scholarly journals SYBR green one-step qRT-PCR for the detection of SARS-CoV-2 RNA in saliva

2020 ◽  
Author(s):  
DR Ganguly ◽  
S Rottet ◽  
S Yee ◽  
WY Hee ◽  
AB Smith ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Positive Control ◽  
Heat Inactivation ◽  
Median Viral Load ◽  
Qrt Pcr ◽  
Pcr Efficiency ◽  
Rna Purification ◽  
Reverse Transcription Reaction ◽  
One Step ◽  
Buffer Composition

AbstractWe describe our efforts at developing a one-step quantitative reverse-transcription (qRT)-PCR protocol to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA directly from saliva samples, without RNA purification. We find that both heat and the presence of saliva impairs the ability to detect synthetic SARS-CoV-2 RNA. Buffer composition (for saliva dilution) was also crucial to effective PCR detection. Using the SG2 primer pair, designed by Sigma-Aldrich, we were able to detect the equivalent of 1.7×106 viral copies per mL of saliva after heat inactivation; approximately equivalent to the median viral load in symptomatic patients. This would make our assay potentially useful for rapid detection of high-shedding infected individuals. We also provide a comparison of the PCR efficiency and specificity, which varied considerably, across 9 reported primer pairs for SARS-CoV-2 detection. Primer pairs SG2 and CCDC-N showed highest specificity and PCR efficiency. Finally, we provide an alternate primer pair to use as a positive control for human RNA detection in SARS-CoV-2 assays, as we found that the widely used US CDC primers (targeting human RPP30) do not span an exon-exon junction and therefore does not provide an adequate control for the reverse transcription reaction.

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

Diagnostics ◽  
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.

scholarly journals Development and Assessment of a Quantitative Reverse Transcription-PCR Assay for Simultaneous Measurement of Four Amplicons

2003 ◽  
Vol 49 (9) ◽  
pp. 1467-1475 ◽  
Author(s):  
Marcy B Grace ◽  
Christopher B McLeland ◽  
Steven J Gagliardi ◽  
Jeffrey M Smith ◽  
William E Jackson ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Whole Blood ◽  
18S Rrna ◽  
Ex Vivo ◽  
Detection System ◽  
Simultaneous Detection ◽  
Positive Control ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Pcr Assays

Abstract Background: High-throughput and forward-deployable biological dosimetry capabilities are required for tactical and medical decisions after radiologic events. We previously reported a quantitative reverse transcription (QRT)-PCR assay for human radiation-responsive gene targets using a whole-blood ex vivo irradiation model, but we needed a multitarget assay on a smaller, less costly, real-time PCR detection system. Methods: We developed a quadruplex QRT-PCR assay in a 96-well, closed-plate format suitable for use with RNA extracted from whole blood. Four cDNA targets were simultaneously amplified in a sealed tube by hybridization to exonuclease probes, each conjugated to distinct fluorogenic reporters. A novel primer-limited 18S rRNA reference target was validated from serial dilutions of human total RNA. To test assay precision, we incorporated a positive-control cDNA mimic into duplex and quadruplex PCR reactions. The master mixture was supplemented with more enzyme, MgCl2, and deoxyribonucleotides. Simultaneous detection of four targets was evaluated in comparison with respective duplex QRT-PCR assays. Results: The simultaneous detection of three radiation-responsive genes by quadruplex QRT-PCR was quantitative, with gene expression changes similar to those observed with optimized duplex and triplex QRT-PCR assays. The 18S rRNA and GADD45 calibration curves (threshold cycle vs log10 cDNA) were linear and reproducible and showed optimal PCR efficiencies as indicated by slopes statistically equivalent to the theoretical value of −3.322. Conclusions: This is the first study of a quadruplex QRT-PCR assay. Our approach has diagnostic utility in the detection of biomarkers, biological and toxicologic agents, and genes of inherited diseases and cancer.

scholarly journals Temperature-controlled Primer Limit for Multiplexing of Rapid, Quantitative Reverse Transcription-PCR Assays: Application to Intraoperative Cancer Diagnostics

2002 ◽  
Vol 48 (8) ◽  
pp. 1329-1337 ◽  
Author(s):  
Siva Raja ◽  
Talal El-Hefnawy ◽  
Lori A Kelly ◽  
Melissa L Chestney ◽  
James D Luketich ◽  
...  
Keyword(s):  
Lymph Nodes ◽  
Reverse Transcription ◽  
Dynamic Range ◽  
Positive Control ◽  
Cancer Diagnostics ◽  
Qrt Pcr ◽  
Quantitative Reverse Transcription Pcr ◽  
Reverse Transcription Pcr ◽  
Temperature Controlled ◽  
Pcr Assays

Abstract Background: Rapid-cycling, real-time PCR instruments bring the opportunity for improved intraoperative detection of metastasis to sentinel lymph nodes. Rapid, standardized, and internally controlled assays need to be developed that are sensitive and accurate. Methods: We describe rapid, multiplexed, internally controlled, quantitative reverse transcription-PCR (QRT-PCR) assays for tyrosinase and carcinoembryonic antigen mRNAs on the SmartCycler (Cepheid). We used a temperature-controlled primer-limiting approach to eliminate amplification of the endogenous control gene as soon as its signal had reached threshold. Positive-control oligonucleotide mimics were incorporated into all reactions to differentiate failed reactions from true negative samples. Results: The optimized assays for rapid QRT-PCR yielded results with threshold cycle values that were only 1–2 cycles higher than slower, more conventional protocols. In rapid PCR, the temperature-controlled multiplex assay was quantitative over a dynamic range of at least 15 cycles, compared with only 6 cycles for conventional multiplexing methods. All histologically positive lymph nodes examined were also QRT-PCR positive for the appropriate marker, and the exogenous, internal positive-control mimics produced signals in all negative samples. Conclusion: Internally controlled, rapid QRT-PCR assays can be performed in an intraoperative time frame and with sufficient sensitivity to detect histologically identified metastases to lymph nodes.

scholarly journals Development and validation of a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of ZIKV in patient samples from Brazil

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Severino Jefferson Ribeiro da Silva ◽  
Keith Pardee ◽  
Udeni B. R. Balasuriya ◽  
Lindomar Pena
Keyword(s):  
Reverse Transcription ◽  
Rapid Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Serum Samples ◽  
Low Resource ◽  
Loop Mediated Isothermal Amplification ◽  
One Step

AbstractWe have previously developed and validated a one-step assay based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of the Zika virus (ZIKV) from mosquito samples. Patient diagnosis of ZIKV is currently carried out in centralized laboratories using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR), which, while the gold standard molecular method, has several drawbacks for use in remote and low-resource settings, such as high cost and the need of specialized equipment. Point-of-care (POC) diagnostic platforms have the potential to overcome these limitations, especially in low-resource countries where ZIKV is endemic. With this in mind, here we optimized and validated our RT-LAMP assay for rapid detection of ZIKV from patient samples. We found that the assay detected ZIKV from diverse sample types (serum, urine, saliva, and semen) in as little as 20 min, without RNA extraction. The RT-LAMP assay was highly specific and up to 100 times more sensitive than RT-qPCR. We then validated the assay using 100 patient serum samples collected from suspected cases of arbovirus infection in the state of Pernambuco, which was at the epicenter of the last Zika epidemic. Analysis of the results, in comparison to RT-qPCR, found that the ZIKV RT-LAMP assay provided sensitivity of 100%, specificity of 93.75%, and an overall accuracy of 95.00%. Taken together, the RT-LAMP assay provides a straightforward and inexpensive alternative for the diagnosis of ZIKV from patients and has the potential to increase diagnostic capacity in ZIKV-affected areas, particularly in low and middle-income countries.

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.

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

ACS Omega ◽  
2021 ◽  
Author(s):  
Masateru Takahashi ◽  
Muhammad Tehseen ◽  
Rahul Salunke ◽  
Etsuko Takahashi ◽  
Sara Mfarrej ◽  
...  
Keyword(s):  
Qrt Pcr ◽  
One Step

scholarly journals Rapid electrochemical detection of coronavirus SARS-CoV-2

2021 ◽  
Vol 12 (1) ◽  
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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