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Droplet microfluidic-based loop-mediated isothermal amplification (dLAMP) for simultaneous quantification of multiple targets.

ABSTRACT: The quantification of trace nucleic acids in biological samples is a frequent requirement in experimental and clinical diagnostics. Here, we present a protocol for the digital quantification of multiple nucleic acid targets with droplet microfluidics-based loop-mediated isothermal amplification (dLAMP). Our protocol provides a fundamental platform for the absolute quantification of multiple nucleic acid targets with high specificity, allowing readily adaption in various in vitro diagnostic settings. For complete details on the use and execution of this protocol, please refer to Tan et al. (2021a, 2021b).

SUBMITTER: Tan YL

PROVIDER: S-EPMC9043755 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Droplet microfluidic-based loop-mediated isothermal amplification (dLAMP) for simultaneous quantification of multiple targets.

Tan Ya-Ling YL Wang Tao T He Jianjun J Jiang Jian-Hui JH

STAR protocols 20220418 2

The quantification of trace nucleic acids in biological samples is a frequent requirement in experimental and clinical diagnostics. Here, we present a protocol for the digital quantification of multiple nucleic acid targets with droplet microfluidics-based loop-mediated isothermal amplification (dLAMP). Our protocol provides a fundamental platform for the absolute quantification of multiple nucleic acid targets with high specificity, allowing readily adaption in various <i>in vitro</i> diagnosti ...[more]

PMID: 35496787

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Project description:BackgroundFemale 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.ResultsStandard 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.ConclusionOur 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.

Dataset Information

Droplet microfluidic-based loop-mediated isothermal amplification (dLAMP) for simultaneous quantification of multiple targets.

Publications

Droplet microfluidic-based loop-mediated isothermal amplification (dLAMP) for simultaneous quantification of multiple targets.

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