Development of two multiplex PCR assays for rapid detection of eleven Gram-negative bacteria in children with septicemia

Abstract

Aim: This study aimed to develop a multiplex PCR assay for simultaneous detection of major Gram-negative etiologies of septicemia and evaluate its performance.

Methods: Multiplex PCR (mPCR) assays were developed targeting 11 bacterial strains. Species-specific primers were confirmed using known clinical isolates and standard strains. Gradient PCR was performed on each primer against its target bacterial gene to determine its optimal amplification condition. The minimum detectable DNA concentration of the two assays was evaluated by adjusting bacterial DNA concentration to 100 ng/μL and, tenfold serially diluting it up to 10 pg/μL with DNAse-free water. The diagnostic accuracy of mPCR assays was established by subjecting the assays to 60 clinical blood samples.

Results: Two mPCR assays were developed. Optimal primer annealing temperature of 55 °C was established and utilized in the final amplification conditions. The assays detected all targeted bacteria, with a 100 pg minimum detectable DNA concentration. Pathogens were not detected directly from whole blood, but after 4 h and 8 h of incubation, 41% (5/12) and 100% (12/12) of the bacteria were detected in culture fluids, respectively. The assays also identified Salmonella spp. and Klebsiella pneumoniae co-infections and extra pathogens (1 E. coli and 2 K. pneumoniae) compared with culture. The sensitivity and specificity of the mPCR were 100.0% (71.7-100.0) and 98.0% (90.7-99.0), respectively. The area under the ROC curve was 1.00 (1.00-1.00).

Conclusions: The mPCR assays demonstrated substantial potential as a rapid tool for septicemia diagnosis alongside the traditional blood culture method. Notably, it was able to identify additional isolates, detect co-infections, and efficiently detect low bacterial DNA loads with high sensitivity, implying its value in enhancing efficiency of diagnosis of septicemia.

Keywords: Diagnosis; Gram-negative bacteria; Septicemia; mPCR.

Fig. 1

Gels showing target genes amplified at the optimized annealing temperature (55 °C). a Multipex A: M = Marker, Lane 1 (Vibrio cholerae, (883 bP), 2. Acinetobacter baumanii (320 bp), 3. Salmonella spp. (284 bp), 4. Pseudomonas aeruginosa (190 bp), 5. Shigella spp. (128 bp), 6. E.coli(128 bp), 7. Negative control. b Multiplex B: M = Marker, 1. Enterobacter spp. (1181 bp), 2. Aeromonas spp. (891 bp), 3. Camphylobacter spp. (855 bp), 4. Providentia alcalifacience (515 bp), 5. K. pneumoniae (174 bp), 6. Negative control

Fig. 2

Gels showing specificity of developed Mpcr. a Multipex A: l = Ladder, Lane 1. DNA mix, Lane 2 (Vibrio cholerae, 883 bP), 3. Acinetobacter baumanii (320 bp), 4. Salmonella spp. (284 bp), 5. Pseudomonas aeruginosa (190 bp), 6. E. coli (128 bp), 7–11. Off targets (Table 1) 12. Negative control. b Multiplex B: L = Ladder, Lane 1 Mixed DNA, 2. Enterobacter spp. (1181 bp), 3. Aeromonas spp. (891 bp), 4. Camphylobacter spp. (855 bp), 5. Providentia alcalifacience (515 bp), 6. K. pneumoniae (174 bp), 7–11. Off targets (Table 1) 12. Negative control

Fig. 3

Gels showing the minimum detectable mixed bacterial DNA concentration. a Multiplex A mixed bacterial DNA and b multiplex B mixed bacterial DNA. a, b M (marker) lane 1. (100 ng), lane 2. (10 ng), lane 3. (1 ng), (lane 4. 100 pg), lane 5. (10 pg); lane 6. NC

Fig. 4

Bacterial pathogens detected in culture fluids at different incubation period