Development of a reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) that detects enteroviruses by targeting the highly conserved 5′-UTR region

Daskou, M.; Dimitriou, Tilemachos G.; Kouklamani-Giannouli, G; Nikolaidis, Marios; Mossialos, Dimitris; Amoutzias, Grigoris D.; Markoulatos, Panayotis

doi:10.1007/s11262-020-01732-w

Cited by 3 publications

(4 citation statements)

References 34 publications

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“…LAMP is a novel, rapid, specific, sensitive, and simple nucleic acid amplification technique located in six regions in the target gene using four primers and amplified RNA at a constant temperature. The most significant one is its rapidity; LAMP allows immediate diagnosis in just 30–50 min 36 . Recently, LAMP has been utilized for the rapid detection of some pathogens, and their results showed high accuracy and availability in the diagnosis of the corresponding pathogens 37‐39 .…”

Section: Discussionmentioning

confidence: 99%

Pooled analysis of LAMP assay for the diagnosis of norovirus infection

Qian

Duan

Huang

et al. 2021

Clinical Laboratory Analysis

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Background Rapid laboratory detection is essential to diagnose norovirus infection. LAMP has many advantages compared with RT‐PCR for detecting norovirus, including high sensitivity, high specificity, rapidity, low cost, and intuitive results, which can be easily read with the naked eye with the help of color‐based reporters. In this study, we intend to analyze the accuracy of LAMP methods for the diagnosis of norovirus infection. Methods Two researchers independently retrieved relevant literature up to January 2021 (PubMed, Web of Science, Cochrane Library, Embase, CNKI, Wan Fang, and VIP). The researchers screened all articles and extracted their research data for meta‐analysis. QUADAS‐2 tool was used to evaluate the quality of the included studies by Review Manager 5.3. Forest plots were performed by Meta‐DiSc 1.4 to evaluate the accuracy of the test. Deeks’ funnel plot symmetry tests were conducted by Stata 15.0 to check the potential publication bias. Results Eleven sets of data extracted from the eight included studies were included for meta‐analysis. For the detection of norovirus, the pooled sensitivity, specificity, positive LR, negative LR, diagnostic OR, and their 95% CI were 0.96 (0.95–0.97), 0.99 (0.99–1.00), 91.14 (31.88–260.56), 0.06 (0.04–0.09), and 1473.68 (562.96–3857.70), respectively. Besides, AUC in the SROC curve was 0.9920. Conclusion LAMP had high sensitivity and specificity in terms of the diagnosis of norovirus infection. However, further extension of this approach should be researched to ensure the accuracy and practicability of this hopeful test in the future.

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Section: Discussionmentioning

confidence: 99%

Pooled analysis of LAMP assay for the diagnosis of norovirus infection

Qian

Duan

Huang

et al. 2021

Clinical Laboratory Analysis

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“…Compared to the previous report, the present assay demonstrates excellent sensitivity regarding Enterovirus B and D ( 0·30 CCID 50 assay −1 versus 0·75 CCID 50 assay −1 ) and a smaller one regarding Enterovirus A (3·00 CCID 50 assay −1 versus 0·75 CCID 50 assay −1 ) and Enterovirus C (0·30 CCID 50 assay −1 versus 0·075 CCID 50 assay −1 ) (Daskou et al . 2020) The design of a primer set for the detection of all the human Enterovirus species ( EV A–D ) is particularly challenging and for that reason all the enterovirus species, were not detected with the same sensitivity.…”

Section: Discussionmentioning

confidence: 99%

“…In a recent study by our group (Daskou et al . 2020), we established a conventional isothermal reverse transcription assay (RT‐Loop‐Mediated Amplification, RT‐LAMP) for the detection of all four human Enterovirus species ( EV A‐D ) by targeting the 5′ UTR region. This assay was performed in three separate stages with a set of four primers (without loop primers) and the analysis of LAMP products was performed through agarose gel electrophoresis.…”

Section: Introductionmentioning

confidence: 99%

WarmStart colorimetric RT‐LAMP for the rapid, sensitive and specific detection of Enteroviruses A–D targeting the 5′UTR region

Daskou

Dimitriou

Alexopoulou

et al. 2020

J. Appl. Microbiol.

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Aims: The aim of the present study was to develop a colorimetric LAMP assay for the detection of enteroviruses belonging to species AD targeting the 5 0 untranslated region (5 0 UTR) of enteroviruses genome. Methods and Results: The RNA was converted to cDNA by the reverse transcriptase and then amplified via LAMP by the WarmStartâBst DNA polymerase, simultaneously in a single reaction tube, so we shortened the reaction time to 50 min. The sensitivity of the assay regarding Enterovirus B, C and D was determined to be 0Á30 CCID 50 assay À1 while the sensitivity for Enterovirus A was 3Á00 CCID 50 assay À1. The assay demonstrated high specificity and sensitivity for the detection of 45 reference strains of Enteroviruses AD and validated on 20 clinical isolates. Conclusions: This assay can be used as a diagnostic tool for the rapid, sensitive and specific detection of enteroviruses, easily implemented in small clinical and research laboratories since LAMP amplicons were visualized by colour changes eliminating the requirement for post-amplification processing steps. Significance and Impact of the Study: We developed a colorimetric assay ideal for field situations for the detection of enteroviruses, by targeting the 5 0 UTR. This assay demonstrated high specificity and sensitivity, based on its performance on 45 EV AD reference strains, on 20 EV B clinical isolates and on three non-enteroviral RNA viruses.

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“…The great burden of viral gastroenteritis on health care due to related illness and hospitalization highlights the need for fast, sensitive, and reliable diagnostic assays to guide infection control measures. The loop-mediated isothermal amplification (LAMP) assay has been successfully implemented for the detection of a wide range of pathogens of infectious diseases, including SARS-CoV-2 ( 10 ), Zika virus ( 11 , 12 ), enteroviruses ( 13 ), Dengue virus ( 14 ), tuberculosis ( 15 , 16 ), Ebola virus ( 17 ), influenza virus ( 18 ), Helicobacter pylori ( 19 ), Plasmodium falciparum ( 20 ), Leishmania ( 20 ), Trypanosoma ( 20 ), Treponema pallidum ( 21 ), and Haemophilus ducreyi ( 21 ). LAMP is a highly specific and sensitive reaction of DNA amplification, as LAMP significantly reduces the amplification time from 2 to 3 h to 15 to 30 min compared to that of PCR.…”

Section: Introductionmentioning

confidence: 99%