Improvement in the sensitivity of viroid detection by adapting the reverse transcription step in one-step RT-qPCR assays

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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Detection of influenza A virus from live-bird market poultry swab samples in China by a pan-IAV, one-step reverse-transcription FRET-PCR

Scientific Reports ◽

10.1038/srep30015 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 6

Author(s):

Lu Luan ◽

Zhihao Sun ◽

Bernhard Kaltenboeck ◽

Ke Huang ◽

Min Li ◽

...

Keyword(s):

Influenza A Virus ◽

Reverse Transcription ◽

Influenza A ◽

Live Bird Market ◽

One Step ◽

Live Bird

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One-Step Reverse Transcription-Polymerase Chain Reaction for Ebola and Marburg Viruses

Osong Public Health and Research Perspectives ◽

10.1016/j.phrp.2016.04.004 ◽

2016 ◽

Vol 7 (3) ◽

pp. 205-209 ◽

Cited By ~ 4

Author(s):

Sun-Whan Park ◽

Ye-Ji Lee ◽

Won-Ja Lee ◽

Youngmee Jee ◽

WooYoung Choi

Keyword(s):

Polymerase Chain Reaction ◽

Reverse Transcription ◽

Chain Reaction ◽

One Step ◽

Polymerase Chain

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

Frontiers in Veterinary Science ◽

10.3389/fvets.2020.579432 ◽

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.

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Robust Saliva-Based RNA Extraction-Free One-Step Nucleic Acid Amplification Test for Mass SARS-CoV-2 Monitoring

Molecules ◽

10.3390/molecules26216617 ◽

2021 ◽

Vol 26 (21) ◽

pp. 6617

Author(s):

Eva Rajh ◽

Tina Šket ◽

Arne Praznik ◽

Petra Sušjan ◽

Alenka Šmid ◽

...

Keyword(s):

Oral Cavity ◽

Reverse Transcription ◽

Screening Program ◽

Quantitative Polymerase Chain Reaction ◽

Rna Extraction ◽

Diagnostic Sensitivity ◽

Nucleic Acid Amplification ◽

Nasopharyngeal Swab ◽

Viral Spread ◽

One Step

Early diagnosis with rapid detection of the virus plays a key role in preventing the spread of infection and in treating patients effectively. In order to address the need for a straightforward detection of SARS-CoV-2 infection and assessment of viral spread, we developed rapid, sensitive, extraction-free one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) tests for detecting SARS-CoV-2 in saliva. We analyzed over 700 matched pairs of saliva and nasopharyngeal swab (NSB) specimens from asymptomatic and symptomatic individuals. Saliva, as either an oral cavity swab or passive drool, was collected in an RNA stabilization buffer. The stabilized saliva specimens were heat-treated and directly analyzed without RNA extraction. The diagnostic sensitivity of saliva-based RT-qPCR was at least 95% in individuals with subclinical infection and outperformed RT-LAMP, which had at least 70% sensitivity when compared to NSBs analyzed with a clinical RT-qPCR test. The diagnostic sensitivity for passive drool saliva was higher than that of oral cavity swab specimens (95% and 87%, respectively). A rapid, sensitive one-step extraction-free RT-qPCR test for detecting SARS-CoV-2 in passive drool saliva is operationally simple and can be easily implemented using existing testing sites, thus allowing high-throughput, rapid, and repeated testing of large populations. Furthermore, saliva testing is adequate to detect individuals in an asymptomatic screening program and can help improve voluntary screening compliance for those individuals averse to various forms of nasal collections.

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