scholarly journals Rapid Detection of SARS-CoV-2 Using Reverse transcription RT-LAMP method

2020 ◽  
Author(s):  
Weihua Yang ◽  
Xiaofei Dang ◽  
Qingxi Wang ◽  
Mingjie Xu ◽  
Qianqian Zhao ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Reverse Transcription ◽  
Virus Disease ◽  
Lamp Assay ◽  
Nucleic Acid Amplification ◽  
N Gene ◽  
Nucleic Acid Detection ◽  
Lamp Method ◽  
Rt Pcr ◽  
Rapid Amplification

AbstractCorona Virus Disease 2019 (COVID-19) is a recently emerged life-threatening disease caused by SARS-CoV-2. Real-time fluorescent PCR (RT-PCR) is the clinical standard for SARS-CoV-2 nucleic acid detection. To detect SARS-CoV-2 early and control the disease spreading on time, a faster and more convenient method for SARS-CoV-2 nucleic acid detecting, RT-LAMP method (reverse transcription loop-mediated isothermal amplification) was developed. RNA reverse transcription and nucleic acid amplification were performed in one step at 63 °C isothermal conditions, and the results can be obtained within 30 minutes. ORF1ab gene, E gene and N gene were detected at the same time. ORF1ab gene was very specific and N gene was very sensitivity, so they can guarantee both sensitivity and specificity for SARS-CoV-2. The sensitivity of RT-LAMP assay is similar to RT-PCR, and specificity was 99% as detecting 208 clinical specimens. The RT-LAMP assay reported here has the advantages of rapid amplification, simple operation, and easy detection, which is useful for the rapid and reliable clinical diagnosis of SARS-CoV-2.

scholarly journals Rapid and accurate detection of three genes in SARS-CoV-2 using RT-LAMP method

2020 ◽  
Author(s):  
Weihua Yang ◽  
Xiaofei Dang ◽  
Qingxi Wang ◽  
Mingjie Xu ◽  
Qianqian Zhao ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Sensitivity And Specificity ◽  
Reverse Transcription ◽  
Lamp Assay ◽  
High Specificity ◽  
N Gene ◽  
Clinical Samples ◽  
Lamp Method ◽  
E Gene ◽  
Primer Sets

Abstract Background Corona Virus Disease 2019 (COVID-19) is a recently emerged life-threatening disease caused by SARS-CoV-2. It has the characteristics of rapid transmission, difficult treatment, rapid deterioration, and high mortality rate. Reliable and rapid diagnosis of SARS-CoV-2 infection is critical to treat patients and control transmission in time. Methods To detect SARS-CoV-2 nucleic acid faster and more convenient, RT-LAMP method (reverse transcription loop-mediated isothermal amplification) was developed. ORF1ab gene, E gene and N gene were selected as the target genes, because ORF1ab gene is the most specific and N gene is the most sensitive, while E gene has both sensitivity and specificity. Over thirty primer sets for them were designed, then the primer sets with rapid response and high specificity were screened out and their loop primers were designed and selected in order to improve the reaction speed further. Using these primers, RNA reverse transcription and nucleic acid amplification were performed in one step at 63 ℃ isothermal conditions, and the results can be judged by naked eyes through color change within 30 minutes. Finally, the optimal primer set for each gene was verified with over 208 clinical samples. Results ORF1ab gene, E gene and N gene were detected simultaneously by this method. ORF1ab gene has high specificity and the same sensitivity as RT-PCR currently used in clinic. Although N gene is less specific than ORF1ab, it is 80 times more sensitive than ORF1ab. The sensitivity and specificity of E gene are between them. Simultaneous detection of these three genes can neither cause false positives nor miss samples with low virus concentration, ensuring the sensitivity and specificity of SARS-CoV-2 detection. BLAST comparing results showed the primers for the three genes were highly specific for SARS-CoV-2. And the sequencing results of RT-LAMP products showed that the amplified fragments were unique to SARS-CoV-2. The accuracy of RT-LAMP assay was 99% as detecting 208 clinical specimens. Conclusions The RT-LAMP assay reported here has the advantages of rapid amplification, simple operation, and easy detection, which is useful for the rapid and reliable clinical diagnosis of SARS-CoV-2.

scholarly journals Schnellnachweis von SARS-CoV-2 mit recombinase polymerase amplification

BIOspektrum ◽  
2020 ◽  
Vol 26 (6) ◽  
pp. 624-627
Author(s):  
Ole Behrmann ◽  
Iris Bachmann ◽  
Frank Hufert ◽  
Gregory Dame
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Dna Polymerase ◽  
Fluorescent Probes ◽  
Reverse Transcription ◽  
Nucleic Acid Detection ◽  
Detection Scheme ◽  
Recombination Proteins ◽  
Target Dna ◽  
Rapid Amplification

Abstract The COVID-19 pandemic highlights the need for fast and simple assays for nucleic acid detection. As an isothermal alternative to RT-qPCR, we outline the development of a detection scheme for SARS-CoV-2 RNA based on reverse transcription recombinase polymerase amplification (RT-RPA) technology. RPA uses recombination proteins in combination with a DNA polymerase for rapid amplification of target DNA at a constant temperature (39–42 °C) within 10 to 20 minutes and can be monitored in real-time with fluorescent probes.

scholarly journals IsoPCR: An Analytically Sensitive, Nested, Multiplex Nucleic Acid Amplification Method

2013 ◽  
Vol 59 (2) ◽  
pp. 436-439 ◽  
Author(s):  
Martin Jensen Søe ◽  
Mikkel Rohde ◽  
Jens Mikkelsen ◽  
Peter Warthoe
Keyword(s):  
Nucleic Acid ◽  
Candida Glabrata ◽  
Simultaneous Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Qpcr Assay ◽  
Nucleic Acid Amplification ◽  
Molecular Diagnostic ◽  
Nucleic Acid Detection ◽  
Amplification Method

BACKGROUND Nucleic acid tests that can simultaneously detect multiple targets with high sensitivity, specificity, and speed are highly desirable. To meet this need, we developed a new approach we call the isoPCR method. METHODS The isoPCR method is a 2-stage nested-like nucleic acid amplification method that combines a single multiplex preamplification PCR with subsequent distinct detection of specific targets by use of isothermal amplification. We compared isoPCR to nested quantitative PCR (qPCR), loop-mediated isothermal amplification (LAMP), and nested LAMP (PCR followed by LAMP), for detection of DNA from Candida glabrata. We evaluated the method's multiplex capability for detecting low copy numbers of pathogens commonly involved in sepsis. RESULTS IsoPCR provided detection of 1 copy of Candida glabrata, an LOD that was 5-fold lower than a nested qPCR assay (5 copies), while the amplification time was simultaneously halved. Similarly, the LOD for isoPCR was lower than that for a LAMP assay (1000 copies) and a nested LAMP assay (5 copies). IsoPCR required recognition of 6 regions for detection, thereby providing a theoretically higher specificity compared to nested qPCR (4 regions). The isoPCR multiplexing capability was demonstrated by simultaneous detection of 4 pathogens with individual LODs of 10 copies or fewer. Furthermore, the specificity of isoPCR was demonstrated by successful pathogen detection from samples with more than 1 pathogen present. CONCLUSIONS IsoPCR provides a molecular diagnostic tool for multiplex nucleic acid detection, with an LOD down to 1 copy, high theoretical specificity, and halving of the amplification time compared to a nested qPCR assay.

scholarly journals Rapid direct nucleic acid amplification test without RNA extraction for SARS-CoV-2 using a portable PCR thermocycler

2020 ◽  
Author(s):  
Soon Keong Wee ◽  
Suppiah Paramalingam Sivalingam ◽  
Eric Peng Huat Yap
Keyword(s):  
Nucleic Acid ◽  
Reverse Transcription ◽  
Rna Extraction ◽  
Cost Effective ◽  
Turnaround Time ◽  
Nucleic Acid Amplification ◽  
Detection Methods ◽  
N Gene ◽  
Direct Pcr ◽  
Viral Lysis

There is an ongoing worldwide coronavirus disease 2019 (Covid-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At present, confirmatory diagnosis is by reverse transcription polymerase chain reaction (RT-PCR), typically taking several hours and requiring a molecular laboratory to perform. There is an urgent need for rapid, simplified and cost-effective detection methods. We have developed and analytically validated a protocol for direct rapid extraction-free PCR (DIRECT-PCR) detection of SARS-CoV-2 without the need for nucleic acid purification. As few as 6 RNA copies per reaction of viral nucleocapsid (N) gene from respiratory samples such as sputum and nasal exudate can be detected directly using our one-step inhibitor-resistant assay. The performance of this assay was validated on a commercially available portable PCR thermocycler. Viral lysis, reverse transcription, amplification and detection are achieved in a single-tube homogeneous reaction within 36 minutes. This minimized hands-on time, reduces turnaround-time for sample-to-result and obviates the need for RNA purification reagents. It could enable wider use of Covid-19 testing for diagnosis, screening and research in countries and regions where laboratory capabilities are limiting.

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 Rapid Direct Nucleic Acid Amplification Test without RNA Extraction for SARS-CoV-2 Using a Portable PCR Thermocycler

Genes ◽  
2020 ◽  
Vol 11 (6) ◽  
pp. 664 ◽  
Author(s):  
Soon Keong Wee ◽  
Suppiah Paramalingam Sivalingam ◽  
Eric Peng Huat Yap
Keyword(s):  
Nucleic Acid ◽  
Reverse Transcription ◽  
Rna Extraction ◽  
Cost Effective ◽  
Turnaround Time ◽  
Nucleic Acid Amplification ◽  
Detection Methods ◽  
N Gene ◽  
Direct Pcr ◽  
Viral Lysis

There is an ongoing worldwide coronavirus disease 2019 (Covid-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At present, confirmatory diagnosis is by reverse transcription polymerase chain reaction (RT-PCR), typically taking several hours and requiring a molecular laboratory to perform. There is an urgent need for rapid, simplified, and cost-effective detection methods. We have developed and analytically validated a protocol for direct rapid extraction-free PCR (DIRECT-PCR) detection of SARS-CoV-2 without the need for nucleic acid purification. As few as six RNA copies per reaction of viral nucleocapsid (N) gene from respiratory samples such as sputum and nasal exudate can be detected directly using our one-step inhibitor-resistant assay. The performance of this assay was validated on a commercially available portable PCR thermocycler. Viral lysis, reverse transcription, amplification, and detection are achieved in a single-tube homogeneous reaction within 36 min. This minimizes hands-on time, reduces turnaround-time for sample-to-result, and obviates the need for RNA purification reagents. It could enable wider use of Covid-19 testing for diagnosis, screening, and research in countries and regions where laboratory capabilities are limiting.

scholarly journals Assessment of the Diagnostic Ability of Four Detection Methods Using Three Sample Types of COVID-19 Patients

2021 ◽  
Vol 11 ◽  
Author(s):  
Fei Yu ◽  
Guoliang Xie ◽  
Shufa Zheng ◽  
Dongsheng Han ◽  
Jiaqi Bao ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Reverse Transcription ◽  
Detection Methods ◽  
Nucleic Acid Detection ◽  
Sample Type ◽  
Rt Pcr ◽  
Chain Reactions ◽  
Polymerase Chain Reactions ◽  
Oropharyngeal Swab ◽  
And Control

BackgroundViral nucleic acid detection is considered the gold standard for the diagnosis of coronavirus disease 2019 (COVID-19), which is caused by SARS-CoV-2 infection. However, unsuitable sample types and laboratory detection kits/methods lead to misdiagnosis, which delays the prevention and control of the pandemic.MethodsWe compared four nucleic acid detection methods [two kinds of reverse transcription polymerase chain reactions (RT-PCR A: ORF1ab and N testing; RT-PCRB: only ORF1ab testing), reverse transcription recombinase aided amplification (RT-RAA) and droplet digital RT-PCR (dd-RT-PCR)] using 404 samples of 72 hospitalized COVID-19 patients, including oropharyngeal swab (OPS), nasopharyngeal swabs (NPS) and saliva after deep cough, to evaluate the best sample type and method for SARS-CoV-2 detection.ResultsAmong the four methods, dd-RT-PCR exhibited the highest positivity rate (93.0%), followed by RT-PCR B (91.2%) and RT-RAA (91.2%), while the positivity rate of RT-PCR A was only 71.9%. The viral load in OPS [24.90 copies/test (IQR 15.58-129.85)] was significantly lower than that in saliva [292.30 copies/test (IQR 20.20-8628.55)] and NPS [274.40 copies/test (IQR 33.10-2836.45)]. In addition, if OPS samples were tested alone by RT-PCR A, only 21.4% of the COVID-19 patients would be considered positive. The accuracy of all methods reached nearly 100% when saliva and NPS samples from the same patient were tested simultaneously.ConclusionsSARS-CoV-2 nucleic acid detection methods should be fully evaluated before use. High-positivity rate methods such as RT-RAA and dd-RT-PCR should be considered when possible. Furthermore, saliva after deep cough and NPS can greatly improve the accuracy of the diagnosis, and testing OPS alone is not recommended.

scholarly journals Development and comparison of novel multiple cross displacement amplification (MCDA) assays with other nucleic acid amplification methods for SARS-CoV-2 detection

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Laurence Don Wai Luu ◽  
Michael Payne ◽  
Xiaomei Zhang ◽  
Lijuan Luo ◽  
Ruiting Lan
Keyword(s):  
Nucleic Acid ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
N Gene ◽  
Rt Pcr ◽  
Amplification Method ◽  
Multiple Cross ◽  
Time To Detection

AbstractThe development of alternative isothermal amplification assays including multiple cross displacement amplification (MCDA) may address speed and portability limitations of real-time PCR (rt-PCR) methods for SARS-CoV-2 detection. We developed a novel SARS-CoV-2 MCDA assay and compared its speed and sensitivity to loop-mediated isothermal amplification (LAMP) and rt-PCR. Two MCDA assays targeting SARS-CoV-2 N gene and ORF1ab were designed. The fastest time to detection and sensitivity of MCDA was compared to LAMP and rt-PCR using DNA standards and transcribed RNA. For the N gene, MCDA was faster than LAMP and rt-PCR by 10 and 20 min, respectively with fastest time to detection at 5.2 min. rt-PCR had the highest sensitivity with the limit of detection at 10 copies/µl compared with MCDA (100 copies/µl) and LAMP (500 copies/µl). For ORF1ab, MCDA and LAMP had similar speed with fastest time to detection at 9.7 and 8.4 min, respectively. LAMP was more sensitive for ORF1ab detection with 50 copies/µl compared to MCDA (500 copies/µl). In conclusion, different nucleic acid amplification methods provide different advantages. MCDA is the fastest nucleic acid amplification method for SARS-CoV-2 while rt-PCR is the most sensitive. These advantages should be considered when determining the most suitable nucleic acid amplification methods for different applications.

scholarly journals Rapid detection of SARS-CoV-2 and other respiratory viruses by using LAMP method with Nanopore Flongle workflow

2020 ◽  
Author(s):  
Jingjing Li ◽  
Weipeng Quan ◽  
Shuge Yan ◽  
Shuangju Wu ◽  
Jianhu Qin ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Respiratory Viruses ◽  
Nucleic Acid Amplification ◽  
Lamp Method ◽  
Global Public Health ◽  
One Step ◽  
Rapid Amplification ◽  
Novel Coronavirus ◽  
Amplification Reaction

AbstractThe ongoing novel coronavirus (COVID-19) outbreak as a global public health emergency infected by SARC-CoV-2 has caused devastating loss around the world. Currently, a lot of diagnosis methods have been used to detect the infection. The nucleic acid (NA) testing is reported to be the clinical standard for COVID-19 infection. Evidence shows that a faster and more convenient method to detect in the early phase will control the spreading of SARS-CoV-2. Here, we propose a method to detect SARC-Cov-2 infection within two hours combined with Loop-mediated Isothermal Amplification (LAMP) reaction and nanopore Flongle workflow. In this approach, RNA reverse transcription and nucleic acid amplification reaction with one step in 30 minutes at 60-65°C constant temperature environment, nanopore Flongle rapidly adapter ligated within 10 minutes. Flongle flow cell sequencing and analysis in real-time. This method described here has the advantages of rapid amplification, convenient operation and real-time detection which is the most important for rapid and reliable clinical diagnosis of COVID-19. Moreover, this approach not only can be used for SARS-CoV-2 detection but also can be extended to other respiratory viruses and pathogens.

scholarly journals Development and comparison of a novel multiple cross displacement amplification (MCDA) assay with other nucleic acid amplification methods for SARS-CoV-2 detection

2020 ◽  
Author(s):  
Laurence Don Wai Luu ◽  
Michael Payne ◽  
Xiaomei Zhang ◽  
Lijuan Luo ◽  
Ruiting Lan
Keyword(s):  
Nucleic Acid ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
N Gene ◽  
Rt Pcr ◽  
Amplification Method ◽  
Multiple Cross ◽  
Time To Detection

AbstractThe development of alternative isothermal amplification assays including multiple cross displacement amplification (MCDA) may address speed and portability limitations of real-time PCR (rt-PCR) methods for SARS-CoV-2 detection. We developed a novel SARS-CoV-2 MCDA assay and compared its speed and sensitivity to loop-mediated isothermal amplification (LAMP) and rt-PCR. Two MCDA assays targeting SARS-CoV-2 N gene and ORF1ab was designed. The fastest time to detection and sensitivity of MCDA was compared to LAMP and rt-PCR using DNA standards and transcribed RNA. For N gene, MCDA was faster than LAMP and rt-PCR by 10 and 20 minutes, respectively with fastest time to detection at 5.2 minutes. rt-PCR had highest sensitivity with limit of detection at 10 copies/µl compared with MCDA (100 copies/µl) and LAMP (500 copies/µl). For ORF1ab, MCDA and LAMP had similar speed with fastest time to detection at 9.7 and 8.4 minutes, respectively. LAMP was more sensitive for ORF1ab detection with 50 copies/µl compared to MCDA (500 copies/µl). In conclusion, different nucleic acid amplification methods provide different advantages. MCDA is the fastest nucleic acid amplification method for SARS-CoV-2 while rt-PCR is the most sensitive. These advantages should be considered when determining the most suitable nucleic acid amplification methods for different applications.

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