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Nowadays, rapid and accurate diagnosis of respiratory tract viruses is an urgent need to prevent another epidemic outbreak. To overcome this problem, we have developed a clustered, regularly interspaced short palindromic repeats (CRISPR) loop mediated amplification (LAMP) technology to detect influenza A virus, influenza B virus, respiratory syncytial A virus, respiratory syncytial B virus, and severe acute respiratory syndrome coronavirus 2, including variants of concern (B.1.1.7), which utilized CRISPR-associated protein 12a (Cas12a) to advance LAMP technology with the sensitivity increased 10 times. To reduce aerosol contamination in CRISPR-LAMP technology, an uracil-DNA-glycosylase-reverse transcription-LAMP system was also developed which can effectively remove dUTP-incorporated LAMP amplicons. In vitro Cas12a cleavage reaction with 28 crRNAs showed that there were no position constraints for Cas12a/CRISPR RNA (crRNA) recognition and cleavage in LAMP amplicons, and even the looped position of LAMP amplicons could be effectively recognized and cleaved. Wild-type or spike N501Y can be detected with a limit of detection of 10 copies/μL (wild-type) even at a 1% ratio level on the background (spike N501Y). Combining UDG-RT-LAMP technology, CRISPR-LAMP design, and mutation detection design, we developed a CRISPR-LAMP detection platform that can precisely diagnose pathogens with better stability and significantly improved point mutation detection efficiency.

Additional file 1: Table S1. Characteristics of HF patients in the discovery phase (n=40). Table S2. Characteristics of HF patients in the training phase (n=30) and validation phase (n=50). Table S3. Home made circulating miRNA panel which was used for the miRNA screening analysis in the discovery phase. Table S4. The features of 11 overlapped target genes of miR-30a-5p in Heart Failure (ICM or DCM). Table S5. The features of 9 overlapped target genes of miR-654-5p between Heart Failure (ICM or DCM). Figure S1. Expression patterns of circulating miR-100, miR-320a, miR-433 and miR-499b-3p in each patient in the discovery phase with paired plasma samples collected at times of check-in and check-out. The p-values showed significant change of the four miRNAs (p < 0.05). Figure S2. Expression patterns and change trends of the rest of 83 circulating miRNAs in all patients in the discovery phase. The p-values of these miRNAs did not show significant change. Figure S3. Correlation analysis between miR-30a-5p, miR-654-5p and NT-proBNP in the 40 HF patients enrolled in the discovery phase before and after treatment. Figure S4. Correlation analysis between the differential miRNA expression change and the NT-proBNP reduction by comparing 5 patients with ~80% NT-proBNP reduction and 5 patients with ~40% NT-proBNP reduction upon medical therapy (n=5, *p < 0.05).

Additional file 1.

Additional file 2: Table S1. Primers for qRT-PCR.

Lower respiratory tract infections (LRTIs) are a leading cause of morbidity and mortality worldwide and lack a rapid diagnostic method. To improve the diagnosis of LRTIs, we established an available loop-mediated isothermal amplification (LAMP) assay for the detection of eight common lower respiratory pathogens, including Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli, Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis. The whole process can be achieved within 1 h (sample to results read out). We established an extraction free isothermal system. 528 sputum samples collected from patients suspected to have LRTIs were analyzed by the system (8 tests in each sample, a total of 4224 tests) and compared with the standard culture method (SCM). The samples with inconsistent results were further verified by Sanger sequencing and High-throughput sequencing (NGS). The detection limits of the LAMP assay for the 8 pathogens ranged from 103 to 104 CFU/mL. Upon testing 528 samples, the Kappa coefficients of all pathogens ranged between 0.5 and 0.7 indicated a moderate agreement between the LAMP assay and the SCM. All inconsistent samples were further verified by Sanger sequencing, we found that the developed LAMP assay had a higher consistency level with Sanger sequencing than the SCM for all pathogens. Additionally, when the NGS was set to a diagnostic gold standard, the specificity and sensitivity of the LAMP assay for LRTIs were 94.49% and 75.00%. The present study demonstrated that the developed LAMP has high consistency with the sequencing methods. Meanwhile, the LAMP assay has a higher detection rate compared to the SCM. It may be a powerful tool for rapid and reliable clinical diagnosis of LRTIs in primary hospitals.