Prevalence of Sugarcane yellow leaf virus in Sugarcane-Producing Regions in Kenya Revealed by Reverse-Transcription Loop-Mediated Isothermal Amplification Method

Plant Disease ◽  
2016 ◽  
Vol 100 (2) ◽  
pp. 260-268 ◽  
Author(s):  
Ruth L. Amata ◽  
Emmanuel Fernandez ◽  
Denis Filloux ◽  
Darren P. Martin ◽  
Philippe Rott ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Yellow Leaf ◽  
Rt Pcr ◽  
Pairwise Identity ◽  
Sugarcane Yellow Leaf Virus ◽  
Actual Distribution ◽  
Loop Mediated Isothermal Amplification ◽  
Leaf Virus

Yellow leaf (YL) is a disease of sugarcane that is currently widespread throughout most American and Asian sugarcane-producing countries. However, its actual distribution in Africa remains largely unknown. A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to facilitate and improve the detection of Sugarcane yellow leaf virus (SCYLV), the causal agent of YL. The RT-LAMP assay was found to be comparable with or superior to conventional RT-polymerase chain reaction (PCR) for the detection of SCYLV genotypes CUB and BRA in infected sugarcane ‘C132-81’ and ‘SP71-6163’, respectively. Additionally, 68 sugarcane samples that tested negative by RT-PCR were found positive by RT-LAMP, whereas the RT-LAMP assay failed to detect SCYLV in only 5 samples that tested positive by RT-PCR. Combining RT-PCR and RT-LAMP data enabled the detection of SCYLV in 86 of 183 Kenyan sugarcane plants, indicating high SCYLV prevalence throughout the country (ranging from 36 to 64% in individual counties). Seminested PCR assays were developed that enabled the amplification of a fragment encompassing the capsid protein coding region gene and its flanking 5′ and 3′ genomic regions. Sequences of this fragment for four Kenyan SCYLV isolates indicated that they shared 99.2 to 99.6% pairwise identity with one another and clearly clustered phylogenetically with SCYLV-BRA genotype isolates. To our knowledge, this is the first report of SCYLV in Kenya.

scholarly journals Rapid Detection of Peste des Petits Ruminants Virus (PPRV) Nucleic Acid Using a Novel Low-Cost Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for Future Use in Nascent PPR Eradication Programme

Viruses ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 699 ◽  
Author(s):  
Mahapatra ◽  
Howson ◽  
Fowler ◽  
Batten ◽  
Flannery ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Reverse Transcription ◽  
Lamp Assay ◽  
Early Warning Systems ◽  
Isothermal Amplification ◽  
Effective Control ◽  
Peste Des Petits Ruminants ◽  
Rt Pcr ◽  
Eradication Programme ◽  
Loop Mediated Isothermal Amplification

Peste des petits ruminants (PPR) is a disease of small ruminants caused by peste des petits ruminants virus (PPRV), and is endemic in Asia, the Middle East and Africa. Effective control combines the application of early warning systems, accurate laboratory diagnosis and reporting, animal movement restrictions, suitable vaccination and surveillance programs, and the coordination of all these measures by efficient veterinary services. Molecular assays, including conventional reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR (RT-qPCR) have improved the sensitivity and rapidity of diagnosing PPR. However, currently these assays are only performed within laboratory settings; therefore, the development of field diagnostics for PPR would improve the fast implementation of control policies, particularly when PPR has been targeted to be eradicated by 2030. Loop-mediated isothermal amplification (LAMP) assays are simple to use, rapid, and have sensitivity and specificity within the range of RT-qPCR; and can be performed in the field using disposable consumables and portable equipment. This study describes the development of a novel RT-LAMP assay for the detection of PPRV nucleic acid by targeting the N-protein gene. The RT-LAMP assay was evaluated using cell culture propagated PPRVs, field samples from clinically infected animals and samples from experimentally infected animals encompassing all four lineages (I-IV) of PPRV. The test displayed 100% concordance with RT-qPCR when considering an RT-qPCR cut-off value of CT >40. Further, the RT-LAMP assay was evaluated using experimental and outbreak samples without prior RNA extraction making it more time and cost-effective. This assay provides a solution for a pen-side, rapid and inexpensive PPR diagnostic for use in the field in nascent PPR eradication programme.

scholarly journals Comparative Diagnostic Performance of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Kit for the Rapid Detection of SARS-CoV-2

Pathogens ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1629
Author(s):  
Alexander Domnich ◽  
Andrea Orsi ◽  
Donatella Panatto ◽  
Vanessa De Pace ◽  
Valentina Ricucci ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Diagnostic Performance ◽  
Point Of Care ◽  
Lamp Assay ◽  
Laboratory Diagnosis ◽  
Isothermal Amplification ◽  
Molecular Diagnostic ◽  
Rt Pcr ◽  
Laboratory Equipment ◽  
Loop Mediated Isothermal Amplification

Although the reverse transcription-polymerase chain reaction (RT-PCR) is considered a standard-of-care assay for the laboratory diagnosis of SARS-CoV-2, several limitations of this method have been described. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is an alternative molecular assay and is potentially able to overcome some intrinsic shortcomings of RT-PCR. In this study, we evaluated the diagnostic performance of the novel HG COVID-19 RT-LAMP assay. In this retrospective analysis, a total of 400 routinely collected leftover nasopharyngeal samples with a known RT-PCR result were tested by means of the HG COVID-19 RT-LAMP assay. The overall sensitivity and specificity values of HG COVID-19 RT-LAMP versus RT-PCR were 97.0% (95% CI: 93.6–98.9%) and 98.5% (95% CI: 95.7–99.7%), respectively. Inter-assay agreement was almost perfect (κ = 0.96). Concordance was perfect in samples with high viral loads (cycle threshold < 30). The average time to a positive result on RT-LAMP was 17 min. HG COVID-19 RT-LAMP is a reliable molecular diagnostic kit for detecting SARS-CoV-2, and its performance is comparable to that of RT-PCR. Shorter turnaround times and the possibility of performing molecular diagnostics in the point-of-care setting make it a valuable option for facilities without sophisticated laboratory equipment.

scholarly journals High diagnostic performance of a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) kit for the rapid detection of SARS-CoV-2

2021 ◽  
Author(s):  
Alexander Domnich ◽  
Andrea Orsi ◽  
Donatella Panatto ◽  
Vanessa De Pace ◽  
Valentina Ricucci ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Diagnostic Performance ◽  
Point Of Care ◽  
Lamp Assay ◽  
Laboratory Diagnosis ◽  
Isothermal Amplification ◽  
Molecular Diagnostic ◽  
Rt Pcr ◽  
Laboratory Equipment ◽  
Loop Mediated Isothermal Amplification

Abstract Although the reverse transcription polymerase chain reaction (RT-PCR) is considered a standard-of-care assay for the laboratory diagnosis of SARS-CoV-2, several limitations of this method have been described. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is an alternative molecular assay and is potentially able to overcome some intrinsic shortcomings of RT-PCR. In this study, we evaluated the diagnostic performance of the novel HG COVID-19 RT-LAMP assay. In this retrospective analysis, a total of 400 routinely collected leftover nasopharyngeal samples with a known RT-PCR result were tested by means of the HG COVID-19 RT-LAMP assay. The overall sensitivity and specificity values of HG COVID-19 RT-LAMP versus RT-PCR were 97.0% (95% CI: 93.6–98.9%) and 98.5% (95% CI: 95.7–99.7%), respectively. Inter-assay agreement was almost perfect (κ = 0.96). Concordance was perfect in samples with high viral loads (cycle threshold <30). The average time to a positive result on RT-LAMP was 17 min. HG COVID-19 RT-LAMP is a reliable molecular diagnostic kit for detecting SARS-CoV-2, and its performance is comparable to that of RT-PCR. Shorter turnaround times and the possibility of performing molecular diagnostics in the point-of-care setting make it a valuable option for facilities without sophisticated laboratory equipment.

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 Colorimetric Reverse Transcription–Loop-Mediated Isothermal Amplification Assay for Rapid Detection of SARS-CoV-2

2021 ◽  
Author(s):  
Meng Yee Lai ◽  
Soo Nee Tang ◽  
Yee Ling Lau
Keyword(s):  
Sensitivity And Specificity ◽  
Reverse Transcription ◽  
Virus Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Sensitivity ◽  
Loop Mediated Isothermal Amplification ◽  
Resource Limited ◽  
Detection Technology ◽  
Simple Detection

Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been spreading rapidly all over the world. In the absence of effective treatments or a vaccine, there is an urgent need to develop a more rapid and simple detection technology of COVID-19. We describe a WarmStart colorimetric reverse transcription–loop-mediated isothermal amplification (RT-LAMP) assay for the detection of SARS-CoV-2. The detection limit for this assay was 1 copy/µL SARS-CoV-2. To test the clinical sensitivity and specificity of the assay, 37 positive and 20 negative samples were used. The WarmStart colorimetric RT-LAMP had 100% sensitivity and specificity. End products were detected by direct observation, thereby eliminating the need for post-amplification processing steps. WarmStart colorimetric RT-LAMP provides an opportunity to facilitate virus detection in resource-limited settings without a sophisticated diagnostic infrastructure.

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