Digital Droplet PCR (ddPCR) for Detection and Quantitation of Hepatitis Delt Virus

2021 ◽  
Author(s):  
Ling Xu ◽  
Xiangying Zhang ◽  
Yaling Cao ◽  
Zihao Fan ◽  
Yuan Tian ◽  
...  
Keyword(s):  
Liver Failure ◽  
Lower Limit ◽  
Limit Of Detection ◽  
Quantitative Detection ◽  
Rt Pcr ◽  
Rna Detection ◽  
Detection Rates ◽  
Digital Droplet Pcr ◽  
Droplet Pcr ◽  
Hdv Infection

Abstract Background & Aims The prevalence of hepatitis delt virus (HDV) far exceeds our expected level, there remains a lack of reliable quantitative assays for HDV RNA detection. We sought to develop a new method based on digital droplet PCR (ddPCR) for HDV RNA quantitative detection. Methods With plasmid (pMD19T) containing HDV full-genome, we determined the method for ddPCR-based HDV RNA quantification. To compare various assays for HDV detection, 30 cases diagnosed hepatitis D and 14 controls were examined by ELISA, RT-PCR and ddPCR. 728 HBV-related patients including 182 chronic hepatitis B (CHB), 182 liver cirrhosis (LC), 182 hepatocellular carcinoma (HCC) and 182 liver failure (LF) were screened for HDV infection. Results The limit of detection of ddPCR for HDV is significantly low, which lower limit of detection (LLoD) and lower limit of quantitation (LLoQ) to be 5.51 copies/reaction (95% CI: 1.15–6.4*105) and 0.18 copies/reaction (95% CI: 0.0012151- 0.76436), respectively. Among the 44 samples, ELISA detected 30 cases positive for anti-HDV, ddPCR reported 24 samples and RT-PCR reported 10 samples positive for HDV RNA. Moreover, the positive rates of anti-HDV IgG were 1.1%, 3.3%, 2.7% and 7.1% in patients with CHB, LC, HCC, and LF; the detection rates of RT-PCR in HDV RNA were 0%, 16.67%, 15.4% and 20%, however, the detection rates of ddPCR were 0%, 33.33%, 30.77% and 60%. Conclusion We establish a high sensitivity and high specificity quantitative HDV RNA detection method based on ddPCR compared to RT-PCR. HBV-related end-stage liver disease, especially liver failure, are associated with a remarkably high rate of HDV infection.

scholarly journals Microfluidic Nano-Scale qPCR Enables Ultra-Sensitive and Quantitative Detection of SARS-CoV-2

Processes ◽  
2020 ◽  
Vol 8 (11) ◽  
pp. 1425
Author(s):  
Xin Xie ◽  
Tamara Gjorgjieva ◽  
Zaynoun Attieh ◽  
Mame Massar Dieng ◽  
Marc Arnoux ◽  
...  
Keyword(s):  
Viral Infections ◽  
Limit Of Detection ◽  
False Negative ◽  
False Negative Rate ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
Rt Pcr ◽  
Viral Loads ◽  
Nano Scale ◽  
Negative Rate

A major challenge in controlling the COVID-19 pandemic is the high false-negative rate of the commonly used RT-PCR methods for SARS-CoV-2 detection in clinical samples. Accurate detection is particularly challenging in samples with low viral loads that are below the limit of detection (LoD) of standard one- or two-step RT-PCR methods. In this study, we implemented a three-step approach for SARS-CoV-2 detection and quantification that employs reverse transcription, targeted cDNA preamplification, and nano-scale qPCR based on a commercially available microfluidic chip. Using SARS-CoV-2 synthetic RNA and plasmid controls, we demonstrate that the addition of a preamplification step enhances the LoD of this microfluidic RT-qPCR by 1000-fold, enabling detection below 1 copy/µL. We applied this method to analyze 182 clinical NP swab samples previously diagnosed using a standard RT-qPCR protocol (91 positive, 91 negative) and demonstrate reproducible and quantitative detection of SARS-CoV-2 over five orders of magnitude (<1 to 106 viral copies/µL). Crucially, we detect SARS-CoV-2 with relatively low viral load estimates (<1 to 40 viral copies/µL) in 17 samples with negative clinical diagnosis, indicating a potential false-negative rate of 18.7% by clinical diagnostic procedures. In summary, this three-step nano-scale RT-qPCR method can robustly detect SARS-CoV-2 in samples with relatively low viral loads (<1 viral copy/µL) and has the potential to reduce the false-negative rate of standard RT-PCR-based diagnostic tests for SARS-CoV-2 and other viral infections.

scholarly journals Comparison of SARS-CoV2 N gene real-time RT-PCR targets and commercially available mastermixes

2020 ◽  
Author(s):  
Julianne R Brown ◽  
Denise O’Sullivan ◽  
Rui PA Pereira ◽  
Alexandra S Whale ◽  
Eloise Busby ◽  
...  
Keyword(s):  
Real Time ◽  
Lower Limit ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
N Gene ◽  
Rt Pcr ◽  
One Step ◽  
Improved Performance ◽  
Rna Transcript ◽  
Nose And Throat

ABSTRACTWe aim to test four one-step RT real-time mastermix options for use in SARS-CoV2 real-time PCR, with three primer/probe assays targeting the N gene. The lower limit of detection is determined using a SARS CoV2 N gene RNA transcript dilution series (to 1 copy/µl) and verified using 74 nose and throat swabs.The N2 assay demonstrates the most sensitive detection of SARS-Cov-2 RNA. Three of the four mastermixes performed well, with the Takara One Step PrimeScript™ III RT-PCR Kit mastermix demonstrating improved performance at the lower limit of detection.

scholarly journals Establishment of a quantitative RT-PCR detection of SARS-CoV-2 virus

2021 ◽  
Vol 26 (1) ◽  
Author(s):  
Yushen Jiang ◽  
Shanming Zhang ◽  
Hong Qin ◽  
Shuai Meng ◽  
Xuyi Deng ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Virus Detection ◽  
Quantitative Detection ◽  
Envelope Gene ◽  
Rt Pcr ◽  
Limit Of Quantification ◽  
Nucleocapsid Gene ◽  
E Gene ◽  
Standard Curve ◽  
Roche Diagnostics

Abstract Background The outbreak of novel coronavirus disease 2019 (COVID-19) has become a public health emergency of international concern. Quantitative testing of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) virus is demanded in evaluating the efficacy of antiviral drugs and vaccines and RT-PCR can be widely deployed in the clinical assay of viral loads. Here, we developed a quantitative RT-PCR method for SARS-CoV-2 virus detection in this study. Methods RT-PCR kits targeting E (envelope) gene, N (nucleocapsid) gene and RdRP (RNA-dependent RNA polymerase) gene of SARS-CoV-2 from Roche Diagnostics were evaluated and E gene kit was employed for quantitative detection of COVID-19 virus using Cobas Z480. Viral load was calculated according to the standard curve established by series dilution of an E-gene RNA standard provided by Tib-Molbiol (a division of Roche Diagnostics). Assay performance was evaluated. Results The performance of the assay is acceptable with limit of detection (LOD) below 10E1 copies/μL and lower limit of quantification (LLOQ) as 10E2 copies/μL. Conclusion A quantitative detection of the COVID-19 virus based on RT-PCR was established.

scholarly journals Teschoviruses as Indicators of Porcine Fecal Contamination of Surface Water

2003 ◽  
Vol 69 (10) ◽  
pp. 6311-6315 ◽  
Author(s):  
Miguel Angel Jiménez-Clavero ◽  
Carlos Fernández ◽  
José Antonio Ortiz ◽  
Javier Pro ◽  
Gregoria Carbonell ◽  
...  
Keyword(s):  
Water Contamination ◽  
Animal Species ◽  
Detection Method ◽  
Quantitative Detection ◽  
Pig Slurry ◽  
Rt Pcr ◽  
Rna Detection ◽  
Detection Assay ◽  
Pcr Method ◽  
Porcine Origin

ABSTRACT Teschoviruses specifically infect pigs and are shed in pig feces. Hence, their presence in water should indicate contamination with pig fecal residues. To assess this hypothesis, we have developed a real-time reverse transcriptase PCR (RT-PCR) method that allows the quantitative detection of pig teschovirus (PTV) RNA. The method is able to detect 92 fg of PTV RNA per ml of sample. Using this method, we have detected the presence of PTV RNA in water and fecal samples from all pig farms examined (n = 5). Feces from other animal species (cattle, sheep, and goats) were negative in this test. To compare the PTV RNA detection method with conventional chemical determinations currently in use for evaluation of water contamination, we analyzed water samples collected downstream from a pig slurry spillage site. We have found a positive correlation within both types of determinations. The sensitivity of the PTV detection assay was similar to that achieved by unspecific organic matter determination and superior to all other conventional chemical analyses performed. Furthermore, the new method is highly specific, revealing the porcine origin of the contamination, a feature that is lacking in currently available methods for the assessment of water contamination.

scholarly journals Use of dual priming oligonucleotide system-based multiplex RT-PCR assay to detect five diarrhea viruses in pig herds in South China

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Guangbin Si ◽  
Jiawei Niu ◽  
Xia Zhou ◽  
Yongsheng Xie ◽  
Zhifei Chen ◽  
...  
Keyword(s):  
Detection Efficiency ◽  
Limit Of Detection ◽  
Transmissible Gastroenteritis Virus ◽  
Rt Pcr ◽  
Simple Method ◽  
Detection Rates ◽  
Rotavirus A ◽  
Diarrhea Virus ◽  
Dual Priming Oligonucleotide ◽  
Pcr Method

AbstractIn this study, a specific and simple method based on the dual priming oligonucleotide (DPO) system was developed to simultaneously detect transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine rotavirus A (PRV-A), porcine delta coronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV), associated with the major enteric RNA viruses in pigs. The DPO system-based multiplex RT-PCR method simplified the primer design and did not require optimization of the annealing temperature. Specificity analysis revealed that the method could specifically detect TGEV, PEDV, PRV-A, PDCoV, and SADS-CoV without any cross-amplification of other circulating swine viruses. The limit of detection of the method was as low as 103–104 copies/μL plasmid of each virus. The method also had good repeatability, and obvious results were seen in three repeat experiments with an interval of 45 days. This optimized multiplex RT-PCR method was used to evaluate 181 clinical swine samples that were collected from four provinces of China between September 2016 and August 2018. The results showed that the positive detection rates of PEDV, PDCoV, SADS-CoV, PRV-A, and TGEV were 30.94% (56/181), 17.67% (32/181), 11.6% (21/181), 9.39% (17/181), and 0.55% (1/181), respectively. Mixed infection of two or more viruses was also common. The DPO system-based multiplex RT-PCR could be a useful tool for detecting enteric virus infections. This method has the advantages of easy operation, low cost, high detection efficiency, and short running time for early diagnosis in clinical cases.

scholarly journals Optimized qRT-PCR Approach for the Detection of Intra- and Extra-Cellular SARS-CoV-2 RNAs

2020 ◽  
Vol 21 (12) ◽  
pp. 4396 ◽  
Author(s):  
Tuna Toptan ◽  
Sebastian Hoehl ◽  
Sandra Westhaus ◽  
Denisa Bojkova ◽  
Annemarie Berger ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Low Cost ◽  
World Health ◽  
Quantitative Detection ◽  
Rt Pcr ◽  
M Gene ◽  
Control And Prevention ◽  
Pcr Assays ◽  
Novel Coronavirus ◽  
Health Organization

The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. Meanwhile, increased demand for testing has led to a shortage of reagents and supplies and compromised the performance of diagnostic laboratories in many countries. Both the World Health Organization (WHO) and the Center for Disease Control and Prevention (CDC) recommend multi-step RT-PCR assays using multiple primer and probe pairs, which might complicate the interpretation of the test results, especially for borderline cases. In this study, we describe an alternative RT-PCR approach for the detection of SARS-CoV-2 RNA that can be used for the probe-based detection of clinical isolates in diagnostics as well as in research labs using a low-cost SYBR green method. For the evaluation, we used samples from patients with confirmed SARS-CoV-2 infections and performed RT-PCR assays along with successive dilutions of RNA standards to determine the limit of detection. We identified an M-gene binding primer and probe pair highly suitable for the quantitative detection of SARS-CoV-2 RNA for diagnostic and research purposes.

scholarly journals Performance Decay of Molecular Assays Near the Limit of Detection: Probabilistic Modeling using Real-World COVID-19 Data

2021 ◽  
Author(s):  
Thomas J.S. Durant ◽  
Christopher D. Koch ◽  
Christopher A. Kerantzas ◽  
David R. Peaper
Keyword(s):  
Real World ◽  
Limit Of Detection ◽  
Bootstrap Resampling ◽  
Rt Pcr ◽  
Detection Rates ◽  
Viral Burden ◽  
Assay Performance ◽  
Source Data ◽  
The Impact ◽  
Assay Detection

ABSTRACTThe gold standard for diagnosis of COVID-19 is detection of SARS-CoV-2 RNA by RT-PCR. However, the effect of systematic changes in specimen viral burden on the overall assay performance is not quantitatively described. We observed decreased viral burdens in our testing population as the pandemic progressed, with median sample Ct values increasing from 22.7 to 32.8 from weeks 14 and 20, respectively. We developed a method using computer simulations to quantify the implications of variable SARS-CoV-2 viral burden on observed assay performance. We found that overall decreasing viral burden can have profound effects on assay detection rates. When real-world Ct values were used as source data in a bootstrap resampling simulation, the sensitivity of the same hypothetical assay decreased from 97.59 (95% CI 97.3-97.9) in week 12, to 74.42 (95% CI 73.9-75) in week 20. Furthermore, simulated assays with a 3-fold or 10-fold reduced sensitivity would both appear to be >95% sensitive early in the pandemic, but sensitivity would fall to 85.55 (95% CI 84.9-86.2) and 74.38 (95% CI 73.6-75.1) later in the pandemic, respectively. Our modeling approach can be used to better quantitate the impact that specimen viral burden may have on the clinical application of tests and specimens.

scholarly journals Assessment of multiplex digital droplet RT-PCR as a diagnostic tool for SARS-CoV-2 detection in nasopharyngeal swabs and saliva samples

2021 ◽  
Author(s):  
Kévin Cassinari ◽  
Elodie Alessandri-Gradt ◽  
Pascal Chambon ◽  
Françoise Charbonnier ◽  
Ségolène Gracias ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Diagnostic Tool ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Digital Droplet Pcr ◽  
Reverse Transcription Quantitative Pcr ◽  
Droplet Pcr ◽  
Diagnosis Method ◽  
Genomic Regions ◽  
Imperfect Sensitivity

Abstract Background Reverse transcription-quantitative PCR on nasopharyngeal swabs is currently the reference COVID-19 diagnosis method but exhibits imperfect sensitivity. Methods We developed a multiplex reverse transcription-digital droplet PCR (RT-ddPCR) assay, targeting six SARS-CoV-2 genomic regions, and evaluated it on nasopharyngeal swabs and saliva samples collected from 130 COVID-19 positive or negative ambulatory individuals, who presented symptoms suggestive of mild or moderate SARS-CoV2 infection. Results For the nasopharyngeal swab samples, the results obtained using the 6-plex RT-ddPCR and RT-qPCR assays were all concordant. The 6-plex RT-ddPCR assay was more sensitive than RT-qPCR (85% versus 62%) on saliva samples from patients with positive nasopharyngeal swabs. Conclusion Multiplex RT-ddPCR represents an alternative and complementary tool for the diagnosis of COVID-19, in particular to control RT-qPCR ambiguous results. It can also be applied to saliva for repetitive sampling and testing individuals for whom nasopharyngeal swabbing is not possible.

scholarly journals Does sampling saliva increase detection of SARS-CoV-2 by RT-PCR? Comparing saliva with oro-nasopharyngeal swabs

2020 ◽  
Author(s):  
Ozlem Akgun Dogan ◽  
Betsi Kose ◽  
Nihat Bugra Agaoglu ◽  
Jale Yildiz ◽  
Gizem Alkurt ◽  
...  
Keyword(s):  
Gold Standard ◽  
Viral Rna ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Standard Samples ◽  
Rna Detection ◽  
Detection Rates ◽  
Gold Standard Method ◽  
Non Invasive ◽  
Isolation Period

The gold standard method in the diagnosis of SARS-CoV-2 infection is the detection of viral RNA in nasopharyngeal sample by RT-PCR. Recently, saliva samples has been suggested as an alternative due to being fast, reliable and non-invasive, rather than nasopharyngeal samples. We compared RT-PCR results in nasopharyngeal, oro-nasopharyngeal and saliva samples of COVID-19 patients. 98 of 200 patients were positive in RT-PCR analysis performed before the hospitalization. In day 0, at least one sample was positive in 67% of 98 patients. Positivity rate was 83% for both oro-nasopharyngeal and nasopharyngeal samples, while it was 63% for saliva samples (p<0.001). On day 5, RT-PCR was performed in 59 patients, 34% had at least one positive result. The positivity rate was 55% for saliva and nasopharyngeal samples, while it was 60% for oro-nasopharyngeal samples. Our study shows that the sampling saliva does not increase the sensitivity of RT-PCR tests at early stages of infection. However, on 5th day, viral RNA detection rates in saliva were similar to nasopharyngeal and oro-nasopharyngeal samples. In conclusion, we suggest that, in patients receiving treatment, virus presence in saliva, in addition to the standard samples, is important to determine the isolation period and to control the transmission.

scholarly journals Optimized qRT-PCR approach for the detection of intra- and extra-cellular SARS-CoV-2 RNAs

2020 ◽  
Author(s):  
Tuna Toptan ◽  
Sebastian Hoehl ◽  
Sandra Westhaus ◽  
Denisa Bojkova ◽  
Annemarie Berger ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Low Cost ◽  
World Health ◽  
Quantitative Detection ◽  
Rt Pcr ◽  
M Gene ◽  
Control And Prevention ◽  
Pcr Assays ◽  
Novel Coronavirus ◽  
Health Organization

AbstractThe novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19 which has become a global concern due to its rapid spread. Meanwhile, increased demand in testing has led to shortage of reagents, supplies, and compromised the performance of diagnostic laboratories in many countries. Both the world health organization (WHO) and the Center for Disease Control and Prevention (CDC) recommend multi-step RT-PCR assays using multiple primer and probe pairs, which might complicate interpretation of the test results especially for borderline cases. In this study, we describe an alternative RT-PCR approach for the detection of SARS-CoV-2 RNA that can be used for the probe-based detection of clinical isolates in the diagnostics as well as in research labs using a low cost SYBR green method. For the evaluation, we used samples from patients with confirmed SARS-CoV-2 infection and performed RT-PCR assays along with successive dilutions of RNA standards to determine the limit of detection. We identified an M-gene binding primer and probe pair highly suitable for quantitative detection of SARS-CoV-2 RNA for diagnostic and research purposes.

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