Quick and Easy Assembly of a One-Step qRT-PCR Kit for COVID-19 Diagnostics Using In-House Enzymes

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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Rapid electrochemical detection of coronavirus SARS-CoV-2

Nature Communications ◽

10.1038/s41467-021-21121-7 ◽

2021 ◽

Vol 12 (1) ◽

Cited By ~ 2

Author(s):

Thanyarat Chaibun ◽

Jiratchaya Puenpa ◽

Tatchanun Ngamdee ◽

Nimaradee Boonapatcharoen ◽

Pornpat Athamanolap ◽

...

Keyword(s):

Electrochemical Biosensor ◽

Rolling Circle Amplification ◽

Clinical Samples ◽

Rolling Circle ◽

Qrt Pcr ◽

Reverse Transcription Pcr ◽

Sandwich Hybridization ◽

Redox Active ◽

One Step ◽

The One

AbstractCoronavirus disease 2019 (COVID-19) is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Diagnosis of COVID-19 depends on quantitative reverse transcription PCR (qRT-PCR), which is time-consuming and requires expensive instrumentation. Here, we report an ultrasensitive electrochemical biosensor based on isothermal rolling circle amplification (RCA) for rapid detection of SARS-CoV-2. The assay involves the hybridization of the RCA amplicons with probes that were functionalized with redox active labels that are detectable by an electrochemical biosensor. The one-step sandwich hybridization assay could detect as low as 1 copy/μL of N and S genes, in less than 2 h. Sensor evaluation with 106 clinical samples, including 41 SARS-CoV-2 positive and 9 samples positive for other respiratory viruses, gave a 100% concordance result with qRT-PCR, with complete correlation between the biosensor current signals and quantitation cycle (Cq) values. In summary, this biosensor could be used as an on-site, real-time diagnostic test for COVID-19.

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Development of novel AllGlo-probe-based one-step multiplex qRT-PCR assay for rapid identification of avian influenza virus H7N9

Archives of Virology ◽

10.1007/s00705-014-1979-5 ◽

2014 ◽

Vol 159 (7) ◽

pp. 1707-1713 ◽

Cited By ~ 5

Author(s):

Yanjun Zhang ◽

Haiyan Mao ◽

Juying Yan ◽

Xinying Wang ◽

Lei Zhang ◽

...

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Avian Influenza Virus ◽

Rapid Identification ◽

Pcr Assay ◽

Qrt Pcr ◽

One Step

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Leveraging Automation toward Development of a High-Throughput Gene Expression Profiling Platform

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555220956593 ◽

2020 ◽

pp. 247255522095659

Author(s):

Jing Chen ◽

Alan Futran ◽

Austin Crithary ◽

Sha Li ◽

Alex Wolicki ◽

...

Keyword(s):

Gene Expression ◽

Drug Discovery ◽

High Throughput ◽

Screening Strategy ◽

Qrt Pcr ◽

Reverse Transcription Pcr ◽

Expression Platform ◽

Program Support ◽

Drug Discovery Program ◽

One Step

We previously developed a panel of one-step real-time quantitative reverse transcription PCR (one-step qRT-PCR; hereafter referred to as qRT-PCR) assays to assess compound efficacy. However, these high-cost, conventional qRT-PCR manual assays are not amenable to high-throughput screen (HTS) analysis in a time-sensitive and complex drug discovery process. Here, we report the establishment of an automated gene expression platform using in-house lysis conditions that allows the study of various cell lines, including primary T cells. This process innovation provides the opportunity to perform genotypic profiling in both immunology and oncology therapeutic areas with quantitative studies as part of routine drug discovery program support. This newly instituted platform also enables a panel screening strategy to efficiently connect HTS, lead identification, and lead optimization in parallel.

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Novel Approach for Detecting Prohibited Species-Specific Central Nervous System Tissue Contamination in Meat by One-Step Real-Time Reverse Transcriptase PCR

Journal of Food Protection ◽

10.4315/0362-028x-72.5.1063 ◽

2009 ◽

Vol 72 (5) ◽

pp. 1063-1069 ◽

Cited By ~ 4

Author(s):

M. M. NAGARAJAN ◽

D. LONGTIN ◽

C. SIMARD

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Reverse Transcriptase ◽

Real Time ◽

Compliance Monitoring ◽

Reverse Transcriptase Pcr ◽

Qrt Pcr ◽

Tissue Contamination ◽

One Step ◽

Species Specific

The dissemination of prohibited species-specific central nervous system (CNS) tissue contamination in meat must be tracked to mitigate human health risk associated with bovine spongiform encephalopathy. The efficiency of compliance monitoring and risk control measures taken by concerned regulatory authorities at meat production facilities to avoid such contamination depends on the ability to detect CNS tissue with a reliable and adequately sensitive quantitative method. A rapid and convenient one-step real-time quantitative reverse transcriptase PCR (qRT-PCR) assay was developed based on the absolute quantification of glial fibrillary acidic protein (GFAP) mRNA as a marker for CNS tissue contamination in meat. The GFAP RNA quantity corresponding to a percentage of CNS tissue in artificially spiked meat was determined using an appropriate in vitro transcribed target GFAP RNA as a calibration standard in the assay. The assay had a linear dynamic range of 102 to 109 copies of target RNA and was able to detect 0.01% CNS contamination in meat. Further evaluation consisted of an analysis of 272 random meat cuts from carcasses and 109 ground meat samples received from a federally inspected abattoir and two meat processing facilities, respectively, over a 5-month period. The analyzed samples were all negative for CNS tissue contamination at an arbitrarily set lower threshold of 0.025%. Overall, the newly developed one-step qRT-PCR may be useful as an objective quantitative compliance monitoring tool and for setting an acceptable low tolerance threshold for such contamination in meat.

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An Ultrafast One-Step qRT-PCR Assay for Detection of SARS-CoV-2

10.21203/rs.3.rs-582897/v1 ◽

2021 ◽

Author(s):

Jadranka Milosevic ◽

Mengrou Lu ◽

Wallace Greene ◽

Hong-Zhang He ◽

Si-Yang Zheng

Keyword(s):

Qpcr Assay ◽

Nasopharyngeal Swab ◽

Pcr Assay ◽

Running Time ◽

Clinical Sensitivity ◽

Qrt Pcr ◽

One Step

Abstract We developed an ultrafast one-step RT-qPCR assay for SARS-CoV-2 detection, which can be completed in only 30 mins on benchtop Bio-Rad CFX96. The assay significantly reduces the running time of conventional RT-qPCR: reduced RT step from 10 to 1 min, and reduced PCR cycle of denaturation from 10 to 1 s and extension from 30 to 1 s. A cohort of 60 nasopharyngeal swab samples testing showed that the assay had a clinical sensitivity of 100% and a clinical specificity of 100%.

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One-step RT-qPCR assay for ZIKV RNA detection in Aedes aegypti samples: A protocol to study infection & gene expression during ZIKV infection

10.21203/rs.2.19173/v1 ◽

2019 ◽

Author(s):

Ricardo Vieira Araujo ◽

Fabiana Feitosa-Suntheimer ◽

Alexander S. Gold ◽

Berlin Londono-Renteria ◽

Tonya Michelle Colpitts

Keyword(s):

Gene Expression ◽

Mammalian Cells ◽

Infected Mosquito ◽

Zikv Infection ◽

Rna Detection ◽

High Background ◽

Qrt Pcr ◽

One Step ◽

Nonspecific Amplification ◽

Lower Background

Abstract Background: Zika virus (ZIKV) is transmitted to humans during the bite of an infected mosquito. In a scenario of globalization and climate change, the frequency of outbreaks has and will increase in areas with competent vectors, revealing a need for continuous improvement of ZIKV detection tools in vector populations. A simple, rapid and sensitive assay for viral detection is qRT-PCR, yet oligos optimized for ZIKV detection in mammalian cells and samples have repeatedly shown high background when used on mosquito RNA. In this work we present a one-step qRT-PCR protocol that allows for the detection of ZIKV in mosquitoes and for the evaluation of gene expression from the same mosquito sample and RNA. This assay is a less expensive qRT-PCR approach than that most frequently used in the literature and has a much lower background, allowing for confident detection.Methods: Our new oligo design to detect ZIKV RNA included in silicoanalysis of both viral and mosquito (Ae. aegyptiand Ae. albopictus)genomes, targeting sequences conserved between Asian and African ZIKV lineages, but not matching Aedesgenomes. This assay will allow researchers to avoid nonspecific amplification in insect samples due to viral integration into the mosquito genome, a phenomenon known to happen in wild and colonized populations of mosquitoes.Standard curves constructed with in vitrotranscribed ZIKV RNA were used to optimize the sensitivity, efficiency and reproducibility of the assay.Results: Finally, the assay was used with success to detect both ZIKV RNA in infected mosquitoes and to detect expression of the Defensin A gene, an antimicrobial peptide (AMP) involved in Aedes aegyptiimmune response to virus infection.Conclusions: The experimental approach to detect ZIKV RNA in Aedes aegyptipresented here has demonstrated to be specific, sensitive and reliable, and additionally it allows for the analysis of mosquito gene expression during ZIKV infection.

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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

10.21203/rs.3.rs-74067/v1 ◽

2020 ◽

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 30–60%. 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 LoD (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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A one-step TaqMan real-time qRT-PCR assay for the specific detection and quantitation of the Spanish goat encephalitis virus (SGEV)

Journal of Virological Methods ◽

10.1016/j.jviromet.2018.02.017 ◽

2018 ◽

Vol 255 ◽

pp. 98-100

Author(s):

Ana del Cerro ◽

Ana Balseiro ◽

Rosa Casais ◽

Kevin P. Dalton ◽

Luis Salinas ◽

...

Keyword(s):

Real Time ◽

Encephalitis Virus ◽

Specific Detection ◽

Pcr Assay ◽

Qrt Pcr ◽

One Step ◽

Spanish Goat

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Application of One-Step Reverse Transcription Droplet Digital PCR for Dengue Virus Detection and Quantification in Clinical Specimens

Diagnostics ◽

10.3390/diagnostics11040639 ◽

2021 ◽

Vol 11 (4) ◽

pp. 639

Author(s):

Dumrong Mairiang ◽

Adisak Songjaeng ◽

Prachya Hansuealueang ◽

Yuwares Malila ◽

Paphavee Lertsethtakarn ◽

...

Keyword(s):

Dengue Virus ◽

Lower Limit ◽

Reverse Transcription ◽

Limit Of Detection ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Clinical Samples ◽

Qrt Pcr ◽

One Step ◽

Detection And Quantification

Detection and quantification of viruses in laboratory and clinical samples are standard assays in dengue virus (DENV) studies. The quantitative reverse transcription polymerase chain reaction (qRT-PCR) is considered to be the standard for DENV detection and quantification due to its high sensitivity. However, qRT-PCR offers only quantification relative to a standard curve and consists of several “in-house” components resulting in interlaboratory variations. We developed and optimized a protocol for applying one-step RT-droplet digital PCR (RT-ddPCR) for DENV detection and quantification. The lower limit of detection (LLOD95) and the lower limit of quantification (LLOQ) for RT-ddPCR were estimated to be 1.851 log10-copies/reaction and 2.337 log10-copies/reaction, respectively. The sensitivity of RT-ddPCR was found to be superior to qRT-PCR (94.87% vs. 90.38%, p = 0.039) while no false positives were detected. Quantification of DENV in clinical samples was independently performed in three laboratories showing interlaboratory variations with biases <0.5 log10-copies/mL. The RT-ddPCR protocol presented here could help harmonize DENV quantification results and improve findings in the field such as identifying a DENV titer threshold correlating with disease severity.

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