Development of Loop-Mediated Isothermal Amplification Assay Targeting lytA and psaA Genes for Rapid and Visual Diagnosis of Streptococcus pneumoniae Pneumonia in Children

Abstract

Streptococcus pneumoniae (S. pneumoniae) is a common major human pathogen associated with community-acquired pneumonia, septicemia, meningitis, and otitis media. It is difficult to isolate and identify S. pneumoniae form clinical samples. To evaluate a novel, rapid, sensitive, and specific loop-mediated isothermal amplification (LAMP) assay to detect S. pneumoniae pneumonia in children, we designed specific LAMP primers targeting lytA and psaA genes. We optimized the reaction time and reaction system, and evaluated its sensitivity and specificity of detection using real-time turbidity monitoring and visual observation. We also analyzed the molecular characteristics of the isolates obtained from the positive samples. The primer sets LytA-1 and PsaA-2 amplified the genes in the shortest times, and 63°C was confirmed as the optimum reaction temperature. The detection sensitivity of each reaction was 10 and 100 copies/μL with primer sets LytA-1 and PsaA-2, respectively. This LAMP assay showed no cross-reactivity with other 27 pathogens. To describe the availability of this method, we collected 748 clinical samples from children with pneumonia. Among them, 135 were confirmed to be S. pneumoniae positive by LAMP. The sensitivity was 100% (95% CI 96.4-100%), specificity 99.0% (95% CI 97.8-99.6%). Including them, 50 were co-infected with Mycoplasma pneumoniae. This LAMP assay detected S. pneumoniae in 1 h and the results can be identified with visual naked eyes. Thus, it will be a powerful tool for S. pneumoniae early diagnosis and effective antibiotic therapy.

Keywords: Streptococcus pneumoniae; children; detection; loop-mediated isothermal amplification; visual.

FIGURE 1

Selection of specific regions and primer positions. (A) Primers to amplify the lytA gene. (B) Primers to amplify the psaA gene. F3: outer forward primer; B3: outer backward primer; FIP: forward inner primer, F1c-F2; BIP: backward inner primer, B1c-B2; LF: loop forward primer; LB: loop backward primer.

FIGURE 2

The most suitable primers and reaction temperature used for loop-mediated isothermal amplification (LAMP) analysis. (A) The most suitable primer pair for LAMP analysis to amplify the lytA gene. (B) The optimal reaction temperature for LAMP analysis using primer LytA-1. (C) The most suitable primer set for LAMP analysis to amplify the psaA gene. (D) The optimal reaction temperature for LAMP determination using primer PsaA-2. The reaction volume is 25 μL, contains 2 μL of DNA template, and the template concentration was 2 ng/μL.

FIGURE 3

The sensitivity of loop-mediated isothermal amplification (LAMP) analysis and conventional PCR to the detection of S. pneumoniae. (A,B) The sensitivity of LAMP analysis using the primer set LytA-1 to target the lytA gene. (C,D) Sensitivity of LAMP assay using primer set PsaA-2 to target psaA gene. (E) The sensitivity of conventional PCR detection for lytA and psaA gene. Use Loopamp real-time turbidity meter to monitor and detect turbidity (A,C), and judge with the naked eye based on the color change from orange to green (B,D). Lanes 1–9: 10⁸, 10⁷, 10⁶, 10⁵, 10⁴, 10³, 10², 10¹, and 10⁰copies/μL.

FIGURE 4

Specificity of the loop-mediated isothermal amplification assay for S. pneumoniae detection. (A) Specificity of the LAMP assay with primer set LytA-1 targeted the lytA gene. (B) Specificity of the LAMP assay with primer set PsaA-2 targeted the psaA gene. The detection was monitored by turbidity using a Loopamp real-time turbidimeter. S1: influenza A, S2: influenza B, S3: parainfluenza viruses, S4: adenoviruses, S5: respiratory syncytial virus, S6: human metapneumovirus, S7: human bocavirus, S8: rhinovirus, S9: Mycoplasma pneumoniae M129, S10: Mycoplasma pneumoniae FH, S11: Haemophilus influenzae, S12: Legionella pneumophila, S13: Listeria monocytogenes, S14: Staphylococcus aureus, S15: Klebsiella pneumoniae, S16: Pseudomonas aeruginosa, S17: Escherichia coli, S18: Acinetobacter baumannii, S19: Mycobacterium tuberculosis, S20: Bordetella pertussis, S21: Streptococcus mitis, S22: Streptococcus oralis, S23: Streptococcus agalactiae, S24: Streptococcus mutans, S25: Streptococcus parasanguinis, S26:Streptococcus sanguinis, S27:Streptococcus salivarius.