Literature Express | naica® Droplet Chip Digital PCR System Facilitates Highly Sensitive Detection of Klebsiella Pneumoniae

In the expert consensus on the diagnosis and treatment of cough caused by novel coronavirus infection (2023, 46), coughing up yellow purulent sputum or increased peripheral blood leukocytes indicates possible bacterial infection. In a clinical analysis of 95 COVID-19 patients with bacterial and fungal infections published in 2021, the results showed that critically ill COVID-19 patients are prone to concurrent bacterial and fungal infections such as Acinetobacter baumannii and Klebsiella pneumoniae. Klebsiella pneumoniae belongs to the genus Klebsiella of the family Enterobacteriaceae and is an important Gram-negative conditional pathogen of nosocomial infection. It is easily resistant to most antimicrobial drugs and accounts for 13% of clinical isolates, making it the second largest pathogen of nosocomial infection after Escherichia coli (19%).

Scientists from the State Key Laboratory of Microbial Metabolism, School of Life Sciences and Technology, Shanghai Jiao Tong University, established a digital PCR detection method for Klebsiella pneumoniae based on the naica® droplet chip digital PCR system. The lowest detection limit of digital PCR detection sensitivity can reach 3.37 copies/μL; the relative standard deviation (RSD) of the method is less than 25%; this study used the optimized digital PCR method to detect a total of 28 clinical strains, 14 of which were Klebsiella pneumoniae and 14 were other species. This method has good specificity, high sensitivity and high accuracy, and is suitable for nucleic acid detection and quantitative analysis of Klebsiella pneumoniae. It also provides a new technical reference for the molecular detection of other clinical pathogens.

Application highlights:

▶ Digital PCR (dPCR), as the third generation of PCR technology, is simple, fast, highly sensitive and accurate, and can detect trace amounts of bacterial nucleic acid molecules.

▶ Compared with traditional qPCR, dPCR is not affected by amplification efficiency, and can be absolutely quantified without relying on amplification curves or standard curves. It is also more accurate and reliable for low-concentration nucleic acid quantification.

Experimental results:

1. Through the detection range and sensitivity experiments of digital PCR, cdPCR samples can be subsequently tested within the detection range of S5-S9 samples.

▲Figure 1. Sensitivity test results of digital PCR for different dilution standards. Blue: positive droplets; gray: negative droplets; NTC: negative control

2. The detection range and sensitivity experiments of digital PCR and fluorescent quantitative PCR showed that cdPCR is more accurate in detecting low-concentration samples. In addition, the minimum detection limit of cdPCR (3.37 copies/μL) has higher detection sensitivity than qPCR (194.9 copies/μL).

▲Figure 2: Standard curves of digital PCR (A) and fluorescence quantitative PCR (B) of pUC57-16S 2B Ct values ​​of different dilution multiples of standard samples: S1: 9.84; S2: 12.89; S3: 16.21; S4: 19.74; S5: 22.34; S6: 25.69; S7: 28.11; S8: 32.56; S9: 35.42

3. Specific evaluation and verification of 3 strains of Klebsiella pneumoniae and 4 other strains were performed at the same time. The cdPCR method was effective in the specific detection of Klebsiella pneumoniae.

4. 28 clinical strains were detected by cdPCR, 14 of which were Klebsiella pneumoniae and 14 were other strains, indicating that cdPCR has the potential for clinical strain detection.

In summary, this study established a digital PCR method for detecting Klebsiella pneumoniae. Compared with qPCR technology, cdPCR can accurately perform absolute quantitative analysis of nucleic acids, with a detection limit as low as a single copy, independent of the standard curve, good data reproducibility, and unique advantages in the detection of rare or trace samples. Therefore, this method provides a new method for improving the early nucleic acid detection of Klebsiella pneumoniae and also provides new data support for the absolute quantification of nucleic acids.

At the same time, the naica® six-channel digital PCR system provides the possibility for multiple detection of new coronavirus combined with bacterial infection.