scholarly journals Correlating qRT-PCR, dPCR and Viral Titration for the Identification and Quantification of SARS-CoV-2: A New Approach for Infection Management

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1022
Author(s):  
Martina Brandolini ◽  
Francesca Taddei ◽  
Maria Michela Marino ◽  
Laura Grumiro ◽  
Agata Scalcione ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Cell Cultures ◽  
Rna Extraction ◽  
Research Field ◽  
Chain Reaction ◽  
Diagnostic Assays ◽  
Qrt Pcr ◽  
Extraction And Purification ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in Wuhan, China, in late 2019 and is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) represents the gold standard for diagnostic assays even if it cannot precisely quantify viral RNA copies. Thus, we decided to compare qRT-PCR with digital polymerase chain reaction (dPCR), which is able to give an accurate number of RNA copies that can be found in a specimen. However, the aforementioned methods are not capable to discriminate if the detected RNA is infectious or not. For this purpose, it is necessary to perform an endpoint titration on cell cultures, which is largely used in the research field and provides a tissue culture infecting dose per mL (TCID50/mL) value. Both research and diagnostics call for a model that allows the comparison between the results obtained employing different analytical methods. The aim of this study is to define a comparison among two qRT-PCR protocols (one with preliminary RNA extraction and purification and an extraction-free qRT-PCR), a dPCR and a titration on cell cultures. The resulting correlations yield a faithful estimation of the total number of RNA copies and of the infectious viral burden from a Ct value obtained with diagnostic routine tests. All these estimations take into consideration methodological errors linked to the qRT-PCR, dPCR and titration assays.

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”.

scholarly journals Digital polymerase chain reaction in an array of microfluidic printed droplets

2019 ◽  
Author(s):  
Yongfan Men ◽  
Jiannan Li ◽  
Tingting Ao ◽  
Zhihao Li ◽  
Bizhu Wu ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Cost Effective ◽  
Digital Pcr ◽  
Research Field ◽  
Clinical Diagnostics ◽  
Future Market ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
New Perspective ◽  
Digital Polymerase Chain Reaction

AbstractDigital polymerase chain reaction (PCR) is a fast-developed technology, which makes it possible to provide absolute quantitative results. However, this technology has not been widely used in research field or clinical diagnostics. Although digital PCR has been born for two decades, the products on this subject still suffer from either high cost or cumbersome user experience, hence very few labs have the willingness or budget to routinely use such product; On the other hand, the unique sensitivity of dPCR over traditional qPCR shows great potential applications. Here, a cost-effective digital PCR method based on a microfluidic printing system was introduced, trying to overcome those shortcomings. The microfluidic droplet printing technology was utilized in this study to directly generate droplet array containing PCR reaction solution onto the simple glass substrate for the subsequent PCR and imaging, which could be done with any regular flat-panel PCR machine and microscope. The method introduces a new perspective in droplet-based digital PCR in that the droplets generated with this method aligns well in an array without touch with each other, therefore the regular glass and oil could be used without any special surfactant. With simple analysis, the data generated with this method showed reliable quality, which followed the Poisson distribution trend. Compared with other expensive digital PCR methods, this system is more affordable and simpler to integrate, especially for those biological or medical labs which are in need for the digital PCR options but short in budget. Therefore, this method is believed to have the great potential in the future market application.

scholarly journals Digital Droplet PCR for SARS-CoV-2 Resolves Borderline Cases

2021 ◽  
Author(s):  
Jing Xu ◽  
Timothy Kirtek ◽  
Yan Xu ◽  
Hui Zheng ◽  
Huiyu Yao ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Limit Of Detection ◽  
False Negative ◽  
Accurate Method ◽  
Viral Envelope ◽  
Chain Reaction ◽  
Borderline Cases ◽  
Droplet Pcr ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

Abstract Objectives The Bio-Rad SARS-CoV-2 ddPCR Kit (Bio-Rad Laboratories) was the first droplet digital polymerase chain reaction (ddPCR) assay to receive Food and Drug Administration (FDA) Emergency Use Authorization approval, but it has not been evaluated clinically. We describe the performance of ddPCR—in particular, its ability to confirm weak-positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) results. Methods We clinically validated the Bio-Rad Triplex Probe ddPCR Assay. The limit of detection was determined by using serial dilutions of SARS-CoV-2 RNA in an artificial viral envelope. The ddPCR assay was performed according to the manufacturer’s specifications on specimens confirmed to be positive (n = 48) or negative (n = 30) by an FDA-validated reverse transcription–polymerase chain reaction assay on the m2000 RealTime system (Abbott). Ten borderline positive cases were also evaluated. Results The limit of detection was 50 copies/mL (19 of 20 positive). Forty-seven specimens spanning a range of quantification cycles (2.9-25.9 cycle numbers) were positive by this assay (47 of 48; 97.9% positive precent agreement), and 30 negative samples were confirmed as negative (30 of 30; 100% negative percent agreement). Nine of 10 borderline cases were positive when tested in triplicate. Conclusions The ddPCR of SARS-CoV-2 is an accurate method, with superior sensitivity for viral RNA detection. It could provide definitive evaluation of borderline positive cases or suspected false-negative cases.

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

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