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PLoS ONE ◽  
2022 ◽  
Vol 17 (1) ◽  
pp. e0261853
Author(s):  
Margaret G. Mills ◽  
Emily Bruce ◽  
Meei-Li Huang ◽  
Jessica W. Crothers ◽  
Ollivier Hyrien ◽  
...  

Reverse transcription–quantitative polymerase chain reaction (RT-qPCR) is used worldwide to test and trace the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). “Extraction-less” or “direct” real time–reverse transcription polymerase chain reaction (RT-PCR) is a transparent and accessible qualitative method for SARS-CoV-2 detection from nasopharyngeal or oral pharyngeal samples with the potential to generate actionable data more quickly, at a lower cost, and with fewer experimental resources than full RT-qPCR. This study engaged 10 global testing sites, including laboratories currently experiencing testing limitations due to reagent or equipment shortages, in an international interlaboratory ring trial. Participating laboratories were provided a common protocol, common reagents, aliquots of identical pooled clinical samples, and purified nucleic acids and used their existing in-house equipment. We observed 100% concordance across laboratories in the correct identification of all positive and negative samples, with highly similar cycle threshold values. The test also performed well when applied to locally collected patient nasopharyngeal samples, provided the viral transport media did not contain charcoal or guanidine, both of which appeared to potently inhibit the RT-PCR reaction. Our results suggest that direct RT-PCR assay methods can be clearly translated across sites utilizing readily available equipment and expertise and are thus a feasible option for more efficient COVID-19 coronavirus disease testing as demanded by the continuing pandemic.


2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Qian-Qian Yang ◽  
Xing-Xing Zhao ◽  
Dao Wang ◽  
Peng-Jun Zhang ◽  
Xue-Nan Hu ◽  
...  

AbstractBean pod mottle virus (BPMV) is a destructive virus that causes serious economic losses in many countries every year, highlighting the importance of its effective detection. In this study, we developed a fast reverse transcription-cross-priming amplification (RT-CPA) coupled with lateral flow dipstick (LFD) diagnostic method for BPMV detection. The RT-CPA-LFD assay that targets the coat protein gene of BPMV was highly specific against diagnosing four other common viruses transmitted by soybean seeds, i.e., Southern bean mosaic virus (SBMV), Tomato ringspot virus (ToRSV), Arabis mosaic virus (ArMV), and Tobacco ringspot virus (TRSV). The sensitivities of the real-time fluorescent RT-CPA and the RT-CPA-LFD assay were at least 50 pg/μl and 500 pg/μl, respectively. Despite a compromise in the limit of detection of the RT-CPA method compared with TaqMan-MGB real-time RT-PCR, our results demonstrated a notably better performance in the detection of field samples of BPMV-infested soybean seeds. With the advantages of efficiency and convenience by visual determination, the RT-CPA-LFD assay presents a potential application for the rapid and accurate detection of BPMV in routine tests.


2022 ◽  
Vol 66 (6) ◽  
pp. 417-424
Author(s):  
V. G. Akimkin ◽  
V. V. Petrov ◽  
K. V. Krasovitov ◽  
N. I. Borisova ◽  
I. A. Kotov ◽  
...  

Introduction. Currently, the basis for molecular diagnostics of most infections is the use of reverse transcription polymerase chain reaction (RT-PCR). Technologies based on reverse transcription isothermal loop amplification (RT-LAMP) can be used as an alternative to RT-PCR for diagnostic purposes. In this study, we compared the RTLAMP and RT-PCR methods in order to analyze both the advantages and disadvantages of the two approaches.Material and methods. For the study, we used reagent kits based on RT-PCR and RT-LAMP. The biological material obtained by taking swabs from the mucous membrane of the oropharynx and nasopharynx in patients with symptoms of a new coronavirus infection was used.Results. We tested 381 RNA samples of the SARS-CoV-2 virus (Coronaviridae: Coronavirinae: Betacoronavirus; Sarbecovirus) from various patients. The obtained values of the threshold cycle (Ct) for RT-PCR averaged 20.0 ± 3.7 s (1530 ± 300 s), and for RT-LAMP 12.8 ± 3.7 s (550 ± 160 s). Proceeding from the theoretical assumptions, a linear relationship between values obtained in two kits was proposed as a hypothesis; the correlation coefficient was approximately 0.827. At the same time, for samples with a low viral load (VL), the higher Ct values in RT-LAMP did not always correlated with those obtained in RT-PCR.Discussion. We noted a significant gain in time for analysis using RT-LAMP compared to RT-PCR, which can be important in the context of testing a large number of samples. Being easy to use and boasting short turnaround time, RT-LAMP-based test systems can be used for mass screening in order to identify persons with medium and high VLs who pose the greatest threat of the spread of SARS-CoV-2, while RT-PCR-based diagnostic methods are also suitable for estimation of VL and its dynamics in patients with COVID-19.


Author(s):  
Dongyan Xiong ◽  
Xiaoxu Zhang ◽  
Mengjuan Shi ◽  
Nuo Wang ◽  
Ping He ◽  
...  

The current stage of the pandemic, led by SARS-CoV-2 variants of concern (VOCs), underscores the necessity to develop a cost-effective and rapid molecular diagnosis assay to differentiate the VOCs. In this study, over 1 million SARS-CoV-2 genomic sequences of high quality from GISAID were analyzed and a network of the common mutations of the lineages was constructed.


2022 ◽  
Vol 50 (1) ◽  
Author(s):  
Meng Yee Lai ◽  
Jeyanthi Suppiah ◽  
Ravindran Thayan ◽  
Ilyiana Ismail ◽  
Nur Izati Mustapa ◽  
...  

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.


2022 ◽  
Vol 14 (11) ◽  
Author(s):  
Dong Chen ◽  
Fangju Tian ◽  
Wanyu Liu ◽  
Jingyi Yu ◽  
Dafeng Song

Background: Salmonella is one of the main foodborne bacterial pathogens, causing diseases and death. The study used reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect Salmonella. Objectives: To design six primers and detect Salmonella using RT-LAMP to facilitate the rapid detection of pathogenic bacteria in food. Methods: We designed six primers based on the gene coding sequences of inv A, specific to Salmonella. Each reaction solution contained 6.0 mM MgSO4, 1 M betaine, 1.6 mM dNTPs, 160 U/mL Bst DNA polymerase, 0.2 μM of both external primers, 0.8 μM of both internal primers, and 0.2 μM of both loop primers. The reaction temperature was 65°C. Results: Our amplified products were separated by 2% agarose gel electrophoresis. The detection limit was 10 CFU per reaction. Conclusions: RT-LAMP exhibited the same accuracy as the GB assay in detecting Salmonella in foods. RT-LAMP was highly specific and sensitive; hence, it may serve as an effective tool in detecting Salmonella.


2022 ◽  
Vol 9 (1) ◽  
pp. 1-20
Author(s):  
Mathieu Durand ◽  
Philippe Thibault ◽  
Simon Lévesque ◽  
Ariane Brault ◽  
Alex Carignan ◽  
...  

The early diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is required to identify and isolate contagious patients to prevent further transmission of SARS-CoV-2. In this study, we present a multitarget real-time TaqMan reverse transcription PCR (rRT-PCR) assay for the quantitative detection of SARS-CoV-2 and some of its circulating variants harboring mutations that give the virus a selective advantage. Seven different primer-probe sets that included probes containing locked nucleic acid (LNA) nucleotides were designed to amplify specific wild-type and mutant sequences in Orf1ab, Envelope (E), Spike (S), and Nucleocapsid (N) genes. Furthermore, a newly developed primer-probe set targeted human β2-microglobulin (B2M) as a highly sensitive internal control for RT efficacy. All singleplex and fourplex assays detected £ 14 copies/reaction of quantified synthetic RNA transcripts, with a linear amplification range of nine logarithmic orders. Primer-probe sets for detection of SARS-CoV-2 exhibited no false-positive amplifications with other common respiratory pathogens, including human coronaviruses NL63, 229E, OC43, and HKU-1. Fourplex assays were evaluated using 160 clinical samples positive for SARS-CoV-2. Results showed that SARS-CoV-2 viral RNA was detected in all samples, including viral strains harboring mutations in the Spike coding sequence that became dominant in the pandemic. Given the emergence of SARS-CoV-2 variants and their rapid spread in some populations, fourplex rRT-PCR assay containing four primer-probe sets represents a reliable approach to allow quicker detection of circulating relevant variants in a single reaction.


2022 ◽  
pp. 114455
Author(s):  
Lara-Simone Pretorius ◽  
Kerri A. Chandra ◽  
Anna E.C. Jooste ◽  
Lebogang C. Motaung ◽  
Louisamarie E. Parkinson ◽  
...  

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