Rapid and simultaneous detection of multiple pathogens in the lower reproductive tract during pregnancy based on loop-mediated isothermal amplification-microfluidic chip

Abstract

Background: Female reproductive tract infection (RTI) is the common source of varied diseases, especially as an important risk factor for pregnancy outcomes, therefore the rapid, accurate and simultaneous detection of multiple pathogens is in urgent need for assisting the diagnosis and treatment of RTI in pregnant women. Streptococcus agalactiae (S. agalactiae), Enterococcus faecalis (E. faecalis), Gardnerella vaginalis (G. vaginalis), Candida albicans (C. albicans) and Chlamydia trachomatis (C. trachomatis) are five main pathogens in lower genital tract with high risk, serious consequences and clinical demands. The combination of loop-mediated isothermal amplification (LAMP) and microfluidic technology was used to develop the LAMP-microfluidic chip for rapid, simple, sensitive and simultaneous detection of the five target pathogens above.

Results: Standard strains and clinical isolates were used for the establishment of the novel LAMP method in tube and LAMP-microfluidic chip, followed by the chip detection on 103 clinical samples and PCR verification partially. The sensitivities of LAMP of S. agalactiae, E. faecalis, G. vaginalis, and C. albicans in tube were 22.0, 76.0, 13.2, 1.11 CFU/μL, respectively, and C. trachomatis was 41.3 copies/μL; on LAMP-microfluidic chip they were 260, 154, 3.9 and 7.53 CFU/μL, respectively, and C. trachomatis was 120 copies/μL. The positive coincidence rates of clinical stains in tube and on chip experiments were 100%. Compared with the classic culture method performed in hospitals, the positive coincidence rate of the 103 clinical samples detected by LAMP-microfluidic chip were 100%. For the six inconsistent ones, including four G. vaginalis and two C. albicans positive samples tested by LAMP-microfluidic chip and verified by PCR were negative by culturing method in hospitals, indicating the lack of efficient detection by the classic culturing method.

Conclusion: Our study suggested that the LAMP-microfluidic chips could simultaneously, efficiently, and accurately detect multiple main pathogens, including S. agalactiae, E. faecalis, G. vaginalis, C. albicans and C. trachomatis, in clinical samples of female RTI to give a great clinical value. Accordingly, this novel method has the potential to provide a valuable reference for female RTI screening and early diagnosis during pregnancy.

Keywords: LAMP-microfluidic chip; Loop-mediated isothermal amplification; Microfluidic technology; Pathogen; Rapid and simultaneous detection; Reproductive tract infection.

Fig. 1

Amplification curves and melting curves of S. agalactiae, E. faecalis, and C. trachomatis. A, C, E Amplification curves of S. agalactiae, E. faecalis, and C. trachomatis. B, D, F Melting curves of S. agalactiae, E. faecalis, and C. trachomatis. The red curves represent the target pathogens and the gray curves represent the blank control

Fig. 2

Specificity of LAMP reaction for five target pathogens. A Streptococcus agalactiae, B Enterococcus faecalis, C Gardnerella vaginalis, D Candida albicans, E Chlamydia trachomatis

Fig. 3

Sensitivity of LAMP detection for A S. agalactiae, B E. faecalis, C G. vaginalis, D C. albicans, E C. trachomatis

Fig. 4

The specificity and positive amplification curves of five target pathogens on LAMP-microfluidic chip. A The specificity test of LAMP reaction on chip. B ~ F Positive amplification of GBS, EF, GV, CA and CT on chip. PQC, positive quality control. CA, C. albicans. CT, C. trachomatis. EF, E. faecalis. GBS, S. agalactiae. GV, G. vaginalis

Fig. 5

Agarose gel electrophoresis of PCR products in the validation experiments of clinical samples with positive chip results for (A) C. albicans and (B) G. vaginalis. M, 100 bp DNA ladder. CK1 and CK3, sterile water as amplification templates. CK2 and CK4, nucleic acid extraction reagent as amplification templates. CA1 and CA2, C. albicans PCR products of sample 1 and 80. GV1 ~ GV4, G. vaginalis PCR products of sample 5, 11, 33, 72. The gels were cropped and the original gels were presented in Additional file 2 and 3