| Target Pathogen | Pathogen Name | NCBI Taxonomy ID | Order | Family | Genus | Species | Pathogen type |
|---|---|---|---|---|---|---|---|
| Angiostrongylus cantonensis | Angiostrongylus cantonensis,Pulmonema cantonensis | 6313 | Rhabditida | Metastrongylidae | Angiostrongylus | Angiostrongylus cantonensis | Eukaryota |
| Primer Name | Sequence(5'-3') | Length(bp) | Primer Final Concentration(μM） | GC Content（%） | Predicted Melting Temperature(℃） | Molecular Weight(g/moles) | Positions in GenBank accession number |
|---|---|---|---|---|---|---|---|
| F | 5′-GTT GCT TTC GAA GCT ATG TAC ATG AAA CAC CTC AA-3′ | 35 | \ | 40.00 | 61.32 | 10714.04 | \ |
| R | 5′-GCA ACA GTT TCA GCG CAA ATC TGA CGT TC-3′ | 29 | \ | 48.28 | 62.66 | 8861.82 | \ |
| P | 5'-CAT GAA ACA CCT CAA ATG TGC TTC GAA GTC\iFluorT\\idSp\\iBHQ-1dT\A AAA TTA GCG CGT AAT\3SpC3\-3' | 46 | \ | 39.13 | 65.06 | 14134.27 | \ |
| Application | Assay | Primer Designing Software | Reaction Time(min) | Assay Temperature(℃) | Readout System(s) | Limit of Detection(LoD) | Sensitivity(%) | Specificity(%) |
|---|---|---|---|---|---|---|---|---|
| In conclusion, RPAcan3990 is an RPA designed to target a DNA sequence predicted in silico to be highly repeated within the A. cantonensis genome. The data suggest that a limit of detection of 1 fg/μL of A. cantonensis genomic DNA can be achieved using a simplified reaction preparation, a visualizable fluorescent readout, and incubation using the human body as the only heat source. | RPAcan3990 | \ | 30 min | 40°C | \ | 1 fg/μL | \ | \ |
| Year of Publication | Title | Author(s) | Journal | PMID | DOI | ||
|---|---|---|---|---|---|---|---|
| 2021 | RPAcan3990: an Ultrasensitive Recombinase Polymerase Assay To Detect Angiostrongylus cantonensis DNA | William J Sears,Yvonne Qvarnstrom,Thomas B Nutman | Journal of clinical microbiology | 34132583 | 10.1128/JCM.01185-21 | ||
RPAcan3990: an Ultrasensitive Recombinase Polymerase Assay To Detect Angiostrongylus cantonensis DNAAuthor(s):William J Sears,Yvonne Qvarnstrom,Thomas B NutmanJournal:Journal of clinical microbiologyYear:2021Abstract:Angiostrongylus cantonensis is one of the leading causes of eosinophilic meningitis worldwide. A field-deployable molecular detection method could enhance both environmental surveillance and clinical diagnosis of this emerging pathogen. Accordingly, RPAcan3990, a recombinase polymerase assay (RPA), was developed to target a region predicted to be highly repeated in the A. cantonensis genome. The assay was then adapted to produce a visually interpretable fluorescent readout using an orange camera lens filter and a blue light. Using A. cantonensis genomic DNA, the limit of detection was found to be 1 fg/μl by both fluorometer measurement and visual reading. All clinical samples known to be positive for A. cantonensis from various areas of the globe were positive by RPAcan3990. Cerebrospinal fluid samples from other etiologies of eosinophilic meningitis (i.e., Toxocara sp. and Gnathostoma sp.) were negative in the RPAcan3990 assay. The optimal incubation temperature range for the reaction was between 35°C and 40°C. The assay successfully detected 1 fg/μl of A. cantonensis genomic DNA after incubation at human body temperature (in a shirt pocket). In conclusion, these data suggest RPAcan3990 is potentially a point-of-contact molecular assay capable of sensitively detecting A. cantonensis by producing visually interpretable results with minimal instrumentation.PMID:34132583
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