (8) ◽  
pp. 1005-1015 ◽  
Author(s):  
KOSALA G. WEERAKOON ◽  
CATHERINE A. GORDON ◽  
PENGFEI CAI ◽  
GEOFFREY N. GOBERT ◽  
MARY DUKE ◽  
...  
Keyword(s):  
Mouse Model ◽  
Large Scale ◽  
Public Health Problem ◽  
Digital Pcr ◽  
Disease Diagnosis ◽  
World Health ◽  
Schistosomiasis Japonica ◽  
Detection Methods ◽  
Clinical Samples ◽  
Additional Tool

SUMMARYThe current World Health Organization strategic plan targets the elimination of schistosomiasis as a public health problem by 2025 and accurate diagnostics will play a pivotal role in achieving this goal. DNA-based detection methods provide a viable alternative to some of the commonly used tests, notably microscopy and serology, for the diagnosis of schistosomiasis. The detection of parasite cell-free DNA in different clinical samples is a recent valuable advance, which provides significant benefits for accurate disease diagnosis. Here we validated a novel duplex droplet digital PCR assay for the diagnosis of Chinese (SjC) and Philippine (SjP) strains of Schistosoma japonicum infection in a mouse model. The assay proved applicable for both SjC and SjP infections and capable of detecting infection at a very early intra-mammalian stage in conveniently obtainable samples (urine and saliva) as well as in serum and feces. The target DNA copy numbers obtained in the assay showed a positive correlation with the infection burden assessed by direct traditional parasitology. The potential to detect parasite DNA in urine and saliva has important practical implications for large-scale epidemiological screening programmes in the future, particularly in terms of logistical convenience, and the assay has the potential to be a valuable additional tool for the diagnosis of schistosomiasis japonica.

scholarly journals Comparative Performance of Reagents and Platforms for Quantitation of Cytomegalovirus DNA by Digital PCR

2016 ◽  
Vol 54 (10) ◽  
pp. 2602-2608 ◽  
Author(s):  
R. T. Hayden ◽  
Z. Gu ◽  
S. S. Sam ◽  
Y. Sun ◽  
L. Tang ◽  
...  
Keyword(s):  
Viral Load ◽  
Digital Pcr ◽  
Pairwise Comparisons ◽  
Comparative Performance ◽  
Standard Testing ◽  
Viral Loads ◽  
Quantitative Correlation ◽  
Quantitative Accuracy ◽  
Quantitative Results ◽  
High Degree

A potential benefit of digital PCR is a reduction in result variability across assays and platforms. Three sets of PCR reagents were tested on two digital PCR systems (Bio-Rad and RainDance), using three different sets of PCR reagents for quantitation of cytomegalovirus (CMV). Both commercial quantitative viral standards and 16 patient samples (n= 16) were tested. Quantitative accuracy (compared to nominal values) and variability were determined based on viral standard testing results. Quantitative correlation and variability were assessed with pairwise comparisons across all reagent-platform combinations for clinical plasma sample results. The three reagent sets, when used to assay quantitative standards on the Bio-Rad system, all showed a high degree of accuracy, low variability, and close agreement with one another. When used on the RainDance system, one of the three reagent sets appeared to have a much better correlation to nominal values than did the other two. Quantitative results for patient samples showed good correlation in most pairwise comparisons, with some showing poorer correlations when testing samples with low viral loads. Digital PCR is a robust method for measuring CMV viral load. Some degree of result variation may be seen, depending on platform and reagents used; this variation appears to be greater in samples with low viral load values.

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