Design and Evaluation of Multiplex One-Step Reverse Transcription PCR-Dipstick Chromatography Method for the Analysis of Seven Respiratory Pathogens

Abstract

Influenza A, influenza B, severe acute respiratory syndrome coronavirus 2, adenovirus, respiratory syncytial virus, Mycoplasma pneumoniae, and Chlamydophila pneumoniae are common pathogens that can cause severe pneumonia and other symptoms, resulting in acute lower respiratory tract infections. The objective of this study was to design and evaluate a sensitive and specific multiplex one-step reverse transcription PCR (RT-PCR)-dipstick chromatography method for simultaneous rapid detection of these seven pathogens. Streptavidin-coated blue latex particles were used to read out a positive signal. Based on the DNA-DNA hybridization of oligonucleotide sequences (Tag) for forward primer with the complementary oligonucleotide sequence (cTag) on the dipstick and biotin-streptavidin interactions, PCR products were able to be illuminated visually on the dipstick. The specificity and the limit of detection (LOD) were also evaluated. Moreover, the clinical performance of this method was compared with Sanger sequencing for 896 samples. No cross reaction with other pathogens was found, confirming the high specificity of this method. The LOD was 10 copies/µL for each of the tested pathogens, and the whole procedure took less than 40 min. Using 896 samples, the sensitivity and specificity were shown to be no lower than 94.5%. The positive predictive value was higher than 82.1%, and the negative predictive value was higher than 99.5%. The kappa value between the PCR-dipstick chromatography method and Sanger sequencing ranged from 0.869 to 0.940. In summary, our one-step RT-PCR-dipstick chromatography method is a sensitive and specific tool for rapidly detecting multiplex respiratory pathogens.

Fig. 1

Schematic diagram of the PCR–dipstick chromatography method. a Making a mixture. b Inserting a dipstick strip into the mixture. c A blue test line appeared on the dipstick strip. Tag: oligonucleotide; cTag: complementary; C3: C3 Spacer (linker); IC: Internal control; Arrow: chromatography direction (Color figure online)

Fig. 2

Detection of seven pathogens by the PCR–dipstick chromatography method. 1. Severe acute respiratory syndrome coronavirus 2; 2. Influenza A; 3. Influenza B; 4. Respiratory syncytial virus; 5. Adenovirus; 6. Mycoplasma pneumoniae; 7. Chlamydophila pneumoniae; NC: negative control

Fig. 3

Specificity of the PCR–dipstick chromatography method. PC1: multiplex detection of Severe acute respiratory syndrome coronavirus 2, Influenza A, and Influenza B; PC2: multiplex detection of Respiratory syncytial virus, Adenovirus, Mycoplasma pneumoniae, and Chlamydophila pneumoniae; 1. Parainfluenza virus 1; 2. Parainfluenza virus 2; 3. Parainfluenza virus 3; 4. Parainfluenza virus 4; 5. Coronavirus 229E; 6. Coronavirus NL63; 7. Coronavirus OC43; 8. Coronavirus HKU1; 9. Metapneumovirus; 10. Boca virus; 11. Rhinovirus; NC: Negative control

Fig. 4

Specificity of the PCR–dipstick chromatography method. PC1: multiplex detection of Severe acute respiratory syndrome coronavirus 2, Influenza A, and Influenza B; PC2: multiplex detection of Respiratory syncytial virus, Adenovirus, Mycoplasma pneumoniae, and Chlamydophila pneumoniae; 1. Escherichia coli; 2. Staphylococcus aureus; 3. Pseudomonas aeruginosa; 4. Acinetobacter baumannii; 5. Streptococcus pneumoniae; 6. Streptococcus pyogenes; 7. Staphylococcus epidermidis; 8. Haemophilus; 9. Klebsiella pneumoniae; 10. Candida albicans; 11. Legionella pneumophila; 12. Bordetella pertussis; NC: Negative control

Fig. 5

Limit of detection determination of the PCR–dipstick chromatography method. a Severe acute respiratory syndrome coronavirus 2; b Influenza A, c Influenza B; d Respiratory syncytial virus; e Adenovirus; f Mycoplasma pneumoniae; g Chlamydophila pneumoniae. The concentration of plasmids 1–6 were 10⁵, 10⁴, 10³, 100, 10, 5 copies/μL, and negative control (NC)