Acute respiratory infections are common respiratory illnesses in children, often caused by viruses. Respiratory syncytial virus (RSV) and respiratory adenovirus (ADV) are two common pathogens causing acute respiratory infections in children. Mild cases can cause common cold symptoms such as cough and runny nose, while severe cases can cause severe complications such as pneumonia and otitis media. According to the recommendations of the World Health Organization, the detection of pathogens needs to be fast, accurate, sensitive, and specific and should not rely on specialized technicians or large instruments. Therefore, it is particularly important to develop a detection method for RSV and ADV that can meet the above detection requirements.
The research group of Director Ma Dongli of the Institute of Pediatrics of Shenzhen Children's Hospital published an article "A multiplex recombinase polymerase amplification assay combined with CRISPR/ Cas12a for the detection of respiratory syncytial virus and respiratory adenovirus" in the journal "Scientific Reports", with an impact factor of 4.6.
Research overview
The multi-enzyme isothermal rapid nucleic acid amplification technology MIRA is combined with the CRISPR/Cas detection system. By designing MIRA primers and CRISPR RNAs (crRNAs), performing multiple sequence comparisons, optimizing reaction conditions, and using fluorescence numerical detection devices to verify sensitivity and specificity.
And using the qPCR results as a comparison control, the consistency of the test results of 160 clinical throat swab samples was studied.
Experimental results
1. Sensitivity and specificity of MIRA-CRISPR/Cas12a detection method
In the MIRA method detection, the RSV-F1/R1 primer pair is used to amplify nucleic acids from throat swab samples of RSV, ADV, PIV, RhV, MP, CP, FluB, hMPV, FluA, or RSV/ADV co-infected patients. Except for RSV and ADV, which had amplified bands, the other results were negative, indicating that the selected MIRA primers did not cross-react with the other eight pathogens tested and were well conserved.
Subsequently, the RSV and ADV recombinant plasmids were diluted 10times using sterile water, and the detection limits for RSV and ADV were both 106 copies/mL.
(1-10: Throat swab samples from patients with RSV, RSV/ADV co-infection, and other respiratory pathogen samples respectively)
2. Compare with qPCR
Compared with the qPCR method, the detection limits of RSV and ADV are both 103 copies/mL.
(Sensitivity of qPCR detection of RSV and ADV)
The MIRA-CRISPR method is more suitable for grassroots laboratories, with short detection time, simple operation, and no need for large-scale qPCR instruments to complete experimental operations.
3. Comparison of clinical samples
Throat swab samples were collected from 160 children with acute respiratory infections. At the same time, MIRA-CRISPR method and qPCR method were used for detection, and the results were compared with clinical trial results.
Compared with the qPCR method, the sensitivity of the MIRA-CRISPR method for RSV and ADV were 98.1% and 91.4% respectively, and the detection specificity was 100%. The Kappa value is greater than 0.95, indicating a high degree of consistency.
The MIRA-CRISPR method has the advantages of rapid, sensitive and specific detection of RSV and ADV. It can detect viral infections promptly and accurately, and is more suitable for use in areas with scarce medical resources.
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