Comparative analysis of loop-mediated isothermal amplification combined with microfluidic chip technology and q-PCR in the detection of clinical infectious pathogens

Abstract

Background: Rapid diagnosis of infectious pathogens at an early stage is crucial to stabilize the patient's condition, reduce medical costs, and shorten hospital stays. Currently, some point-of-care tests have their own shortcomings. Therefore, we built a microfluidic chip based on loop-mediated isothermal amplification to can quickly and sensitively detect infectious pathogens.

Methods: We extracted the DNA of S. aureus, MRSA, Shigella and Klebsiella pneumoniae. Then, the DNA samples were diluted by 10-fold and examined by two methods: LAMP-microfluidic chip and q-PCR, the sensitivity of whom was also compared. In addition, the specificity of the two was also examined by detecting the target bacteria and other microorganisms using the same methods. Finally, we extracted and tested the DNA of clinically infected humoral samples to determine the coincidence rate between the two methods and the bacterial culture method.

Results: For S. aureus, MRSA, Shigella, and Klebsiella pneumoniae, the detection limits of the chip were 2.25 × 10³ copies/μl, 5.32 × 10³ copies/μl, 2.89 × 10³ copies/μl, 6.53 × 10² copies/μl, and the detection limits of q-PCR were 2.25 × 10² copies/μl, 5.32 × 10¹ copies/μl, 2.89 × 10² copies/μl, 6.53 × 10¹ copies/μl, respectively. In terms of detection specificity, neither method cross-reacted with other strains. For the detection of infectious humoral samples, the total coincidence rate between the q-PCR and bacterial culture method was 85.7%, 95%, 95%, and 95.5%, and the total coincidence rate between the chip and bacterial culture method was 81%, 95%, 90%, and 86.4%, respectively.

Conclusion: LAMP-microfluidic chip provides a simple, sensitive, specific, convenient, and rapid pathogen detection method for clinically infected humoral samples without relying on expensive equipment or technical personnels.

Keywords: bacteria; chip; detection; loop-mediated isothermal amplification; microfluidics.

FIGURE 1

The dilution multiple and sensitivity result of four bacteria detected by q‐PCR

FIGURE 2

The dilution multiple and positive results of four bacteria were detected by microfluidic chip method. The blue curve is the AC quality control line, and the positive signal indicates that the test is effective; TP (min) represents the jump time of the reaction curve signal

FIGURE 3

Specificity of detecting S. aureus by qPCR

FIGURE 4

Specificity of detecting MRSA by qPCR

FIGURE 5

Specificity of detecting Shigella by qPCR

FIGURE 6

Specificity of detecting Klebsiella pneumoniae by qPCR