An ultra‐sensitive and highly specific detection method for a newly discovered Oncotshavirus using one‐step reverse transcription droplet‐based digital PCR

2021 ◽  
Author(s):  
Xiaoyu Chen ◽  
Pengyu Zhu ◽  
Kaiyue Duan ◽  
Jian Zheng ◽  
Xuan Zhou ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Detection Method ◽  
Digital Pcr ◽  
Specific Detection ◽  
One Step

scholarly journals Simple and Visible Detection of Novel Astroviruses Causing Fatal Gout in Goslings Using One-Step Reverse Transcription Polymerase Spiral Reaction Method

2020 ◽  
Vol 7 ◽  
Author(s):  
Jun Ji ◽  
Qinxi Chen ◽  
Zhengli Yu ◽  
Xin Xu ◽  
Xinhao Mu ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Cost Effective ◽  
Pcr Primers ◽  
Probit Regression ◽  
Conventional Pcr ◽  
Specific Detection ◽  
Isothermal Method ◽  
Naked Eye ◽  
One Step ◽  
The One

In this study, a one-step isothermal method combining polymerase spiral reaction (PSR) with reverse transcription (RT-PSR) was established for rapid and specific detection of novel astroviruses causing fatal gout in goslings (N-GoAstV). The one-step RT-PSR was accomplished at the optimal temperature of 62°C and time of 40 min and used primers simply designed as conventional PCR primers, and the results of detection were visible to the naked eye. The detection limit of PSR was above 34.7 copies/μL at a 95% probability level according to probit regression analysis. The assay specifically detected N-GoAstV, and no other reference viruses were detected. These results suggest that the newly established RT-PSR assay could, in one step, accomplish reverse-transcription, amplification, and result determination providing a visible, convenient, rapid, and cost-effective test that can be carried out onsite, in order to ensure timely quarantine of N-GoAstV-infected birds, leading to effective disease control.

scholarly journals One-step triplex reverse-transcription PCR detection of porcine epidemic diarrhea virus, porcine sapelovirus, and porcine sapovirus

2019 ◽  
Vol 31 (6) ◽  
pp. 909-912 ◽  
Author(s):  
Chunyan Jiang ◽  
Haijian He ◽  
Chaoying Zhang ◽  
Xiaoju Zhang ◽  
Jianfeng Han ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Porcine Epidemic Diarrhea Virus ◽  
Detection Method ◽  
Rt Pcr ◽  
Diarrhea Virus ◽  
Reverse Transcription Pcr ◽  
Porcine Sapovirus ◽  
One Step ◽  
Porcine Epidemic Diarrhea ◽  
Porcine Sapelovirus

Swine diarrhea can be caused by multiple agents, including porcine epidemic diarrhea virus (PEDV), porcine sapelovirus (PSV), and porcine sapovirus (SaV). We designed a one-step triplex reverse-transcription PCR (RT-PCR) detection method including 3 pairs of primers that focused on the S1 gene of PEDV, a conserved gene of PSV, and the VP1 gene of SaV. The optimal concentrations of upstream and downstream primers in the triplex RT-PCR were 0.24 μM for PEDV, 0.15 μM for PSV, and 0.2 μM for SaV, and the optimal annealing temperature was 55.5°C. Triplex RT-PCR assessment of 402 piglet diarrhea samples was compared with conventional individual RT-PCR. Concordance rates in both tests for individual viruses were 100%, 97.6%, and 94.4% for PEDV, PSV, and SaV, respectively. PEDV, PSV, and SaV were detected in 57.2%, 10.4%, and 9.0% of the samples, respectively. The high sensitivity and specificity of this triplex RT-PCR–based detection method for PEDV, PSV, and SaV could allow rapid detection and analysis of mixed infections by these 3 viruses.

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 Investigation of SARS-CoV-2 on Ocular Surface of Coronavirus Disease 2019 Patients Using One-Step Reverse-Transcription Droplet Digital PCR

2021 ◽  
Vol Volume 14 ◽  
pp. 5395-5401
Author(s):  
Xian Zhang ◽  
Liting Chen ◽  
Gaoxiang Wang ◽  
Liwen Chen ◽  
Lifang Huang ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Ocular Surface ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
One Step

scholarly journals Validation of a One-Step Reverse Transcription-Droplet Digital PCR (RT-ddPCR) Approach to Detect and Quantify SARS-CoV-2 RNA in Nasopharyngeal Swabs

2021 ◽  
Vol 2021 ◽  
pp. 1-6
Author(s):  
Catia Mio ◽  
Adriana Cifù ◽  
Stefania Marzinotto ◽  
Barbara Marcon ◽  
Corrado Pipan ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
University Hospital ◽  
Reverse Transcription Pcr ◽  
Containment Measures ◽  
One Step ◽  
Molecular Biology Method ◽  
Sensitivity Specificity ◽  
The University

Background. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has rapidly spread worldwide from the beginning of 2020. Quantitative reverse transcription-PCR (RT-qPCR) is, to this day, the preferred methodology for viral RNA detection, even if not without problems. To overcome some of the limitations still existing for the detection and quantification of nucleic acids in various applications, the use of one-step reverse transcription-droplet digital PCR (RT-ddPCR) has been established. The purpose of this study was, then, to evaluate the efficacy of ddPCR for the detection of SARS-CoV-2 RNA in nasopharyngeal swabs, optimizing the detection of low-viral load-burdened samples. Methods. The RT-ddPCR workflow was validated for sensitivity, specificity, linearity, reproducibility, and precision using samples from 90 COVID-19-infected patients referred to the Department of Laboratory Medicine of the University Hospital of Udine (Italy). Results. The present study shows that RT-ddPCR allows the detection of as low as 10.3 copies of a SARS-COV-2 E-gene per sample with a higher level of accuracy and precision, especially at low concentration. Conclusion. During the postpeak phase of the SARS-CoV-2 pandemic, it is essential to rely on a highly robust molecular biology method to identify infected subjects, whether they have symptoms or not, in order to prepare appropriate containment measures.

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.

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