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

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.

Keywords: Klebsiella pneumoniae; Staphylococcus aureus; lateral flow dipstick; rapid detection; recombinase-aided amplification.

Figure 1

The working principle of the duplex lateral flow dipstick assay.

Figure 2

The optimization results of the duplex RAA–LFD. Primer concentration (A,D), incubation temperature (B,E), and reaction time (C,F) using genomic DNA of S. aureus and K. pneumoniae. Note: the blue and brown lines were fitted curves additionally generated using gray values.

Figure 3

The specificity of the duplex RAA–LFD. 1—K. pneumoniae G412 and S. aureus AB91093; 2—K. pneumoniae G412; 3—K. pneumoniae G304; 4—K. pneumoniae G305; 5—K. pneumoniae G413; 6—S. aureus AB91093; 7—S. aureus G404; 8—S. aureus G109; 9—S. aureus 115; 10—K. oxytoca G414; 11—S. saprophyticus ATCC BAA-750; 12—S. epidermidis ATCC1228; 13—V. cholerae C606; 14—L. monocytogenes G12; 15—L. monocytogenes ATCC19115; 16—P. aeruginosa H012; 17—E. coli O157:H7 ATCC43889; 18—V. parahaemolyticus ATCC17802; 19—V. harveyi ATCC33842; 20—E. faecalis G401; 21—S. Enteritidis CMCC50041; 22—negative control.

Figure 4

Sensitivity of the duplex RAA–LFD. (A) Sensitivity of the duplex RAA–LFD assay for K. pneumoniae and S. aureus genomic DNA. (B) Sensitivity of the duplex RAA–LFD for pure cultures of K. pneumoniae and S. aureus. NC: negative control.

Figure 5

Growth effect of target bacteria under different conditions. (A) The growth of each bacterium in the SKII medium alone. (B) The growth of each bacterium in the SKII medium under the co-existence of two target bacteria. (C) The growth of target bacteria in the SKII medium in the presence of non-target bacteria.

Figure 6

Detection of K. pneumoniae and S. aureus in simulated samples with the duplex RAA–LFD method; 1~4: 1 mL of 10³, 10², 10¹, and 10⁰ CFU of K. pneumoniae and S. aureus were added to 23 mL of milk and enriched for 0 h (A), 2 h (B), 4 h (C), and 6 h (D). NC: negative control.