scholarly journals A Novel Multiplex qRT-PCR Assay to Detect SARS-CoV-2 Infection: High Sensitivity and Increased Testing Capacity

2020 ◽  
Vol 8 (7) ◽  
pp. 1064 ◽  
Author(s):  
Sara Petrillo ◽  
Giovanna Carrà ◽  
Paolo Bottino ◽  
Elisa Zanotto ◽  
Maria Chiara De Santis ◽  
...  
Keyword(s):  
Internal Control ◽  
False Negative ◽  
Rna Extraction ◽  
Simultaneous Detection ◽  
High Sensitivity ◽  
N Gene ◽  
Qrt Pcr ◽  
P Gene ◽  
Global Pandemic ◽  
Pcr Method

Rapid and sensitive screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential to limit the spread of the global pandemic we are facing. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is currently used for the clinical diagnosis of SARS-CoV-2 infection using nasopharyngeal swabs, tracheal aspirates, or bronchoalveolar lavage (BAL) samples. Despite the high sensitivity of the qRT-PCR method, false negative outcomes might occur, especially in patients with a low viral load. Here, we developed a multiplex qRT-PCR methodology for the simultaneous detection of SARS-CoV-2 genome (N gene) and of the human RNAse P gene as internal control. We found that multiplex qRT-PCR was effective in detecting SARS-Cov-2 infection in human specimens with 100% sensitivity. Notably, patients with few copies of SARS-CoV-2 RNA (<5 copies/reaction) were successfully detected by the novel multiplex qRT-PCR method. Finally, we assessed the efficacy of multiplex qRT-PCR on human nasopharyngeal swabs without RNA extraction. Collectively, our results provide evidence of a novel and reliable tool for SARS-CoV-2 RNA detection in human specimens, which allows the testing capacity to be expanded and the RNA extraction step to be bypassed.

scholarly journals False-negative Molecular Diagnosis of SARS-CoV-2 in Samples with Amplification Inhibitors

2020 ◽  
Author(s):  
Marcelo Fruehwirth ◽  
Açucena Veleh Rivas ◽  
Andressa Faria Rahyn Fitz ◽  
Aline Cristiane Cechinel Assing Batista ◽  
Cleypson Vinicius Silveira ◽  
...  
Keyword(s):  
Internal Control ◽  
False Negative ◽  
Rna Extraction ◽  
N Gene ◽  
Gold Standard Method ◽  
Negative Results ◽  
False Negative Results ◽  
Average Variation ◽  
Wrong Diagnosis ◽  
Inhibitor Compounds

Although rRT-PCR is the gold standard method for SARS-CoV-2 detection, some factors, such as amplification inhibitors presence, lead to false-negative results. Here we describe differences between rRT-PCR results for SARS-CoV-2 infection in normal and diluted samples, simulating the need for dilution due to amplification inhibitors presence. Viral RNA extraction of nasopharyngeal swabs samples from 20 patients previously detected as 'Negative' and 21 patients detected as 'Positive' for SARS-CoV-2 was realized with the EasyExtract DNA-RNA (Interprise&reg;). rRT-PCR was realized with OneStep/COVID-19 (IBMP) kit with normal and diluted (80&micro;l of H₂O RNAse free) samples, totaling 82 tests. The results indicate that there is an average variation (ɑ &lt; 0.05) delaying Cq between the amplification results of internal control (IC), N Gene (NG), and ORF-1ab (OF) of 1.811 Cq, 3.840 Cq, and 3.842 Cq, respectively. The extraction kit does not completely purify the inhibitor compounds, therefore non-amplification by inhibitors may occur. In this study, we obtained a 19.04% false-negative diagnosis after sample dilution, and this process reduces the efficiency of rRT-PCR to 29.80% for detecting SARS-CoV-2. Knowing the rRT-PCR standards of diluted samples can help in the identification of false-negative cases, and consequently avoid a wrong diagnosis.

scholarly journals False-Negative Molecular Diagnosis of SARS-CoV-2 in Samples with Amplification Inhibitors

2020 ◽  
Author(s):  
Marcelo Fruehwirth ◽  
Açucena Veleh Rivas ◽  
Andressa Faria Rahyn Fitz ◽  
Aline Cristiane Cechinel Assing Batista ◽  
Cleypson Vinicius Silveira ◽  
...  
Keyword(s):  
Internal Control ◽  
False Negative ◽  
Rna Extraction ◽  
N Gene ◽  
Gold Standard Method ◽  
Negative Results ◽  
False Negative Results ◽  
Average Variation ◽  
Wrong Diagnosis ◽  
Inhibitor Compounds

Although rRT-PCR is the gold standard method for SARS-CoV-2 detection, some factors, such as amplification inhibitors presence, lead to false-negative results. Here we describe differences between rRT-PCR results for SARS-CoV-2 infection in normal and diluted samples, simulating the need for dilution due to amplification inhibitors presence. Viral RNA extraction of nasopharyngeal swabs samples from 20 patients previously detected as 'Negative' and 21 patients detected as 'Positive' for SARS-CoV-2 was realized with the EasyExtract DNA-RNA (Interprise&reg;) for extraction. rRT-PCR was realized with OneStep/COVID-19 (IBMP) kit with normal and diluted (80&micro;l of H₂O RNAse free) samples, totaling 82 tests. The results indicate that there is an average variation (ɑ &lt; 0.05) delaying Ct between the amplification results of internal control (IC), N Gene (NG), and ORF-1ab (OF) of 1.811Ct, 3.840Ct, and 3.842Ct, respectively. The extraction kit does not completely purify the inhibitor compounds, therefore non-amplification by inhibitors may occur. In this study, we obtained a 19.04% false-negative diagnosis after sample dilution, and this process reduces the efficiency of rRT-PCR to 29.8% for detecting SARS-CoV-2. Knowing the rRT-PCR standards of diluted samples can help in the identification of false-negative cases, and consequently avoid a wrong diagnosis.

scholarly journals Overhauling a faulty control in the CDC-recommended SARS-CoV-2 RT-PCR test panel

2020 ◽  
Author(s):  
Rob J. Dekker ◽  
Wim A. Ensink ◽  
Selina van Leeuwen ◽  
Han Rauwerda ◽  
Timo M. Breit
Keyword(s):  
False Positive ◽  
False Negative ◽  
Rna Extraction ◽  
N Gene ◽  
Rt Pcr ◽  
P Gene ◽  
Reverse Primer ◽  
Probe Set ◽  
Pcr Test ◽  
Test Control

ABSTRACTTo battle the COVID-19 pandemic, widespread testing for the presence of the SARS-CoV-2 virus is worldwide being employed by specific real-time RT-PCR (rRT-PCR) of viral RNA. The CDC has issued a recommended panel of PCR-based test sets that entail several primer/probe sets that target the SARS-CoV-2 N-gene, but also one that targets the human RNase P gene (h-RP) as a positive control for RNA extraction and/or reverse-transcription (RT) efficacy.We discovered that the CDC-recommended h-RP primer/probe set has a faulty design, because both PCR primers are located in the same exon, which allows for unwanted PCR-amplification of background genomic DNA (gDNA). By removing RNA from nose-swab samples by an RNase treatment, we showed that the presence of gDNA in samples resulted in false-positive signals for the h-RP test control. This is rather serious, because it could lead to false-negative test outcomes, since the CDC interpretation of an absent SARS-CoV-2 rRT-PCR signal plus a positive h-RP rRT-PCR signal is interpreted as “2019-nCoV not detected”, whereas a false-positive h-RP rRT-PCR signal resulting from amplification of gDNA should be interpreted as “Invalid Result” and the procedure should be repeated.In order to overhaul the faulty h-RP rRT-PCR primer/probe set with minimal modification, we designed and tested several new h-RP reverse primers. Replacement of the CDC-recommended PCR reverse primer with our selected exon-exon junction reverse primer corrected the problem of false-positive results with this important SARS-CoV-2 RT-PCR test control and thus eliminated the problem of potential false-negative COVID-19 diagnoses.

scholarly journals Efficient SARS-CoV-2 detection in unextracted oro-nasopharyngeal specimens by rRT-PCR with the Seegene Allplex™ 2019-nCoV assay

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Wesley Freppel ◽  
Natacha Merindol ◽  
Fabien Rallu ◽  
Marco Bergevin
Keyword(s):  
Detection Rate ◽  
High Efficiency ◽  
False Negative ◽  
Rna Extraction ◽  
Free Water ◽  
Cost Savings ◽  
Global Scale ◽  
Proteinase K ◽  
N Gene ◽  
Infected People

Abstract Background The fight against the COVID-19 pandemic has created an urgent need to rapidly detect infected people. The challenge for clinical laboratories has been finding a high throughput, cost-efficient, and accurate testing method in the context of extraction reagents shortage on a global scale. To answer this need, we studied SARS-CoV-2 detection in oro-nasopharyngeal (ONP) swabs stored in Universal Transport Media (UTM) or in RNase-free water by rRT-PCR with Seegene Allplex™ 2019-nCoV assay without RNA extraction. Results Optimal results were obtained when swabs stored in UTM were diluted 1/5 and 1/2 in RNase-free water. Thermal lysis before rRT-PCR testing slightly improved detection rate. In addition, proteinase K (PK) treatment allowed for a significant reduction of invalid results and increased sensitivity for detection of low viral load specimens. In a panel of positive samples with all 3 viral genes amplified and N gene Cycle threshold values (Ct values) from 15 to 40, our detection rate was 98.9% with PK and 94.4% without. In a challenging panel of low positive samples with only the N gene being detectable at Ct values > 30, detection rate was increased from 53.3 to 76.7% with the addition of PK, and invalid rate fell off from 18.3 to 0%. Furthermore, we demonstrated that our method reliably detects specimens with Ct values up to 35, whereas false negative samples become frequent above this range. Finally, we show that swabs should be stored at − 70 °C rather than 4 °C when testing cannot be performed within 72 h of collection. Conclusion We successfully optimized the unextracted rRT-PCR process using the Seegene Allplex™ 2019-nCoV assay to detect SARS-CoV-2 RNAs in nasopharyngeal swabs. This improved method offers cost savings and turnaround time advantages compared to automated extraction, with high efficiency of detection that could play an important role in the surveillance of Covid-19.

scholarly journals Multiplex RT-PCR Reaction for Simultaneous Detection of Tomato Torrado Virus and Pepino Mosaic Virus Co-Infecting Solanum Lycopersicum

2013 ◽  
Vol 53 (3) ◽  
pp. 289-294 ◽  
Author(s):  
Przemysław Wieczorek ◽  
Aleksandra Obrępalska-Stęplowska
Keyword(s):  
Mosaic Virus ◽  
Solanum Lycopersicum ◽  
Internal Control ◽  
Production Efficiency ◽  
False Negative ◽  
Simultaneous Detection ◽  
Rt Pcr ◽  
High Resolution Melt ◽  
Pepino Mosaic Virus ◽  
Solanum Lycopersicum L

Abstract The tomato (Solanum lycopersicum L.) is cultivated all over the world and is a vegetable of significant economic importance. However, an increased production of the vegetable is directly connected with an elevated occurrence of pathogens limiting the production efficiency of the vegetable. Both, Tomato torrado virus and Pepino mosaic virus have been found to be serious disease factors. When not controlled, these viruses can significantly decrease tomato cultivation. In this article, we report a multiplex reverse transcription-polymerase chain reaction (RT-PCR) protocol for simultaneous detection of both, Tomato torrado virus (ToTV) and Pepino mosaic virus (PepMV) in virus infected plants. The assay was designed to specifically amplify the conserved regions of genomic ribonucleic acid (RNA) of both viruses. Moreover, the glycerandehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control of amplification to exclude false-negative assay results. High-resolution melt analysis of generated RT-PCR products was additionally performed to increase sensitivity and double-check the specificity of the reaction without the need of subsequent complementary deoxyribonucleic acid (cDNA) sequencing

scholarly journals CovidArray: A Microarray-Based Assay with High Sensitivity for the Detection of Sars-Cov-2 in Nasopharyngeal Swabs

Sensors ◽  
2021 ◽  
Vol 21 (7) ◽  
pp. 2490
Author(s):  
Francesco Damin ◽  
Silvia Galbiati ◽  
Stella Gagliardi ◽  
Cristina Cereda ◽  
Francesca Dragoni ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Solid Phase ◽  
Quantitative Polymerase Chain Reaction ◽  
False Negative ◽  
Rna Extraction ◽  
High Sensitivity ◽  
Great Sensitivity ◽  
Viral Rna ◽  
Analytical Sensitivity ◽  
Negative Results

A new coronavirus (SARS-CoV-2) caused the current coronavirus disease (Covid-19) epidemic. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is used as the gold standard for clinical detection of SARS-CoV-2. Under ideal conditions, RT-qPCR Covid-19 assays have analytical sensitivity and specificity greater than 95%. However, when the sample panel is enlarged including asymptomatic individuals, the sensitivity decreases and false negatives are reported. Moreover, RT-qPCR requires up to 3–6 h with most of the time involved in RNA extraction from swab samples. We introduce CovidArray, a microarray-based assay, to detect SARS-CoV-2 markers N1 and N2 in the nasopharyngeal swabs. The method is based on solid-phase hybridization of fluorescently-labeled amplicons upon RNA extraction and reverse transcription. This approach combines the physical-optical properties of the silicon substrate with the surface chemistry used to coat the substrate to obtain a diagnostic tool of great sensitivity. Furthermore, we used an innovative approach, RNAGEM, to extract and purify viral RNA in less than 15 min. We correctly assigned 12 nasopharyngeal swabs, previously analyzed by RT-qPCR. Thanks to the CovidArray sensitivity we were able to identify a false-negative sample. CovidArray is the first DNA microarray-based assay to detect viral genes in the swabs. Its high sensitivity and the innovative viral RNA extraction by RNAGEM allows the reduction of both the amount of false-negative results and the total analysis time to about 2 h.

scholarly journals Simultaneous Detection of Nine Grapevine Viruses by Multiplex Reverse Transcription-Polymerase Chain Reaction with Coamplification of a Plant RNA as Internal Control

2006 ◽  
Vol 96 (11) ◽  
pp. 1223-1229 ◽  
Author(s):  
Giorgio Gambino ◽  
Ivana Gribaudo
Keyword(s):  
Polymerase Chain Reaction ◽  
Internal Control ◽  
Reverse Transcription ◽  
Rna Extraction ◽  
Simultaneous Detection ◽  
Grapevine Virus ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Arabis Mosaic Virus ◽  
Polymerase Chain

A multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed for simultaneous detection of nine grapevine viruses: Arabis mosaic virus, Grapevine fanleaf virus, Grapevine virus A, Grapevine virus B, Rupestris stem pitting-associated virus, Grapevine fleck virus, Grapevine leafroll-associated virus-1, -2, and -3, in combination with a plant RNA internal control used as an indicator of the effectiveness of RNA extraction and RT-PCR. Primers were designed from conserved regions of each virus and their specificity was confirmed by sequencing PCR products. Two plant total RNA extraction methods (silica capture and modified RNeasy method) and two RT-PCR systems (onestep and two-step) were evaluated to develop a reliable protocol for mRT-PCR. One to nine fragments specific for the viruses were simultaneously amplified from infected samples and identified by their specific molecular sizes in agarose gel electrophoresis. In the two-step mRT-PCR, the detection limits were 10-3 or 10-4 extract dilutions, depending on the virus. Leaves, phloem from dormant cuttings, and in vitro plantlets from 103 naturally infected and healthy grapevines were analyzed. The mRT-PCR provided a reliable and rapid method for detecting grapevine viruses from a large number of samples.

scholarly journals Relative Sensitivity of Common Target Genes For The Detection of SARS-Cov-2 In Real Time-PCR

2021 ◽  
Author(s):  
Reza Valadan ◽  
Soheila Golchin ◽  
Reza Alizadeh-Navaei ◽  
Mohammadreza Haghshenas ◽  
Mehryar Zargari ◽  
...  
Keyword(s):  
Real Time ◽  
Target Genes ◽  
False Negative ◽  
Rna Extraction ◽  
Host Cells ◽  
N Gene ◽  
Clinical Samples ◽  
Human Beings ◽  
Gene Expressions ◽  
Mazandaran Province

Abstract SARS-CoV-2(COVID-19) currently is the main cause of the severe acute respiratory disease and fatal outcomes in human beings worldwide. Several genes are used as targets for the detection of SARS-CoV-2, including the RDRP, N, and E genes. The present study aimed to determine the RDRP, N, and E genes expressions of SARS-CoV- 2 in clinical samples. For this purpose, 100 SARS-CoV-2 positive samples were collected from diagnostic laboratories of Mazandaran province, Iran. After RNA extraction, the real time RT-PCR assay was performed for differential gene expressions’ analysis of N, E, and RDRP. The CT values for N, RDRP, and E targets of 100 clinical samples for identifying SARS-CoV-2 were then evaluated using qRT-PCR. This result suggests N gene as a potential target for the detection of the SARS‐CoV‐2, since it was observed to be highly expressed in the nasopharyngeal or oropharynges of COVID-19 patients (P < 0.0001). Herein, we showed that SARS-CoV- 2 genes were differentially expressed in the host cells. Therefore, to reduce obtaining false negative results and to increase the sensitivity of the available diagnostic tests, the target genes should be carefully selected based on the most expressed genes in the cells.

scholarly journals Processing Hundreds of SARS-CoV-2 Samples with an In-House PCR-Based Method without Robotics

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1712
Author(s):  
Theresa Mair ◽  
Maja Ivankovic ◽  
Christian Paar ◽  
Helmut J. F. Salzer ◽  
Angelika Heissl ◽  
...  
Keyword(s):  
Internal Control ◽  
Multiple Testing ◽  
Rna Extraction ◽  
P Gene ◽  
Human Rnase ◽  
Extraction Step ◽  
One Step ◽  
And Control ◽  
Digestion Step ◽  
Pcr Test

The SARS-CoV-2 pandemic has required the development of multiple testing systems to monitor and control the viral infection. Here, we developed a PCR test to screen COVID-19 infections that can process up to ~180 samples per day without the requirement of robotics. For this purpose, we implemented the use of multichannel pipettes and plate magnetics for the RNA extraction step and combined the reverse transcription with the qPCR within one step. We tested the performance of two RT-qPCR kits as well as different sampling buffers and showed that samples taken in NaCl or PBS are stable and compatible with different COVID-19 testing systems. Finally, we designed a new internal control based on the human RNase P gene that does not require a DNA digestion step. Our protocol is easy to handle and reaches the sensitivity and accuracy of the standardized diagnostic protocols used in the clinic to detect COVID-19 infections.

scholarly journals Putative Internal Control Genes in Bovine Milk Small Extracellular Vesicles Suitable for Normalization in Quantitative Real Time-Polymerase Chain Reaction

Membranes ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 933
Author(s):  
Md. Matiur Rahman ◽  
Shigeo Takashima ◽  
Yuji O. Kamatari ◽  
Yassien Badr ◽  
Kaori Shimizu ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Extracellular Vesicles ◽  
Internal Control ◽  
Rna Extraction ◽  
Bovine Milk ◽  
Chain Reaction ◽  
Qrt Pcr ◽  
Internal Control Genes ◽  
Polymerase Chain

Bovine milk small extracellular vesicles (sEVs) contain many biologically important molecules, including mRNAs. Quantitative real-time polymerase chain reaction (qRT-PCR) is a widely used method for quantifying mRNA in tissues and cells. However, the use, selection, and stability of suitable putative internal control genes in bovine milk sEVs for normalization in qRT-PCR have not yet been identified. Thus, the aim of the present study was to determine suitable putative internal control genes in milk sEVs for the normalization of qRT-PCR data. Milk sEVs were isolated from six healthy Holstein-Friesian cattle, followed by RNA extraction and cDNA synthesis. In total, 17 mRNAs were selected for investigation and quantification using qRT-PCR; they were further evaluated using geNorm, NormFinder, BestKeeper, and ∆CT algorithms to identify those that were highly stable putative internal control genes in milk sEVs. The final ranking of suitable putative internal control genes was determined using RefFinder. The mRNAs from TUB, ACTB, DGKZ, ETFDH, YWHAZ, STATH, DCAF11, and EGFLAM were detected in milk sEVs from six cattle by qRT-PCR. RefFinder demonstrated that TUB, ETFDH, and ACTB were highly stable in milk sEVs, and thus suitable for normalization of qRT-PCR data. The present study suggests that the use of these genes as putative internal control genes may further enhance the robustness of qRT-PCR in bovine milk sEVs. Since these putative internal control genes apply to healthy bovines, it would be helpful to include that the genes were stable in sEVs under “normal or healthy conditions”.

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