An Ultrafast One-Step Quantitative Reverse Transcription–Polymerase Chain Reaction Assay for Detection of SARS-CoV-2

Milošević, Jadranka; Lü, Min; Greene, Wallace; He, Hong‐Zhang; Zheng, Siyang

doi:10.3389/fmicb.2021.749783

Cited by 4 publications

(5 citation statements)

References 22 publications

(17 reference statements)

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“…For the IMRS qPCR, the aim was to lower the amplification time to at most 40 minutes, as opposed to most standard qPCR assays, which takes up to 1 hour on average (Nayab et al, 2008;Bustin and Nolan, 2020). To attain abovementioned objective, the fast-cycling conditions were applied as described by Milosevic et al (2021). In their study, however, Milosevic et al (2021) managed to develop an ultra-sensitive reverse transcriptase (RT) qPCR, which could detect as little as 25 copies of SARS-CoV-2 RNA genome in 30 minutes.…”

Section: Discussionmentioning

confidence: 99%

“…To attain abovementioned objective, the fast-cycling conditions were applied as described by Milosevic et al (2021). In their study, however, Milosevic et al (2021) managed to develop an ultra-sensitive reverse transcriptase (RT) qPCR, which could detect as little as 25 copies of SARS-CoV-2 RNA genome in 30 minutes. For our study, the qPCR detected as little as one genome copy per microliter of Schistosoma genome within 36 minutes.…”

Section: Discussionmentioning

confidence: 99%

“…The difference in polymerase enzyme used accounts for six minutes amplification time difference. The SpeExcelTAR HS DNA Polymerase used by Milosevic et al is optimized for a 10s/kb extension rate, while the standard DNA polymerase has a 60s/kb extension rate (Milosevic et al, 2021). Reducing the amplification time in qPCR will enable processing of many samples in a reasonable time, especially for surveillance studies.…”

Section: Discussionmentioning

confidence: 99%

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Development of a rapid and highly sensitive nucleic acid-based diagnostic test for schistosomes, leveraging on identical multi-repeat sequences

Ally,

Kanoi,

Kamath

et al. 2024

Front. Parasitol.

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IntroductionSchistosomiasis (Bilharzia), a neglected tropical disease caused by Schistosoma parasites, afflicts over 240 million people globally, disproportionately impacting Sub-Saharan Africa. Current diagnostic tests, despite their utility, suffer from limitations like low sensitivity. Polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR) remain the most common and sensitive nucleic acid amplification tests. Still, the sensitivity of nucleic acid amplification tests is significantly affected by the copy number of amplification targets, resulting in underestimation of true Schistosoma infections, especially in low transmission settings. Additionally, lengthy qPCR run times pose challenges when dealing with large sample volumes and limited resources. In this study, the identical multi-repeat sequences (IMRS) were used as a novel approach to enhance the sensitivity of nucleic acid-based Bilharzia diagnosis.MethodsTo identify novel genomic repeat regions, we utilized the IMRS algorithm, with modifications to enable larger target region (100-200bp) identification instead of smaller sequences (18-30bp). These regions enabled customised primer-probe design to suit requirements for qPCR assay. To lower the qPCR amplification times, the assay was conducted using fast cycling condition. Regression analysis, and qPCR data visualization was conducted using Python programming.ResultsUsing Schistosoma mansoni and S. haematobium, we found that IMRS-based qPCR, employing genus-specific primers and TaqMan probes, offers exceptional analytical sensitivity, detecting as little as a single genome copy per microliter within 36 minutes.DiscussionThe lowest concentration of DNA detected using IMRS-based PCR and qPCR represented tenfold improvement over conventional PCR. As part of further development, there is a need to compare IMRS-based qPCR against other qPCR methods for Schistosoma spp. Nonetheless, IMRS-based diagnostics promise a significant advancement in bilharzia diagnosis, particularly in low-transmission settings, potentially facilitating more effective control and treatment strategies.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Development of a rapid and highly sensitive nucleic acid-based diagnostic test for schistosomes, leveraging on identical multi-repeat sequences

Ally,

Kanoi,

Kamath

et al. 2024

Front. Parasitol.

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“…We then evaluated the LoD of FAST RT-qPCR with the Chai Open qPCR machine (ChaiBio, single channel) that provides a maximal ramp rate of 5 °C per second. Specifically, we adopted the method of FAST RT-PCR [44] to set up a 20 µL reaction for a selected RNA target. The reaction uses SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific, 18090010) and SpeedStar HS DNA polymerase (TaKaRa, RR070B) to conduct an ultrafast one-step RT-qPCR.…”

Section: Standard Curves and Limit Of Detection (Lod) Of Rt-qpcrmentioning

confidence: 99%

Characterization of a fieldable process for airborne virus detection

Du,

Bruno,

Overholt

et al. 2023

Preprint

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Rapid, on-site, airborne virus detection is a requirement for timely action against the spread of air-transmissible infectious diseases. This applies both to future threats and to common viral diseases, such as influenza and COVID-19, which hit vulnerable populations yearly with severe consequences. The ultra-low concentrations of virus in the air make airborne virus detection difficult, yet readily infect individuals when breathed. Here, we propose a fieldable process that includes an enrichment step to concentrate collected genetic material in a small volume. The enrichment approach uses capillary electrophoresis and an RT-qPCR-compatible buffer, which allow enrichment of the RNA by about 5-fold within only 10 minutes of operation. Our detection process consists of air sampling through electrostatic precipitation, RNA extraction via heating, RNA enrichment, and RT-qPCR for detection. We optimized each step of the process and estimated a detection sensitivity of 3106±2457 genome copies (gc) per m3of air. We then performed an integration experiment and confirmed a sensitivity of 5654 gc/m3with a detection rate of 100% and a sensitivity of 4221 gc/m3with a detection rate of 78.6%. When using fast RT-qPCR, the latency of the whole process is down to 61 minutes. Given that our sensitivity falls in the low range of influenza and SARS-CoV-2 concentrations reported in indoor spaces, our study shows that, with enrichment, airborne pathogen detection can be made sufficiently sensitive for practical use.

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“…In addition, the use of UF-PCR system can reduce reagent usage by half. Owing to these advantages, the UF-PCR system is being considered for application in various analyses, such as forensic genotyping (Aboud et al, 2013), pathogen screening (Das et al, 2022;Kim et al, 2018;Lee et al, 2014), allergen analysis (Kim et al, 2019), detection of epidemic viruses like SARS CoV-2 (Chen et al, 2022;Milosevic et al, 2021), and authenticity determination (Hong et al, 2021).…”

Section: Introductionmentioning

confidence: 99%

Detection method for genetically modified potato using an ultra-fast PCR system

Shin

Jeon

Koo

2023

Food Sci Biotechnol

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Genetically modified (GM) potatoes having resistance to insects and viral diseases, low reducing sugar contents, and black spots for high quality continue to be developed. However, no GM potato has been approved as food or feed in the Republic of Korea as the country adheres to a zero-tolerance policy to unauthorized genetically modified organisms (GMOs). When the self-sufficiency rate is low, a detection method to assess GMOs in crops or other products is necessary. Therefore, a rapid method for two GM potato events (SPS-Y9 and EH92-527-1) using an ultra-fast PCR (UF-PCR) system has been developed, and its specificity, sensitivity, and applicability were demonstrated. UF-PCR can decrease the runtime of PCR by more than half of that needed in conventional methods. However, UF-PCR is not a common method for GMO analysis. This rapid detection method may be useful for GMO analyses in field conditions.

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An Ultrafast One-Step Quantitative Reverse Transcription–Polymerase Chain Reaction Assay for Detection of SARS-CoV-2

Cited by 4 publications

References 22 publications

Development of a rapid and highly sensitive nucleic acid-based diagnostic test for schistosomes, leveraging on identical multi-repeat sequences

Development of a rapid and highly sensitive nucleic acid-based diagnostic test for schistosomes, leveraging on identical multi-repeat sequences

Characterization of a fieldable process for airborne virus detection

Detection method for genetically modified potato using an ultra-fast PCR system

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