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The lower detection limits of the assays were similar to those of established diagnostic PCR tests for the viruses. A sample preparation method was optimized to eliminate the need for total DNA extraction from fish tissues. The estimated time to perform these RPA assays, from receiving the sample to having a result, is 50 min, compared to 10 and 7 […]
Herein, the detection of mutations in PIK3CA gene, such as p.E545K, and p.H1047L, is presented. The main element was an oligonucleotide (dideoxycytidine functionalized at 3′-end) which matched with wild-type sequence in the target locus.
Blocked recombinase polymerase amplification for mutation analysis of PIK3CA gene Read More
The analytical sensitivity of the assay was 102 copies for gB and gE genes. The dual real-time RPA demonstrated a 100% diagnostic agreement with the real-time PCR on 4 PRV strains and 37 clinical samples.
With incubation at 37°C, the 18S rRNA gene of P. knowlesi was successfully amplified within 12 minutes. By adding a specifically designed probe to the reaction solution, the amplified RPA product can be visualized on a LF strip.
All results were compared with real-time PCR assays directed against the same rickettsial genes. The RPA assays are easy to handle and produced quicker results in comparison to real-time PCR.
Using this enrichment platform, the detection limit was found to be 20 times more sensitive in 10 ml urine with Salmonella and 10 times more sensitive in 10 ml urine with Brucella than that of real-time PCR without the enrichment process.
The assay was specific, as it did not amplify genomes from five other Mycobacterium and five pathogenic enteric bacteria. Then, 612 clinical samples (320 fecal and 292 serum) were assessed by RPA-LFD, qPCR and ELISA assays respectively, also the established RPA-LFD assay yielded 100% sensitivity, 97.63% specificity, and 98.44% concordance rate with the qPCR.
The result showed that the coincidence rate of BEFV LFD-RPA and real-time qPCR was 96.09% (123/128), which was higher than conventional RT-PCR. The RPA combined with LFD assay probably provides a rapid and sensitive alternative for diagnosis of BEFV infections outbreak.
In our platform, real-time RPA (RT-RPA) was integrated on a lab-on-a-disc (LOAD) with on-board power to maintain temperature for DNA amplification. Sputa collected from healthy volunteers were spiked with respective target M. tb samples for testing. A limit of detection of 102 colony-forming unit per millilitre in 15 min was achieved, making early detection and differentiation of M. tb strains highly feasible
The assay performance on acute-phase high fever clinical samples collected from cattle with no vaccine against IBRV, which were suspected to be infected with IBRV, was validated by detecting 24 fecal, 36 blood, 38 nasal swab and 8 tissue specimens, and compared with SYBR Green I based real-time PCR.
