Full
Outline
To establish a one-tube assay based on reverse transcription recombinase polymerase amplification (RT-RPA) and clustered regularly interspaced short palindromic repeats and CRISPR-associated protein system (CRISPR/Cas12a) (OTRRC).
An OTRRC-based detection workflow for influenza A H1N1 virus was established and optimized. According to different signal readout modes, two detection formats were developed, including fluorescence-based detection (OTRRC-FD) and lateral flow immunoassay-based detection (OTRRC-LFIA). The sensitivity and specificity of both methods were systematically evaluated using plasmids and pseudoviruses as templates, followed by validation using clinical samples.
The OTRRC-FD method detected as low as 2.8×10-1 copies/μl of standard plasmid template within 20 min and 1.71×100 copies/μl of pseudovirus template within 30 min. The OTRRC-LFIA method detected 101 copies/μl of standard plasmid template within 25 min and 101 copies/μl of pseudovirus template within 35 min. No cross-reactivity was observed with SARS-CoV-2, IBV-Victoria or respiratory syncytial virus (RSV). In clinical sample testing, the sensitivities of the OTRRC-FD and OTRRC-LFIA methods were 95% and 90%, respectively, with a specificity of 100% for both methods. The agreements with real-time quantitative polymerase chain reaction (RT-qPCR) were 97.19% for OTRRC-FD and 94.56% for OTRRC-LFIA.
In this study, an OTRRC method for the detection of influenza A H1N1 virus was successfully established, and visual readout was achieved using lateral flow strips. This method features rapid detection, high sensitivity, and good specificity, and demonstrated favorable applicability in clinical sample testing. The detection results showed good agreement with the gold standard RT-qPCR, highlighting the potential of this assay as an efficient point-of-care testing (POCT) tool.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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