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 A highly sensitive octopus-like azobenzene fluorescent probe for determination of abamectin B1 in apples

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Zhenlong Guo ◽  
YiFei Su ◽  
Kexin Li ◽  
MengYi Tang ◽  
Qiang Li ◽  
...  
Keyword(s):  
Fluorescent Probe ◽  
Limit Of Detection ◽  
Quantitative Detection ◽  
Fluorescence Signal ◽  
Ionization Mass ◽  
Limit Of Quantification ◽  
Visible Absorption ◽  
Esi Ms ◽  
Relative Standard ◽  
Ft Ir

AbstractThe development of detecting residual level of abamectin B1 in apples is of great importance to public health. Herein, we synthesized a octopus-like azobenzene fluorescent probe 1,3,5-tris (5′-[(E)-(p-phenoxyazo) diazenyl)] benzene-1,3-dicarboxylic acid) benzene (TPB) for preliminary detection of abamectin B1 in apples. The TPB molecule has been characterized by ultraviolet–visible absorption spectrometry, 1H-nuclear magnetic resonance, fourier-transform infrared (FT-IR), electrospray ionization mass spectroscopy (ESI-MS) and fluorescent spectra. A proper determination condition was optimized, with limit of detection and limit of quantification of 1.3 µg L−1 and 4.4 μg L−1, respectively. The mechanism of this probe to identify abamectin B1 was illustrated in terms of undergoing aromatic nucleophilic substitution, by comparing fluorescence changes, FT-IR and ESI-MS. Furthermore, a facile quantitative detection of the residual abamectin B1 in apples was achieved. Good reproducibility was present based on relative standard deviation of 2.2%. Six carboxyl recognition sites, three azo groups and unique fluorescence signal towards abamectin B1 of this fluorescent probe demonstrated reasonable sensitivity, specificity and selectivity. The results indicate that the octopus-like azobenzene fluorescent probe can be expected to be reliable for evaluating abamectin B1 in agricultural foods.

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 359. Clinical implications of SARS-CoV-2 PCR presumptive positive results

2021 ◽  
Vol 8 (Supplement_1) ◽  
pp. S283-S283
Author(s):  
Takeru Yamamoto ◽  
Masako Mizusawa
Keyword(s):  
Target Genes ◽  
Limit Of Detection ◽  
Medical Center ◽  
Laboratory Data ◽  
Rt Pcr ◽  
Gene Target ◽  
Specific Target ◽  
E Gene ◽  
Stable Conditions ◽  
Positive Results

Abstract Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse transcriptase (RT) polymerase chain reaction (PCR) testing is a cornerstone of coronavirus disease 2019 (COVID-19) diagnostics. A number of RT-PCR tests are currently available with different combinations of target genes including the pan-Sarbecovirus E gene target. When the E gene target is positive and the SARS-CoV-2 specific target is negative, the result is reported as presumptive positive which may indicate 1) a sample at concentrations near or below the limit of detection, 2) a mutation in the SARS-CoV-2 specific target, 3) infection with some other Sarbecovirus or 4) other factors. However, what the presumptive positive results mean clinically and whether they should be treated in the same way as the positive results are unknown. Methods We conducted a retrospective review of electronic health records for patients who had the presumptive positive results by Xpert Xpress SARS-CoV-2 (Cepheid, Sunnyvale, CA) or Cobas SARS-CoV-2 (Roche Molecular Systems, Branchburg, NJ) at Truman Medical Center from April 13, 2020 to December 31, 2021. Demographics, comorbidities, symptoms, laboratory data, radiographic data, clinical course and COVID-19 related complications were recorded and analyzed. Results During the study period, 85,267 SARS-CoV-2 RT-PCR tests were performed and 253 (0.3%) presumptive positive results were reported for 243 patients. Symptom information were available for 178 patients and 70% of them were symptomatic at the time of testing. Only 2 patients were admitted for COVID-19 pneumonia with the presumptive positive results. Both of them had low oxygen requirement during hospitalization and were discharged with stable conditions. Conclusion Symptomatic COVID-19 patients who presented with presumptive positive results by Xpert Xpress SARS-CoV-2 or Cobas SARS-CoV-2 had generally mild disease and rarely required hospitalization for COVID-19. Disclosures All Authors: No reported disclosures

scholarly journals Rapid Adaptation and Continuous Performance Evaluation of SARS-CoV-2 Envelope Gene (E-gene) Real-Time RT-PCR Assays to Support the Hospital Surge in Test Demand

2020 ◽  
Author(s):  
Yolanda Iok Ieng Ho ◽  
Ann Han Wong ◽  
Eddie Chi Man Leung ◽  
River Chun Wai Wong ◽  
Raymond Wai Man Lai
Keyword(s):  
Real Time ◽  
Tertiary Hospital ◽  
Envelope Gene ◽  
Rt Pcr ◽  
Continuous Training ◽  
E Gene ◽  
Gene Assay ◽  
Test Platform ◽  
Time Analyzer ◽  
One Step

Abstract We describe timely adaption of both published WHO E-gene protocol and commercially available LightMix Modular E-gene assay to the test platform (ABI 7900 Fast real-time analyzer and TaqMan Fast One-step Virus Master Mix) available in an accredited tertiary hospital laboratory with on-going evaluation to ensure provision of quality service within time constraint. The LightMix Modular E-gene was slightly more sensitive when compared to the WHO E-gene, both analytically and diagnostically. The assay was recommended for screening of SARS-CoV-2 infection. With the availability of technically competent staff through continuous training, provision of round the clock service is feasible despite the test is of high complexity. The shorten turn-around-time of about 4 hours per batch could support surges in testing demand, which is essential to control the current SARS-CoV-2 pandemic, to prevent potential overwhelming of the healthcare system and to optimize utilization of the isolation beds.

scholarly journals Development of a Sensitive Real-Time Fast-qPCR Based on SYBR® Green for Detection and Quantification of Chicken Parvovirus (ChPV)

2018 ◽  
Vol 5 (3) ◽  
pp. 69 ◽  
Author(s):  
Luis Nuñez ◽  
Silvana Santander-Parra ◽  
Lucas Chaible ◽  
David De la Torre ◽  
Marcos Buim ◽  
...  
Keyword(s):  
Real Time ◽  
Viral Infections ◽  
Limit Of Detection ◽  
Melting Curve ◽  
Clinical Signs ◽  
Limit Of Quantification ◽  
Viral Genomes ◽  
Standard Curve ◽  
Ns Gene ◽  
Detection And Quantification

Many viruses have been associated with runting and stunting syndrome (RSS). These viral infections mainly affect young chickens, causing apathy, depression, ruffled feathers, cloacal pasting, and diarrhea. Chicken Parvovirus (ChPV) is such an infection and has been detected in chickens showing signs of enteric diseases worldwide. Therefore, the present study aims to develop a sensitive real-time fast-qPCR assay based on SYBR® Green for detection and quantification of ChPV. A 561-bp non-structural (NS) gene was amplified and cloned, and a pair of primers was designed based on conserved nucleotide sequences on the NS gene of ChPV, the intercalating DNA reagent SYBR® Green was employed, and the Fast mode of a thermocycler was used. The assay detects 109 to 101 copies of the genome (CG). The limit of detection (LoD) was estimated to five CG, and the limit of quantification (LoQ) was estimated at ten CG. The standard curve efficiency was 101.94%, and the melting curve showed a unique clean peak and a melting temperature of 79.3 °C. The assay was specific to amplify the ChPV NS gene, and no amplification was shown from other viral genomes or in the negative controls. A total of 141 samples were tested using the assay, of which 139 samples were found positive. The highest CG value of ChPV was 5.7 × 106 CG/uL of DNA without apparent clinical signs of enteric disturbance, and 4.6 × 106 CG/uL DNA were detected in chickens with RSS.

A highly sensitive octopus-like azobenzene fluorescent probe for determination of abamectin B1 in apples

2020 ◽  
Author(s):  
Zhenlong Guo ◽  
YiFei Su ◽  
Kexin Li ◽  
MengYi Tang ◽  
Qiang Li ◽  
...  
Keyword(s):  
Fluorescent Probe ◽  
Limit Of Detection ◽  
Quantitative Detection ◽  
Fluorescence Signal ◽  
Ionization Mass ◽  
Limit Of Quantification ◽  
Visible Absorption ◽  
Esi Ms ◽  
Relative Standard ◽  
Ft Ir

Abstract The development of detecting residual level of abamectin B1 in apples is of great importance to public health. Herein, we synthesized a octopus-like azobenzene fluorescent probe 1,3,5-tris (5'-[(E)-(p-phenoxyazo) diazenyl)] benzene-1,3-dicarboxylic acid) benzene (TPB) for preliminary detection of abamectin B1 in apples. The TPB molecule was characterized by ultraviolet-visible absorption spectrometry, 1H-nuclear magnetic resonance, fourier-transform infrared (FT-IR), electrospray ionization mass spectroscopy (ESI-MS) and fluorescent spectrum. A proper determination condition was optimized, with limit of detection and limit of quantification of 1.3 µg L-1 and 4.4 μg L-1, respectively. The mechanism of this probe to identify abamectin B1 was illustrated in terms of undergoing aromatic nucleophilic substitution, by comparing fluorescence changes, FT-IR and ESI-MS. Furthermore, a facile quantitative detection of the residual abamectin B1 in apples was achieved. Good reproducibility was shown based on relative standard deviation of 2.20%. Six carboxyl recognition sites, three azo groups and unique fluorescence signal towards abamectin B1 of this fluorescent probe decided ideal sensitivity, specificity and selectivity. The results show that the octopus-like azobenzene fluorescent probe may be promising for evaluating abamectin B1 in agricultural foods.

scholarly journals 1773. Development and Evaluation of an Automated Adenovirus Quantitative Assay Using the Luminex ARIES® System

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S653-S654
Author(s):  
Fann Wu ◽  
Jun Shi ◽  
Shailesh Desai ◽  
Daniel A Green ◽  
Susan Whittier
Keyword(s):  
Limit Of Detection ◽  
Full Range ◽  
Random Access ◽  
Quantitative Assay ◽  
Coefficient Of Determination ◽  
Quantitative Detection ◽  
Plasma Samples ◽  
Limit Of Quantification ◽  
Additional Advantage ◽  
Disseminated Disease

Abstract Background The quantification of circulating human adenovirus (HAdV) DNA is the recommended diagnostic method to predict disseminated disease. Most current HAdV quantitative assays are manually performed and lack the flexibility needed to provide rapid answers when required.The purpose of this study was to evaluate the application of MultiCode® HAdV PCR assay for use with the Luminex ARIES® system (Luminex Corporation, Austin, TX) as an automated and random access test for the quantitative detection of HAdV DNA in plasma. Methods Analytical performance characteristics including assay limit of detection/quantitation (LoD/LoQ), accuracy, and inter-, intra-reproducibility were studied using commercial panels (Exact Diagnostics, Fort Worth, TX). Assay specificity was determined usingHAdV reference strains obtained from the American Type Culture Collection (Manassas, VA) and plasma spiked with related Herpes viruses and pathogens commonly found in the blood. Assay accuracy was verified with analysis of 30 plasma samples spiked with different concentrations of control material that covered the full range of HAdV DNA levels. We also prospectively analyzed 180 plasma samples collected from 102 patients. DNA from all samples were extracted, amplified and detected on a single automated Luminex ARIES® system. Results The assay has a wide linear range from 2.55 to 9.4 log10 HAdV DNA copies/mL (coefficient of determination; R2 = 0.995) with a detection limit of 1.82 log10 (95% positivity rate), and a limit of quantification of 2.55 log10 copies/mL. The assay detected HAdV DNA from Adenovirus groups A-F, although slight shifts in Tm peaks were observed. Inter- and intra-assay reproducibility was evaluated using 6 panels of commercial standards, producing variation coefficients of 5% and 2%, respectively. Assay accuracy results reflected a good correlation with a mean difference of 0.10 log10 copies/mL. The results of the prospective study showed 6/102 (5.8%) patients had HAdV viremia, including 4 (3.9%) patients with a viral load ≥4 log10/mL, which might necessitate therapy. Conclusion The HAdV quantitative assay using the Luminex ARIES® system provides excellent performance for routine testing with the additional advantage of random access capabilities for urgent testing to identify patients at risk for disseminated disease. Disclosures All authors: No reported disclosures.

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 Analysis of the initial lot of the CDC 2019-Novel Coronavirus (2019-nCoV) real-time RT-PCR diagnostic panel

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0260487
Author(s):  
Justin S. Lee ◽  
Jason M. Goldstein ◽  
Jonathan L. Moon ◽  
Owen Herzegh ◽  
Dennis A. Bagarozzi ◽  
...  
Keyword(s):  
Real Time ◽  
False Positive ◽  
Bulk Material ◽  
Virus Detection ◽  
Rt Pcr ◽  
Nucleocapsid Gene ◽  
Positive Reactivity ◽  
Control And Prevention ◽  
Diagnostic Panel ◽  
Novel Coronavirus

At the start of the COVID-19 pandemic, the Centers for Disease Control and Prevention (CDC) designed, manufactured, and distributed the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel for SARS-CoV-2 detection. The diagnostic panel targeted three viral nucleocapsid gene loci (N1, N2, and N3 primers and probes) to maximize sensitivity and to provide redundancy for virus detection if mutations occurred. After the first distribution of the diagnostic panel, state public health laboratories reported fluorescent signal in the absence of viral template (false-positive reactivity) for the N3 component and to a lesser extent for N1. This report describes the findings of an internal investigation conducted by the CDC to identify the cause(s) of the N1 and N3 false-positive reactivity. For N1, results demonstrate that contamination with a synthetic template, that occurred while the “bulk” manufactured materials were located in a research lab for quality assessment, was the cause of false reactivity in the first lot. Base pairing between the 3’ end of the N3 probe and the 3’ end of the N3 reverse primer led to amplification of duplex and larger molecules resulting in false reactivity in the N3 assay component. We conclude that flaws in both assay design and handling of the “bulk” material, caused the problems with the first lot of the 2019-nCoV Real-Time RT-PCR Diagnostic Panel. In addition, within this study, we found that the age of the examined diagnostic panel reagents increases the frequency of false positive results for N3. We discuss these findings in the context of improvements to quality control, quality assurance, and assay validation practices that have since been improved at the CDC.

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