Fast-Flu: RT-RPA-CRISPR/Cas12a assisted one-step platform for rapid influenza B virus detection

Abstract

Influenza B virus (Flu B) is a prevalent respiratory pathogen responsible for seasonal influenza epidemics. Despite its clinical significance, there remains a lack of rapid and accurate diagnostic methods for Flu B detection. In this study, we developed a novel Flu B detection system, named Fast-Flu, by integrating reverse transcription recombinase polymerase amplification (RT-RPA) with the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system (CRISPR/Cas). Through optimization of reaction temperature and adjustment of Cas12a concentrations, we successfully balanced RPA amplification and CRISPR/Cas12a trans-cleavage activity, enabling the establishment of a one-step detection system. The one-step Fast-Flu system demonstrated the ability to specifically identify Flu B within 45 min, with a limit of detection of 58 copies per test. It eliminates the need for uncapping operations and minimizes the risk of cross-contamination, without cross-reactivity with other pathogens. When evaluated using 101 clinical throat swab samples, the one-step Fast-Flu system achieved a sensitivity of 56.25% and a specificity of 100% compared to the PCR-based method, with an overall concordance rate of 93.06% (94/101). The development of this one-step RT-RPA-CRISPR/Cas12a system represents a significant advancement in the rapid, convenient, and accurate detection of Flu B, highlighting its potential for clinical diagnosis. Furthermore, with future technical improvements to enhance sensitivity, this one-step RT-RPA-CRISPR assay holds promise as a versatile tool for the rapid nucleic acid detection of other RNA viruses.

Importance: Influenza B virus (Flu B) is a significant global health concern, and rapid, accurate pathogen diagnosis is crucial for effective influenza prevention and control. The integration of isothermal amplification methods with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has achieved high sensitivity and specificity for nucleic acid detection. Although CRISPR/Cas-based systems have been developed for influenza detection, existing platforms require the transfer of amplified products into the CRISPR/Cas12a detection system through uncapping operations, which increases the risk of cross-contamination. In this study, we developed a one-step reverse transcription recombinase polymerase amplification-CRISPR/Cas12a Flu B detection method using a one-pot detection system. By optimizing the reaction temperature and Cas12a concentration, we achieved a streamlined and contamination-free workflow. This innovative approach not only improves Flu B detection but also serves as a valuable reference for constructing CRISPR/Cas systems for the detection of other pathogens and targets, paving the way for broader applications in molecular diagnostics.

Keywords: CRISPR/Cas12a; Fast-Flu; Flu B detection; RT-RPA; one-step.

Fig 1

The workflow of one-pot and one-step RT-RPA-CRISPR/Cas12a system for Flu B detection. (A) For one-pot Flu B detection system, the RT-RPA assay was prepared at the bottom of the tube, and the CRISPR/Cas12a detection system was set in the cap of the tube. After amplification, the CRISPR/Cas12a reaction was added into the RT-RPA system via a short spin. The Cas12a can be activated in the presence of the target sequence and trans-cleaved FAM-labeled ssDNA probe to release the fluorescence signal. (B) For the one-step Flu B detection system, both the RT-RPA reaction and CRISPR/Cas12a detection system were mixed at the bottom of the tube. Then the reaction was performed at a constant temperature on a fluorescence signal detector. The Flu B-positive samples will induce a remarkable fluorescence signal, while no significant signal can be observed for Flu B-negative samples.

Fig 2

The primer selection for Flu B amplification in RT-RPA reaction. According to the conserved region of Flu B, three forward primers (F1 to F3) were designed and combined with three reverse primers (R1 to R3), respectively. RNA extracted from the Flu B pseudovirus acted as the detection template (1,000 copies per test). RPA amplification was performed at 38°C according to the recommendation by the kit. Their amplification products were examined by performing a 2% agarose gel electrophoresis.

Fig 3

The selection of crRNA for one-pot RT-RPA-CRISPR/Cas12a system. Based on the sequences of the target gene, a total of six crRNAs were designed (crRNA1 to crRNA 6), and they were evaluated and selected for one-pot Flu B detection system. The RNA template (500 copies per test) derived from the Flu B pseudovirus was amplified through the RPA assay in 10 µL of reaction system by incubating at 38°C for 30 min. The CRISPR/Cas12a system was performed in a reaction volume of 10 µL, by maintaining at 48°C for 10 min.

Fig 4

The condition optimization for one-pot Flu B detection system. (A) The optimal working temperature of Lb5Cas12a was selected, with 500 copies per test RNA template used, RT-RPA reaction performed at 38°C for 30 min, CRISPR/Cas12a maintained at 48°C for 10 min (n = 3 replicates, Student’s t-test; *P < 0.05, **P < 0.01, ***P < 0.001; bars represent mean ± SD; ns, no significance). (B) The best concentration of Lb5Cas12a was analyzed, with 500 copies per test RNA template used, RT-RPA reaction performed at 38°C for 30 min, CRISPR/Cas12a maintained at 48°C for 10 min (n = 3 replicates, Student’s t-test; *P < 0.05; bars represent mean ± SD; ns, no significance). (C) The performance of different probes was examined, with 500 copies per test RNA template used, RT-RPA reaction performed at 38°C for 30 min, CRISPR/Cas12a maintained at 48°C for 10 min (n = 3 replicates, Student’s t-test; **P < 0.01, ***P < 0.001, ****P < 0.0001; bars represent mean ± SD; nd, not detected).

Fig 5

The performance test of the one-step Flu B detection system. A total of 400 copies per test of RNA derived from the Flu B pseudovirus was used as template, and the reaction was performed in 10 µL of system. (A) The fluorescence intensity detected in systems at different working temperatures of Lb5Cas12a. (B) The optimal working temperature of Lb5Cas12a was selected (n = 3 replicates, Student’s t-test; *P < 0.05; bars represent mean ± SD; ns, no significance). (C) The best concentration of Lb5Cas12a was analyzed (n = 3 replicates, Student’s t-test; *P < 0.05, **P < 0.01; bars represent mean ± SD; ns, no significance; nd, not detected).

Fig 6

Condition optimization of the one-pot and one-step Flu B detection system. (A) The fluorescence intensities detected in template plasmid at different doses, when performing the one-pot Flu B detection system (n = 10 replicates). (B) The LOD of the one-pot Flu B detection system was predicted by using the sigmoid function. (C) The fluorescence intensities detected in template plasmid at different doses, when performing the one-step Flu B detection system (n = 10 replicates). (D) The LOD of the one-step Flu B detection system was predicted by using the sigmoid function.

Fig 7

The specificity of the RT-RPA-CRISPR/Cas12a system for Flu B detection. Six interfering nucleic acid samples were used to analyze the specificity of the one-step RT-RPA CRISPR/Cas12a system for Flu B detection, with the Flu B pseudoviral nucleic acid serving as the positive control, while the nuclease-free water was used as the NTC.