RPB0285

Pathogen Description

Target Pathogen Pathogen Name NCBI Taxonomy ID Order Family Genus Species Pathogen type
Staphylococcus aureus Staphylococcus aureus, Micrococcus aureus, Staphylococcus pyogenes aureus 1280 Bacillales Staphylococcaceae Staphylococcus Staphylococcus aureus Bacterium

Primer Description

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
nuc-LF2 DIG-TGTATTTTCTTTTTAATATTGCCTTTATGCG 31 100nM 25.81 51.88 9453.2 \
nuc-LR2 Biotin-TTGTCACTGAGTAAAACAGAATCAAATATGC 31 100nM 32.26 54.89 9535.31 \

Gene Description

Target Gene GenBank ID
nuc gene \

Assay Description

Application Assay Primer Designing Software Reaction Time(min) Assay Temperature(℃) Readout System(s) Limit of Detection(LoD) Sensitivity(%) Specificity(%)
a precise, sensitive, and simple field testing technique, can be used to identify S. aureus and K. pneumoniae and is expected to be used for disease diagnosis. RAA-LFD \ 20 mins 39°C lateral flow dipstick (LFD) method 1.78 × 10³CFU\mL 60 fg 1

Publication Description

Year of Publication Title Author(s) Journal PMID DOI
2025 Establishment and Application of Duplex Recombinase-Aided Amplification Combined with Lateral Flow Dipsticks for Rapid and Simultaneous Visual Detection of Klebsiella pneumoniae and Staphylococcus aureus in Milk Ni Zhang,Laiwang Hou,Darong Li,Weiqing Lan,Yong Zhao,Xiaohong Sun Foods 40002017 10.3390/foods14040573

Establishment and Application of Duplex Recombinase-Aided Amplification Combined with Lateral Flow Dipsticks for Rapid and Simultaneous Visual Detection of Klebsiella pneumoniae and Staphylococcus aureus in Milk

Author(s):

Ni Zhang,Laiwang Hou,Darong Li,Weiqing Lan,Yong Zhao,Xiaohong Sun

Journal:

Foods

Year:

2025

Abstract:

Staphylococcus aureus and Klebsiella pneumoniae are significant and prevalent pathogens associated with bovine mastitis on dairy farms worldwide, resulting in severe infections in both dairy cows and, subsequently, human beings. Fast and dependable pathogen diagnostics are essential to minimize the effects of cow mastitis and human infections. The aim of this research was to develop a duplex recombinase-aided amplification (RAA) combined with the lateral flow dipstick (LFD) method, which was used for rapid, simultaneous detection of S. aureus and K. pneumoniae. The SKII culture medium for S. aureus and K. pneumoniae cocultivation was developed in this study. By optimizing the duplex RAA-LFD reaction conditions in terms of primer concentration, amplification temperature, and reaction time, the duplex RAA-LFD assay could successfully detect S. aureus and K. pneumoniae when the reaction was conducted at 39 °C for 20 min. The duplex RAA-LFD method demonstrated good specificity, exhibiting no cross-reactivity with other pathogens. In addition, the detection limit of the duplex RAA-LFD for S. aureus and K. pneumoniae was 60 fg of genomic DNA and 1.78 × 103 and 2.46 × 103 CFU/mL of bacteria in pure culture. Moreover, the duplex RAA-LFD technique is capable of identifying S. aureus and K. pneumoniae in artificially spiked milk samples even at very low initial concentrations of 1.78 × 101 and 2.46 × 100 CFU/mL, respectively, after 6 h of enrichment. The result of the actual samples showed that the total concordance rate of the duplex RAA-LFD method with the biochemical identification method and PCR method could reach 92.98~98.25% with high consistency. The results of this study indicated that the duplex RAA-LFD assay, which is a precise, sensitive, and simple field testing technique, can be used to identify S. aureus and K. pneumoniae and is expected to be used for disease diagnosis.