scholarly journals Isothermal amplification and fluorescent detection of SARS-CoV-2 and SARS-CoV-2 variant virus in nasopharyngeal swabs

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0257563
Author(s):  
Les Jones ◽  
Abhijeet Bakre ◽  
Hemant Naikare ◽  
Ravindra Kolhe ◽  
Susan Sanchez ◽  
...  

The COVID-19 pandemic caused by the SARS-CoV-2 is a serious health threat causing worldwide morbidity and mortality. Real-time reverse transcription PCR (RT-qPCR) is currently the standard for SARS-CoV-2 detection. Although various nucleic acid-based assays have been developed to aid the detection of SARS-CoV-2 from COVID-19 patient samples, the objective of this study was to develop a diagnostic test that can be completed in 30 minutes without having to isolate RNA from the samples. Here, we present an RNA amplification detection method performed using reverse transcription loop-mediated isothermal amplification (RT-LAMP) reactions to achieve specific, rapid (30 min), and sensitive (<100 copies) fluorescent detection in real-time of SARS-CoV-2 directly from patient nasopharyngeal swab (NP) samples. When compared to RT-qPCR, positive NP swab samples assayed by fluorescent RT-LAMP had 98% (n = 41/42) concordance and negative NP swab samples assayed by fluorescent RT-LAMP had 87% (n = 59/68) concordance for the same samples. Importantly, the fluorescent RT-LAMP results were obtained without purification of RNA from the NP swab samples in contrast to RT-qPCR. We also show that the fluorescent RT-LAMP assay can specifically detect live virus directly from cultures of both SARS-CoV-2 wild type (WA1/2020), and a SARS-CoV-2 B.1.1.7 (alpha) variant strain with equal sensitivity to RT-qPCR. RT-LAMP has several advantages over RT-qPCR including isothermal amplification, speed (<30 min), reduced costs, and similar sensitivity and specificity.

2005 ◽  
Vol 54 (11) ◽  
pp. 1037-1041 ◽  
Author(s):  
Ryoichi Saito ◽  
Yoshiki Misawa ◽  
Kyoji Moriya ◽  
Kazuhiko Koike ◽  
Kimiko Ubukata ◽  
...  

A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Mycoplasma pneumoniae was developed and evaluated. The assay specifically amplified only M. pneumoniae sequences, and no cross-reactivity was observed for other Mycoplasma species or respiratory bacterial species. The detection limit for this assay was found to be 2 × 102 copies, corresponding to 2–20 colour changing units of M. pneumoniae in 1 h, as observed in a real-time turbidimeter and electrophoretic analysis. The accuracy of the LAMP reaction was confirmed by restriction endonuclease analysis as well as direct sequencing of the amplified product. The assay was applied to 95 nasopharyngeal swab samples collected from patients or from healthy individuals, and compared to a real-time PCR assay in-house. A concordance of 100 % was observed between the two assays. The LAMP assay is easy to perform, shows a rapid reaction and is inexpensive. It may therefore be applied in the routine diagnosis of M. pneumoniae infection in the clinical laboratory.


2020 ◽  
Vol 21 (8) ◽  
pp. 2826 ◽  
Author(s):  
Renfei Lu ◽  
Xiuming Wu ◽  
Zhenzhou Wan ◽  
Yingxue Li ◽  
Xia Jin ◽  
...  

COVID-19 has become a major global public health burden, currently causing a rapidly growing number of infections and significant morbidity and mortality around the world. Early detection with fast and sensitive assays and timely intervention are crucial for interrupting the spread of the COVID-19 virus (SARS-CoV-2). Using a mismatch-tolerant amplification technique, we developed a simple, rapid, sensitive and visual reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for SARS-CoV-2 detection based on its N gene. The assay has a high specificity and sensitivity, and robust reproducibility, and its results can be monitored using a real-time PCR machine or visualized via colorimetric change from red to yellow. The limit of detection (LOD) of the assay is 118.6 copies of SARS-CoV-2 RNA per 25 μL reaction. The reaction can be completed within 30 min for real-time fluorescence monitoring, or 40 min for visual detection when the template input is more than 200 copies per 25 μL reaction. To evaluate the viability of the assay, a comparison between the RT-LAMP and a commercial RT-qPCR assay was made using 56 clinical samples. The SARS-CoV-2 RT-LAMP assay showed perfect agreement in detection with the RT-qPCR assay. The newly-developed SARS-CoV-2 RT-LAMP assay is a simple and rapid method for COVID-19 surveillance.


2012 ◽  
Vol 84 (11) ◽  
pp. 1771-1778 ◽  
Author(s):  
Vijayalakshmi Reddy ◽  
Vasanthapuram Ravi ◽  
Anita Desai ◽  
Manmohan Parida ◽  
Ann M. Powers ◽  
...  

Author(s):  
Yi Wang ◽  
Xiaoxia Wang ◽  
Hai Chen ◽  
Limei Han ◽  
Licheng Wang ◽  
...  

The ongoing Corona virus disease (COVID-19) outbreak has become a huge global health concern. Here, we reported a novel detection platform based on the loop-mediated isothermal amplification (LAMP), termed real-time reverse transcription LAMP (rRT-LAMP) and applied it for the diagnosis of COVID-19 (COVID-19 rRT-LAMP). rRT-LAMP integrates reverse transcription, LAMP amplification, restriction endonuclease cleavage and real-time fluorescence detection into one-pot reaction, and facilitates the diagnosis of COVID-19 at 64°C for only 35 min. The ORF1ab (opening reading frame 1a/b) and NP (nucleoprotein) genes of SARS-CoV-2 were detected for diagnosing COVID-19. The limit of detection (LoD) of COVID-19 rRT-LAMP assay was 14 copies (for each marker) per vessel, and no positive results were obtained from non-SARS-CoV-2 templates. To demonstrate its feasibility, a total of 33 oropharynx swab samples collected from COVID-19 patients also were diagnosed as SARS-CoV-2 infection using COVID-19 rRT-LAMP protocol. No cross-reactivity was yielded from 41 oropharynx swab samples collected from non-COVID-19 patients. These data suggesting that the COVID-19 rRT-LAMP assay is a potential detection tool for the diagnosis of SARS-CoV-2 infection in clinical, field and disease control laboratories, and will be valuable for controlling the COVID-19 epidemic.


2020 ◽  
Author(s):  
yanxia liu ◽  
zhengan cao ◽  
mei chen ◽  
Yan zhong ◽  
yuhao luo ◽  
...  

Background: Real-time reverse transcription PCR (rRT-PCR) is commonly used to diagnose SARS-CoV-2 infection. Heat inactivation prior to nucleic acid isolation may allow safe testing, while the effects of heat inactivation on SARS-CoV-2 rRT-PCR detection result need to be determined. Methods: 14 positive nasopharyngeal swab specimens were inactivated at 56°C for 30min, 56°C for 60min, 60°C for 30min, 60°C for 75min, and 100°C for 10min, and another 2 positive nasopharyngeal swab specimens were also inactivated at 100°C for 10min, 100°C for 30min, 100°C for 60min, after which the samples were isolated and detected by rRT-PCR. Results: All 14 heat treated samples remained positive. The range of threshold cycle (Ct) values observed when detecting ORF1a/b was 27.228-34.011 in heat-treated samples, while 25.281-34.861 in unheated samples, and the range of threshold cycle (Ct) values observed at the time of detecting N was 25.777-33.351 in heat-treated samples, while 24.1615-35.433 in unheated samples, on basis of which it showed no statistical difference otherwise a good correlation of Ct values between the heat-inactivated samples and the untreated samples. However, the 2 samples inactivated at 100°C 30min, 100°C 60min turned into negative. Conclusions: Heat inactivation at 56°C for 30min, 56°C for 60min, 60°C for 30min, 60°C for 75min, and 100°C for 10min shall not affect the detection results of Real-Time Reverse Transcription PCR of the SARS-COV2. Furthermore, it is recommended to inactive nasopharyngeal swab specimens 10min at 100°C before RNA extraction in consideration of efficiency and reliable results. Key Words: SARS-CoV-2, Heat Inactivation, rRT-PCR, Comparison


PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9278 ◽  
Author(s):  
Yee Ling Lau ◽  
Ilyiana Ismail ◽  
Nur Izati Mustapa ◽  
Meng Yee Lai ◽  
Tuan Suhaila Tuan Soh ◽  
...  

Background Highly sensitive real-time reverse transcription polymerase chain reaction (RT-qPCR) methods have been developed for the detection of SARS-CoV-2. However, they are costly. Loop-mediated isothermal amplification (LAMP) assay has emerged as a novel alternative isothermal amplification method for the detection of nucleic acid. Methods A rapid, sensitive and specific real-time reverse transcription LAMP (RT-LAMP) assay was developed for SARS-CoV-2 detection. Results This assay detected one copy/reaction of SARS-CoV-2 RNA in 30 min. Both the clinical sensitivity and specificity of this assay were 100%. The RT-LAMP showed comparable performance with RT-qPCR. Combining simplicity and cost-effectiveness, this assay is therefore recommended for use in resource resource-limited settings.


2020 ◽  
Author(s):  
Deguo Wang

AbstractBackgroundRapid and reliable diagnostic assays were critical for prevention and control of the coronavirus pneumonia caused by COVID-19.ObjectiveThis study was to establish one-pot real-time reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay and one-pot visual RT-LAMP assay for the detection of COVID-19.MethodsSix specific LAMP primers targeting the N gene of COVID-19 were designed, the RT-LAMP reaction system was optimized with plasmid pUC57 containing N gene sequence, the detection limit was determined with a serial dilution of the plasmid pUC57 containing N gene sequence, and the one-pot real-time RT-LAMP assay and one-pot visual RT-LAMP assay for the detection of COVID-19 were established.ResultsOur results showed that the one-pot RT-LAMP assays can detect COVID-19 with a limit of ≥ 6 copies per μl−1 of pUC57 containing N gene sequence.ConclusionThis study provides rapid, reliable and sensitive tools for facilitating preliminary and cost-effective prevention and control of COVID-19.


Author(s):  
Mohsen Golabi ◽  
Marion Flodrops ◽  
Beatrice Grasland ◽  
Aaydha C. Vinayaka ◽  
Than Linh Quyen ◽  
...  

Avian influenza virus (AIV) outbreaks occur frequently worldwide, causing a potential public health risk and great economic losses to poultry industries. Considering the high mutation rate and frequent genetic reassortment between segments in the genome of AIVs, emerging new strains are a real threat that may infect and spread through the human population, causing a pandemic. Therefore, rapid AIV diagnostic tests are essential tools for surveillance and assessing virus spreading. Real-time reverse transcription PCR (rRT-PCR), targeting the matrix gene, is the main official standard test for AIV detection, but the method requires well-equipped laboratories. Reverse transcription Loop-Mediated Isothermal Amplification (RT-LAMP) has been reported as a rapid method and an alternative to PCR in pathogen detection. The high mutation rate in the AIV genome increases the risk of false negative in nucleic acid amplification methods for detection, such as PCR and LAMP, due to possible mismatched priming. In this study, we analyzed 800 matrix gene sequences of newly isolated AIV in the EU and designed a highly efficient LAMP primer set that covers all AIV subtypes. The designed LAMP primer set was optimized in real-time RT-LAMP (rRT-LAMP) assay. The rRT-LAMP assay detected AIV samples belonging to nine various subtypes with the specificity and sensitivity comparable to the official standard rRT-PCR assay. Further, a two-color visual detection RT-LAMP assay protocol was adapted with the aim to develop on-site diagnostic tests. The on-site testing successfully detected spiked AIV in birds oropharyngeal and cloacal swabs samples at a concentration as low as 100.8 EID50 per reaction within 30 minutes including sample preparation. The results revealed a potential of this newly developed rRT-LAMP assay to detect AIV in complex samples using a simple heat treatment step without the need for RNA extraction.


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