scholarly journals Two extraction-free reverse transcription loop-mediated isothermal amplification assays for detection of SARS-CoV-2

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Meng Yee Lai ◽  
Fatma Diyana Mohd Bukhari ◽  
Nur Zulaikha Zulkefli ◽  
Ilyiana Ismail ◽  
Nur Izati Mustapa ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Virus Isolation ◽  
Direct Detection ◽  
Isothermal Amplification ◽  
Rapid Screening ◽  
Nasopharyngeal Swab ◽  
Purification Step ◽  
Simple Method ◽  
Loop Mediated Isothermal Amplification ◽  
Rna Purification

Abstract Background Current assays for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rely on time consuming, costly and laboratory based methods for virus isolation, purification and removing inhibitors. To address this limitation, we propose a simple method for testing RNA from nasopharyngeal swab samples that bypasses the RNA purification step. Methods In the current project, we have described two extraction-free reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for the detection of SARS-CoV-2 by using E gene and RdRp gene as the targets. Results Here, results showed that reverse transcription loop-mediated isothermal amplification assays with 88.4% sensitive (95% CI: 74.9–96.1%) and 67.4% sensitive (95% CI: 51.5–80.9%) for E gene and RdRp gene, respectively. Conclusion Without the need of RNA purification, our developed RT-LAMP assays for direct detection of SARS-CoV-2 from nasopharyngeal swab samples could be turned into alternatives to qRT-PCR for rapid screening.

scholarly journals Detection and discrimination of multiple strains of Zika virus by reverse transcription-loop-mediated isothermal amplification

2020 ◽  
Vol 48 (1) ◽  
Author(s):  
Hiroka Aonuma ◽  
Itoe Iizuka-Shiota ◽  
Tokio Hoshina ◽  
Shigeru Tajima ◽  
Fumihiro Kato ◽  
...  
Keyword(s):  
Virus Strain ◽  
Reverse Transcription ◽  
Zika Virus ◽  
Virus Disease ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Simple Method ◽  
Loop Mediated Isothermal Amplification ◽  
Specificity And Sensitivity ◽  
Virus Strains

Abstract Background Monitoring both invasion of Zika virus disease into free countries and circulation in endemic countries is essential to avoid a global pandemic. However, the difficulty lies in detecting Zika virus due to the large variety of mutations in its genomic sequence. To develop a rapid and simple method with high accuracy, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) was adopted for the detection of Zika virus strains derived from several countries. Results Common primers for RT-LAMP were designed based on the genomic sequences of two standard Zika strains: African lineage, MR-766, and Asian lineage, PRVABC59. RT-LAMP reactions using a screened primer set, targeting the NS3 region, detected both Zika virus strains. The minimum detectable quantity was 3 × 10−2 ng of virus RNA. Measurable lag of reaction times among strains was observed. The RT-LAMP method amplified the target virus sequence from the urine and serum of a patient with a travel history in the Caribbean Islands and also provided a prediction about which lineage of Zika virus strain was present. Conclusions The RT-LAMP method using a well-optimized primer set demonstrated high specificity and sensitivity for the detection of Zika virus strains with a variety in genomic RNA sequences. In combination with the simplicity of LAMP reaction in isothermal conditions, the optimized primer set established in this study may facilitate rapid and accurate diagnosis of Zika fever patients with virus strain information.

scholarly journals Rapid Detection of Novel Coronavirus (COVID-19) by Reverse Transcription-Loop-Mediated Isothermal Amplification

2020 ◽  
Author(s):  
Laura E. Lamb ◽  
Sarah N. Bartolone ◽  
Elijah Ward ◽  
Michael B. Chancellor
Keyword(s):  
Diagnostic Test ◽  
Reverse Transcription ◽  
Simulated Patient ◽  
Isothermal Amplification ◽  
Rapid Screening ◽  
Health Concern ◽  
Loop Mediated Isothermal Amplification ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Novel Coronavirus

AbstractNovel Corona virus (COVID-19 or 2019-nCoV) is an emerging global health concern that requires a rapid diagnostic test. Quantitative reverse transcription PCR (qRT-PCR) is currently the standard for COVID-19 detection; however, Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) may allow for faster and cheaper field based testing at point-of-risk. The objective of this study was to develop a rapid screening diagnostic test that could be completed in under 30 minutes. Simulated patient samples were generated by spiking serum, urine, saliva, oropharyngeal swabs, and nasopharyngeal swabs with a portion of the COVID-19 nucleic sequence. The samples were tested using RT-LAMP as well as by conventional qRT-PCR. Specificity of the RT-LAMP was evaluated by also testing against other related coronaviruses. RT-LAMP specifically detected COVID-19 in simulated patient samples. This test was performed in under 30 minutes. This approach could be used for monitoring of exposed individuals or potentially aid with screening efforts in the field and potential ports of entry.

scholarly journals RNA purification-free detection of SARS-CoV-2 using reverse transcription loop-mediated isothermal amplification (RT-LAMP)

2022 ◽  
Vol 50 (1) ◽  
Author(s):  
Meng Yee Lai ◽  
Jeyanthi Suppiah ◽  
Ravindran Thayan ◽  
Ilyiana Ismail ◽  
Nur Izati Mustapa ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Processing Speed ◽  
Diagnostic Testing ◽  
Lamp Assay ◽  
Extraction Methods ◽  
Turnaround Time ◽  
Isothermal Amplification ◽  
Chelating Resin ◽  
Loop Mediated Isothermal Amplification ◽  
Rna Purification

Abstract Background Current diagnosis of SARS-CoV-2 infection relies on RNA purification prior to amplification. Typical extraction methods limit the processing speed and turnaround time for SARS-CoV-2 diagnostic testing. Methods Here, we applied reverse transcription loop-mediated isothermal amplification directly onto human clinical swabs samples to amplify the RNA from SARS-CoV-2 swab samples after processing with chelating resin. Results By testing our method on 64 samples, we managed to develop an RT-LAMP assay with 95.9% sensitivity (95% CI 86 to 99.5%) and 100% specificity (95% CI 78.2–100%). Conclusion The entire process including sample processing can be completed in approximately 50 min. This method has promising potential to be applied as a fast, simple and inexpensive diagnostic tool for the detection of SARS-CoV-2.

scholarly journals SARS-CoV-2 On-the-Spot Virus Detection Directly from Patients

2020 ◽  
Author(s):  
Nadav Ben-Assa ◽  
Rawi Naddaf ◽  
Tal Gefen ◽  
Tal Capucha ◽  
Haitham Hajjo ◽  
...  
Keyword(s):  
Standard Method ◽  
Reverse Transcription ◽  
Virus Detection ◽  
Isothermal Amplification ◽  
Purification Step ◽  
Spot Virus ◽  
Quantitative Reverse Transcription Pcr ◽  
Reverse Transcription Pcr ◽  
Rna Purification ◽  
Nose And Throat

AbstractMany countries are currently in a lockdown state due to the SARS-CoV-2 pandemic. One key aspect to transition safely out of lockdown is to continuously test the population for infected subjects. Currently, detection is performed at points of care using quantitative reverse-transcription PCR (RT-qPCR), and requires dedicated professionals and equipment. Here, we developed a protocol based on Reverse Transcribed Loop-Mediated Isothermal Amplification (RT-LAMP) for detection of SARS-CoV-2. This protocol is applied directly on SARS-CoV-2 nose and throat swabs, with no RNA purification step required. We tested this protocol on over 180 suspected patients, and compared its results to the standard method. We further succeeded to apply the protocol on self-sampled saliva from confirmed cases. Since the proposed protocol provides results on-the-spot, and can detect SARS-CoV-2 from saliva, it can allow simple and continuous surveillance of the community.

scholarly journals Preliminary optimisation of a simplified sample preparation method to permit direct detection of SARS-CoV-2 within saliva samples using reverse-transcription loop-mediated isothermal amplification (RT-LAMP)

2020 ◽  
Author(s):  
Emma Howson ◽  
Stephen Kidd ◽  
Jason Sawyer ◽  
Claire Cassar ◽  
David Cross ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Reverse Transcription ◽  
Preparation Method ◽  
Direct Detection ◽  
Saliva Sample ◽  
Rna Extraction ◽  
Isothermal Amplification ◽  
Molecular Testing ◽  
Sample Preparation Method ◽  
Loop Mediated Isothermal Amplification

We describe the optimization of a simplified sample preparation method which permits rapid and direct detection of SARS-CoV-2 RNA within saliva using reverse-transcription loop-mediated isothermal amplification (RT-LAMP). Treatment of saliva samples prior to RT-LAMP by dilution 1:1 in MucolyseTM, followed by dilution (within the range of 1 in 5 to 1 in 40) in 10% (w/v) Chelex 100 Resin and a 98oC heat step for 2 minutes enabled detection of SARS-CoV-2 RNA in all positive saliva samples tested, with no amplification detected in pooled negative saliva. The time to positivity for which SARS-CoV-2 RNA was detected in these positive saliva samples was proportional to the real-time reverse-transcriptase PCR cycle threshold (CT), with SARS-CoV-2 RNA detected in as little as 05:43 (CT 21.08), 07:59 (CT 24.47) and 08:35 (CT 25.27) minutes, respectively. The highest CT where direct RT-LAMP detected SARS-CoV-2 RNA was 31.39 corresponding to a 1 in 40 dilution of a positive saliva sample (1:1 in MucolyseTM) with a starting CT of 25.27. When RT-LAMP was performed on pools of SARS-CoV-2 negative saliva samples spiked with whole inactivated SARS-CoV-2 virus, RNA was detected at dilutions spanning 1 in 5 to 1 in 160 representing CTs spanning 22.49-26.43. Here we describe a simple but critical rapid sample preparation method which can be used up front of RT-LAMP to permit direct detection of SARS-CoV-2 within saliva samples. Saliva is a sample which can be collected non-invasively without the use of highly skilled staff and critically can be obtained from both healthcare and home settings. Critically, this approach overcomes both the requirement and validation of different swabs and the global bottleneck in obtaining RNA extraction robots and reagents to enable molecular testing by PCR. Such testing opens the possibility of public health approaches for effective intervention to control the COVID-19 pandemic through regular SARS-CoV-2 testing at a population scale, combined with isolation and contact tracing for positive cases.

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