| Target Pathogen | Pathogen Name | NCBI Taxonomy ID | Order | Family | Genus | Species | Pathogen type |
|---|---|---|---|---|---|---|---|
| Listeria monocytogenes | Listeria monocytogenes (Murray et al. 1926) Pirie 1940 (Approved Lists 1980),SLCC:53,"Bacterium monocytogenes","Erysipelothrix monocytogenes",Listeria sp. FDA00013359,Listeria sp. FDA00013360,Listeria sp. FDA00013361,Listeria sp. FDA00013362,Listeria sp. FDA00013363,Listeria sp. FDA00013364,Listeria sp. FDA00013365,Listeria sp. FDA00013366,Listeria sp. FDA00013367,Listeria sp. FDA00013503,Listeria sp. FDA00013504,Listeria sp. FDA00013505,Listeria sp. FDA00013506,Listeria sp. FDA00013507,Listeria sp. FDA00013508,Listeria sp. FDA00013509,Listeria sp. FDA00013510,Listeria sp. FDA00013511,Listeria sp. FDA00013512,Listeria sp. FDA00013536,Listeria sp. FDA00013537,Listeria sp. FDA00013538,Listeria sp. FDA00013539,Listeria sp. FDA00013540,Listeria sp. FDA00013541,Listeria sp. FDA00013542,Listeria sp. FDA00013543,Listeria sp. FDA00013544,Listeria sp. FDA00013545,Listeria sp. FDA00013546,Listeria sp. FDA00013547,Listeria sp. FDA00013548,Listeria sp. FDA00013549,Listeria sp. FDA00013550,Listeria sp. FDA00013551,Listeria sp. FDA00013552,Listeria sp. FDA00013553,Listeria sp. FDA00013554,Listeria sp. FDA00013555,Listeria sp. FDA00013556,Listeria sp. FDA00013557,Listeria sp. FDA00013558,Listeria sp. FDA00013559,Listeria sp. FDA00013560,Listeria sp. FDA00013561,Listeria sp. FDA00013562,Listeria sp. FDA00013563,Listeria sp. FDA00013564,Listeria sp. FDA00013565,Listeria sp. FDA00013566,Listeria sp. FDA00013567,Listeria sp. FDA00013568,Listeria sp. FDA00013570,Listeria sp. FDA00013571,Listeria sp. FDA00013572,Listeria sp. FDA00013573,Listeria sp. FDA00013574,Listeria sp. FDA00013575,Listeria sp. FDA00013576,Listeria sp. FDA00013577,Listeria sp. FDA00013578,Listeria sp. FDA00013579,Listeria sp. FDA00013607,"Listerella hepatolytica","Bacterium monocytogenes hominis","Corynebacterium parvulum","Corynebacterium infantisepticum" | 1639 | Bacillales | Listeriaceae | Listeria | Listeria monocytogenes (Murray et al. 1926) Pirie 1940 (Approved Lists 1980) | Bacteria |
| 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 |
|---|---|---|---|---|---|---|---|
| hly-F | TTA CAC TTA TAT TAG TTA GTC TAC CAA TTG CG | 32 | 200 nM | 31.25 | 53.6 | 9763.42 | \ |
| hly-R | TCC AAT CCT TGT ATA TAC TTA TCG ATT TCA TC | 32 | 200 nM | 31.25 | 53.44 | 9674.36 | \ |
| hly-exo-P | TCT GCA TTC AAT AAA GAA AAT TCA ATT TCA 1CZA2G GCA CCA CCA GCA TC | 47 | 150 nM | 38.3 | 65.03 | 14327.41 | \ |
| Application | Assay | Primer Designing Software | Reaction Time(min) | Assay Temperature(℃) | Readout System(s) | Limit of Detection(LoD) | Sensitivity(%) | Specificity(%) |
|---|---|---|---|---|---|---|---|---|
| In summary, in the present study a novel method, combining IMS and qRPA was successfully developed and evaluated. The new method significantly reduced the time of analysis from 48 hr to just one working day. When compared to previously published methodologies based on RPA for the detection of L. monocytogenes, even though the duration of the method was significantly reduced, its overall performance was not compromised. This represents an important improvement for the application of this technique as a fast identification strategy of L. monocytogenes. | IMS-qRPA | \ | 50 min | 39 °C | IMS | LOD50 of 6.3 cfu\25 g | \ | \ |
| Year of Publication | Title | Author(s) | Journal | PMID | DOI | ||
|---|---|---|---|---|---|---|---|
| 2019 | Combination of Immunomagnetic Separation and Real-Time Recombinase Polymerase Amplification (IMS-qRPA) for Specific Detection of Listeria monocytogenes in Smoked Salmon Samples | Alejandro Garrido-Maestu,Sarah Azinheiro,Joana Carvalho,Marta Prado | Journal of food science | 31264719 | 10.1111/1750-3841.14662 | ||
Combination of Immunomagnetic Separation and Real-Time Recombinase Polymerase Amplification (IMS-qRPA) for Specific Detection of Listeria monocytogenes in Smoked Salmon SamplesAuthor(s):Alejandro Garrido-Maestu,Sarah Azinheiro,Joana Carvalho,Marta PradoJournal:Journal of food scienceYear:2019Abstract:Nowadays, Listeria monocytogenes continues to be a major health issue. Therefore, improvements in the speed and reliability of its detection are still needed. In the present study, the combination of real-time Recombinase Polymerase Amplification (qRPA) with immunomagnetic separation (IMS) is described. The proposed methodology was tested against a real-time PCR method, and was successfully applied to 50 smoked salmon samples spiked at levels ranging from 2 to 9.3 × 102 cfu/25 g. L. monocytogenes was detected after a 24 hr pre-enrichment, which represents a great improvement over other previously published RPA methods. Additionally, the evaluation of the method reported a Limit of dDetection 50 (LoD50 ) of 6.3 cfu/25 g, along with relative sensitivity, specificity and accuracy values higher than 90%. Finally, the index of kappa concordance was calculated to be 0.93 which is interpreted as "almost complete concordance" between the reference and alternative method. Overall, the described methodology proved to be faster, specific, and as sensitive as other methods based on RPA or real-time PCR. PRACTICAL APPLICATION: The methodology described in this study significantly reduces the detection time of L. monocytogenes, when compared with culture-based methods, and it requires fewer steps than other molecular methods, making it a reliable and more convenient method for routine testing. Finally, the evaluation of the methodology in spiked food samples, confirms its reliability.PMID:31264719
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