scholarly journals Direct Quantitation of SARS-CoV-2 Using Droplet Digital PCR in Suspected Samples With Very Low Viral Load

2021 ◽  
Author(s):  
Michela Deiana ◽  
Chiara Piubelli ◽  
Antonio Mori ◽  
Gian Paolo Chiecchi ◽  
Giulia La Marca ◽  
...  
Keyword(s):  
Viral Load ◽  
Real Time ◽  
False Negative ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Hospitalized Patients ◽  
Lower Sensitivity ◽  
Rt Pcr ◽  
Viral Genes ◽  
Viral Target

Background: The reference test for SARS-CoV-2 detection is the reverse transcriptase real time PCR (real time RT-PCR). However, evidences reported that real time RT-PCR has a lower sensitivity compared with the droplet digital PCR (ddPCR) leading to possible false negative in low viral load cases. Methods: We used ddPCR for viral genes N1 and N2 on 20 negative (no detection) samples from symptomatic hospitalized COVID-patients presenting fluctuating real time RT-PCR results and 10 suspected samples (Ct value>35) from asymptomatic not hospitalized subjects. Results: ddPCR performed on RNA revealed 65% of positivity for at least one viral target in the hospitalized patients group of samples (35% for N1 and N2, 10% only for N1 and 20% only for N2) and 50% in the suspected cases (30% for N1 and N2, while 20% only for N2). On hospitalized patients’ samples, we applied also a direct ddPCR approach on the swab material, achieving an overall positivity of 83%. Conclusion: ddPCR, in particular the direct quantitation on swabs, shows a sensitivity advantage for the SARS-CoV-2 identification and may be useful to reduce the false negative diagnosis, especially for low viral load suspected samples.

scholarly journals Viral Dynamics and Real-Time RT-PCR Ct Values Correlation with Disease Severity in COVID-19

Diagnostics ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1091
Author(s):  
Ali A. Rabaan ◽  
Raghavendra Tirupathi ◽  
Anupam A Sule ◽  
Jehad Aldali ◽  
Abbas Al Mutair ◽  
...  
Keyword(s):  
Viral Load ◽  
Real Time ◽  
Disease Severity ◽  
False Negative ◽  
Influential Factors ◽  
Confirmatory Test ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Viral Kinetics ◽  
Ct Value

Real-time RT-PCR is considered the gold standard confirmatory test for coronavirus disease 2019 (COVID-19). However, many scientists disagree, and it is essential to understand that several factors and variables can cause a false-negative test. In this context, cycle threshold (Ct) values are being utilized to diagnose or predict SARS-CoV-2 infection. This practice has a significant clinical utility as Ct values can be correlated with the viral load. In addition, Ct values have a strong correlation with multiple haematological and biochemical markers. However, it is essential to consider that Ct values might be affected by pre-analytic, analytic, and post-analytical variables such as collection technique, specimen type, sampling time, viral kinetics, transport and storage conditions, nucleic acid extraction, viral RNA load, primer designing, real-time PCR efficiency, and Ct value determination method. Therefore, understanding the interpretation of Ct values and other influential factors could play a crucial role in interpreting viral load and disease severity. In several clinical studies consisting of small or large sample sizes, several discrepancies exist regarding a significant positive correlation between the Ct value and disease severity in COVID-19. In this context, a revised review of the literature has been conducted to fill the knowledge gaps regarding the correlations between Ct values and severity/fatality rates of patients with COVID-19. Various databases such as PubMed, Science Direct, Medline, Scopus, and Google Scholar were searched up to April 2021 by using keywords including “RT-PCR or viral load”, “SARS-CoV-2 and RT-PCR”, “Ct value and viral load”, “Ct value or COVID-19”. Research articles were extracted and selected independently by the authors and included in the present review based on their relevance to the study. The current narrative review explores the correlation of Ct values with mortality, disease progression, severity, and infectivity. We also discuss the factors that can affect these values, such as collection technique, type of swab, sampling method, etc.

scholarly journals SARS-CoV-2 detection using digital PCR for COVID-19 diagnosis, treatment monitoring and criteria for discharge

2020 ◽  
Author(s):  
Renfei Lu ◽  
Jian Wang ◽  
Min Li ◽  
Yaqi Wang ◽  
Jia Dong ◽  
...  
Keyword(s):  
Viral Load ◽  
Limit Of Detection ◽  
False Negative ◽  
Digital Pcr ◽  
Detection Sensitivity ◽  
Rt Pcr ◽  
Pharyngeal Swab ◽  
Clinical Specimens ◽  
Positive Results ◽  
Negative Detection

SummaryBackgroundSARS-CoV-2 nucleic acid detection by RT-PCR is one of the criteria approved by China FDA for diagnosis of COVID-19. However, inaccurate test results (for example, high false negative rate and some false positive rate) were reported in both China and US CDC using RT-PCR method. Inaccurate results are caused by inadequate detection sensitivity of RT-PCR, low viral load in some patients, difficulty to collect samples from COVID-19 patients, insufficient sample loading during RT-PCR tests, and RNA degradation during sample handling process. False negative detection could subject patients to multiple tests before diagnosis can be made, which burdens health care system. Delayed diagnosis could cause infected patients to miss the best treatment time window. False negative detection could also lead to prematurely releasing infected patients who still carry residual SARS-CoV-2 virus. In this case, these patients could infect many others. A high sensitivity RNA detection method to resolve the existing issues of RT-PCR is in need for more accurate COVID-19 diagnosis.MethodsDigital PCR (dPCR) instrument DropX-2000 and assay kits were used to detect SARS-CoV-2 from 108 clinical specimens from 36 patients including pharyngeal swab, stool and blood from different days during hospitalization. Double-blinded experiment data of 108 clinical specimens by dPCR methods were compared with results from officially approved RT-PCR assay. A total of 109 samples including 108 clinical specimens and 1 negative control sample were tested in this study. All of 109 samples, 26 were from 21patients reported as positive by officially approved clinical RT-PCR detection in local CDC and then hospitalized in Nantong Third Hospital. Among the 109 samples, dPCR detected 30 positive samples on ORFA1ab gene, 47 samples with N gene positive, and 30 samples with double positive on ORFA1ab and N genes.ResultsThe lower limit of detection of the optimize dPCR is at least 10-fold lower than that of RT-PCR. The overall accuracy of dPCR for clinical detection is 96.3%. 4 out 4 of (100 %) negative pharyngeal swab samples checked by RT-PCR were positive judged by dPCR based on the follow-up investigation. 2 of 2 samples in the RT-PCR grey area (Ct value > 37) were confirmed by dPCR with positive results. 1 patient being tested positive by RT-PCR was confirmed to be negative by dPCR. The dPCR results show clear viral loading decrease in 12 patients as treatment proceed, which can be a useful tool for monitoring COVID-19 treatment.ConclusionsDigital PCR shows improved lower limit of detection, sensitivity and accuracy, enabling COVID-19 detection with less false negative and false positive results comparing with RT-PCR, especially for the tests with low viral load specimens. We showed evidences that dPCR is powerful in detecting asymptomatic patients and suspected patients. Digital PCR is capable of checking the negative results caused by insufficient sample loading by quantifying internal reference gene from human RNA in the PCR reactions. Multi-channel fluorescence dPCR system (FAM/HEX/CY5/ROX) is able to detect more target genes in a single multiplex assay, providing quantitative count of viral load in specimens, which is a powerful tool for monitoring COVID-19 treatment.

scholarly journals Droplet Digital PCR – A Superior Complementary Technique for SARS-CoV-2 Detection

2020 ◽  
pp. 12-15
Author(s):  
I. M. Hussaini ◽  
S. Gide ◽  
B. Musa ◽  
M. A. Sulaiman ◽  
A. Usman
Keyword(s):  
Sensitivity And Specificity ◽  
False Negative ◽  
Standard Technique ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Reference Standard ◽  
Rt Pcr ◽  
Negative Results ◽  
False Negative Results ◽  
Current Reference Standard

Accurate and timely SARS-CoV-2 detection in suspected persons is crucial in the fight against its spread. Many techniques have been developed to meet up with the continuously growing demand, however some of these techniques lack the required accuracy, sensitivity and specificity. The current reference standard technique for SARS-CoV-2 detection is RT-PCR, but studies have shown that false-negative results are inevitable and data can be non-reproducible when samples and primers are not appropriately verified and validated. Droplet digital PCR (ddPCR) is a newly introduced technique that performs precise nucleic acid quantification. Researchers have evaluated the efficacy of ddPCR and the technique has shown promising results even in specimens with low viral load. ddPCR has shown increased accuracy, precision, sensitivity and specificity. Furthermore, it is less affected by annealing and amplification inhibitors. This suggests that ddPCR can be used as a complementary detection technique especially in convalescent cases.

scholarly journals Comparison of Real-Time PCR and Droplet Digital PCR for the Determination of JAK2V617F Mutation in Ph'-Negative Myeloproliferative Neoplasms

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5548-5548
Author(s):  
Giulia Fontanelli ◽  
Claudia Baratè ◽  
Elena Ciabatti ◽  
Francesca Guerrini ◽  
Riccardo Morganti ◽  
...  
Keyword(s):  
Real Time ◽  
Standard Method ◽  
Real Time Pcr ◽  
Myeloproliferative Neoplasms ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Jak2v617f Mutation ◽  
Rt Pcr ◽  
Jak2 Mutation ◽  
Allele Burden

Abstract Myeloproliferative neoplasms (MPNs) are a group of clonal diseases associated with JAK2 mutation (V617F) in a percentage varying from 50% (Essential Thrombocytemia ET, and Primary Myelofibrosis PMF) to 95% (Polycitemia Vera, PV). The standard method for determining the V617F mutation is today represented by the Real Time PCR (RT-PCR). However, in recent years some studies have focused attention on a new method, the Droplet Digital PCR (DD-PCR), that allows an absolute quantitation of the mutated allele without any reference curve. The main objective of this study was the comparison of two PCR methods (RT-PCR and DD-PCR) for the analysis of the JAK2V617F mutation in order to assess the sensitivity, specificity and feasibility of the two techniques. In the study 225 patients with MPNs JAK2V617F-positive were enrolled; in 99 cases, DNA was still available for further assays and so DD-PCR tests were performed and compared with the results coming from RT-PCR (MutaQuant kit, Ipsogen, Luminy Biotech, Marseille, France). The results show a good sensitivity for both methods; nevertheless, after appropriate dilution tests, the DD-PCR was able to detect the JAK2V617F mutation in the 5x10-4 dilution versus 5x10-3 for the RT-PCR, in front of a fully comparable specificity. A linear regression analysis showed an absolute correlation between the 2 methods (R2=0.91), and the median mutated allele burden was not different when measured by the DD-PCR or the RT-PCR (p=0.67), with the highest median allele burden observed in secondary myelofibrosis (86% by RT-PCR and 91% by DD-PCR) and the lowest one in ET (23% by RT-PCR and 25% by DD-PCR) (see Figure 1 and Figure 2), as expected. Figure 1 Figure 1. Figure 2 Figure 2. Our study demonstrates that a new molecular technique, the DD-PCR, may represent a new frontier in the world of molecular techniques for the detection of JAK2 mutation with a high sensitivity and specificity. In addition the method allows obtain two types of information, qualitative and quantitative, with one analysis, saving time and at a lower cost than the standard method. Disclosures No relevant conflicts of interest to declare.

scholarly journals Comparison of real-time PCR and droplet digital PCR for the detection of Xylella fastidiosa in plants

2019 ◽  
Author(s):  
Enora Dupas ◽  
Bruno Legendre ◽  
Valérie Olivier ◽  
Françoise Poliakoff ◽  
Charles Manceau ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Xylella Fastidiosa ◽  
False Negative ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Detection Sensitivity ◽  
Plant Sample ◽  
The European Union

AbstractXylella fastidiosa (Xf) is a quarantine plant pathogen bacterium originating from the Americas and that has emerged in Europe in 2013. Xf can be detected directly on plant macerate using molecular methods such as real-time PCR, which is a sensitive technique. However, some plants may contain components that can act as PCR reaction inhibitors, which can lead to false negative results or an underestimation of the bacterial concentration present in the analyzed plant sample. Droplet digital PCR (ddPCR) is an innovative tool based on the partitioning of the PCR reagents and the DNA sample into thousands of droplets, allowing the quantification of the absolute number of target DNA molecules present in a reaction mixture, or an increase of the detection sensitivity. In this study, a real-time PCR protocol, already used for Xf detection in the framework of official surveys in the European Union, was transferred and optimized for Xf detection using ddPCR. This new assay was evaluated and compared to the initial real-time PCR on five plant matrices artificially inoculated and on naturally infected plants. In our conditions, this new ddPCR enabled the detection of Xf on all artificially inoculated plant macerates with a similar limit of detection, or a slight benefit for Quercus ilex. Moreover, ddPCR improved diagnostic sensitivity as it enabled detection of Xf in samples of Polygala myrtifolia or Q. ilex that were categorized as negative or close to the limit of detection using the real-time PCR. Here, we report for the first time a ddPCR assay for the detection of the bacterium Xf.

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.

Detection of SARS-CoV-2 RNA in nasopharyngeal swabs from COVID-19 patients and asymptomatic cases of infection by real-time and digital PCR

2020 ◽  
Vol 65 (12) ◽  
pp. 785-792
Author(s):  
V. A. Ternovoi ◽  
R. Yu. Lutkovsky ◽  
E. P. Ponomareva ◽  
A. V. Gladysheva ◽  
E. V. Chub ◽  
...  
Keyword(s):  
Viral Load ◽  
Real Time ◽  
Real Time Pcr ◽  
Clinical Symptoms ◽  
False Negative ◽  
Digital Pcr ◽  
Viral Rna ◽  
Laboratory Research ◽  
Low Concentration ◽  
Negative Results

In this work we tested two reagent kits developed by us for detecting SARS-CoV-2 RNA using a fragment of the ORF1ab gene in digital PCR and real-time PCR formats. Data were obtained on the detection of SARS-CoV-2 virus RNA in nasopharyngeal swabs of patients with COVID-19 and asymptomatic carriers. The developed reagent kits provided 100% sensitivity and a detection limit of 103 GE / ml for qPCR, and at least 200 copies / ml of viral RNA when performing digital PCR. These methods were tested using a panel of 1,328 samples collected from patients with suspected COVID-19 at the beginning of 2020 in the Russian Federation. It has been shown that dPCR is more sensitive and can be used to analyze samples with low viral load, including those from patients without clinical symptoms. dPCR significantly improves the accuracy of laboratory research and significantly reduces the number of false negative results in the diagnosis of SARS-CoV-2. Determination of the concentration of SARS-CoV-2 RNA in patients with different clinical course of the disease showed that the concentration of viral RNA can sharply decrease in the first days of the disease. A low concentration of viral RNA in samples from patients is also characteristic of asymptomatic disease. Digital PCR provides a higher detection rate for asymptomatic cases, which is approximately 75% of those infected, as opposed to 45% for real-time PCR. The results obtained on the use of the digital PCR method for detecting SARS-CoV-2 RNA showed that this method is especially suitable for detecting RNA in case of its low concentration in contacts, as well as for monitoring changes in viral load in convalescent patients.

Detection of KRAS mutations in colon adenocarcinoma by droplet digital PCR.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e15080-e15080
Author(s):  
Yaroslav S. Enin ◽  
Oleg I. Kit ◽  
Yuriy A. Gevorkyan ◽  
Natalya V. Soldatkina ◽  
Dmitry A. Kharagezov ◽  
...  
Keyword(s):  
Real Time ◽  
Colon Adenocarcinoma ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Analytical Sensitivity ◽  
Rt Pcr ◽  
Kras Gene ◽  
Kras Mutations ◽  
Mutational Status ◽  
Pcr Method

e15080 Background: The screening of mutations in the KRAS gene is a traditional marker of the effectiveness of targeted therapy for colorectal cancer. Mutations in the 12 codon of the second exon of the KRAS gene are present in 40% of colorectal tumors, and monitoring of the mutational status together with the level of mutant DNA is of particular clinical interest. However, the sensitivity level of the traditionally used real-time polymerase chain reaction (RT-PCR) method is insufficient in some cases. Recently, Droplet Digital PCR (DD-PCR) has been considered as an alternative method, which is more sensitive. The purpose of this study was to compare the methods of detection of somatic mutations in the second exon of the KRAS gene using DD-PCR versus RT-PCR. Methods: This study included 134 patients with colon adenocarcinoma. The presence or absence of activating mutations in the second exon of the KRAS gene in samples of FFPE blocks was detected by RT-PCR using the “Real-time-PCR-KRAS-7M reagent kit” (Biolink, Russia) and Digital Droplet PCR using the “KRAS Screening Multiplex kit” (Bio-Rad, USA). Results: For all 134 samples, selected for the study, the WT status of the KRAS gene was identified by the RT-PCR method. According to the results of DD-PCR for 131 samples (96.2%), a positive amplification response of mutant DNA with more than 200 events was obtained. The number of amplicons varied from 312 to 117917 per sample, the median was 2940 copies. According to recent trials, a clinically significant level of mutational events must exceed 5% of the total number of amplicons in a sample. In our study, 12.9% of cases (17 patients) met this criterion. Conclusions: The DD-PCR method demonstrated a much higher analytical sensitivity for the detection of SNP mutations in comparison with RT-PCR that may be of critical importance in the therapeutic decision.

scholarly journals Development and validation of a method for quantification of common wheat, durum wheat, rye and barley by droplet digital PCR

2021 ◽  
Author(s):  
Christian Schulze ◽  
Anne-Catrin Geuthner ◽  
Dietrich Mäde
Keyword(s):  
Durum Wheat ◽  
Real Time ◽  
Bread Wheat ◽  
Common Wheat ◽  
Real Time Pcr ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Food Fraud ◽  
Single Genome ◽  
Cereal Species

AbstractFood fraud is becoming a prominent topic in the food industry. Thus, valid methods for detecting potential adulterations are necessary to identify instances of food fraud in cereal products, a significant component of human diet. In this work, primer–probe systems for real-time PCR and droplet digital PCR (ddPCR) for the detection of these cereal species: bread wheat (together with spelt), durum wheat, rye and barley for real-time PCR and ddPCR were established, optimized and validated. In addition, it was projected to validate a molecular system for differentiation of bread wheat and spelt; however, attempts for molecular differentiation between common wheat and spelt based on the gene GAG56D failed because of the genetic variability of the molecular target. Primer–probe systems were further developed and optimized on the basis of alignments of DNA sequences, as well as already developed PCR systems. The specificity of each system was demonstrated on 10 (spelt), 11 (durum wheat and rye) and 12 (bread wheat) reference samples. Specificity of the barley system was already proved in previous work. The calculated limits of detection (LOD95%) were between 2.43 and 4.07 single genome copies in real-time PCR. Based on the “three droplet rule”, the LOD95% in ddPCR was calculated to be 9.07–13.26 single genome copies. The systems were tested in mixtures of flours (rye and common wheat) and of semolina (durum and common wheat). The methods proved to be robust with regard to the tested conditions in the ddPCR. The developed primer–probe systems for ddPCR proved to be effective in quantitatively detecting the investigated cereal species rye and common wheat in mixtures by taking into account the haploid genome weight and the degree of milling of a flour. This method can correctly detect proportions of 50%, 60% and 90% wholemeal rye flour in a mixture of wholemeal common wheat flour. Quantitative results depend on the DNA content, on ploidy of cereal species and are also influenced by comminution. Hence, the proportion of less processed rye is overestimated in higher processed bread wheat and adulteration of durum wheat by common wheat by 1–5% resulted in underestimation of common wheat.

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