Rapid DNA visual detection of polymicrobial bloodstream infection using filter paper

Yajing Song,Peter Gyarmati

doi:10.1038/s41598-022-08487-4

Abstract

Bloodstream infection (BSI) is a major complication in patients with cancers due to therapy-induced neutropenia and underlying conditions, which increases hospitalization time and mortality rate. Targeted and timely antimicrobial management is crucial to save the patients’ lives and reduce the social and economic burdens. Blood culture is a routine clinical diagnostic method of BSI with a long turnaround time, and generally identifies monomicrobial BSI. Thus, polymicrobial BSI often goes undetected although it occurs more frequently in these patients and results in more severe outcomes compared to monomicrobial BSI. In this work, we apply glutaric anhydride, N-hydroxysuccinimide and N,N′-dicyclohexylcarbodiimide to fabricate a functional surface on cellulose filter paper. Targeting three pathogens (Escherichia coli, Saccharomyces cerevisiae, and human cytomegalovirus) commonly occurring in BSI in neutropenic patients, we demonstrate rapid and accurate triplex pathogen DNA detection using the functionalized paper. All three pathogen DNA was identified in 1–5 min with a detection limit of 0.1–0.5 ng/µL. The developed test tool has the potential to provide rapid polymicrobial BSI diagnosis in support of timely, accurate antimicrobial treatment, and could be integrated into an automatic sample-to-result portable equipment.

Highlights

Materials and methodsGenomic DNA (gDNA) of E. coli, S. cerevisiae, human serum, DNA oligonucleotides (Tables S1–S3), and chemicals including glutaric anhydride (GA), N-hydroxysuccinimide (NHS), and N,N′-dicyclohexylcarbodiimide (DCC), dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), SSC buffer, and SDS were purchased from Sigma-Aldrich
We optimized the length of probe immobilized on the filter paper surface; designed specific primers of one bacterium (E. coli), one fungus (S. cerevisiae), and one virus (HCMV); and confirmed the corresponding optimal concentration of each pair of primers and the universal annealing temperature in one multiplex PCR system
The functionalized filter paper can simultaneously detect the three amplified products from different species of DNA (E. coli, S. cerevisiae, and Human cytomegalovirus (HCMV)) in one test, which are the common pathogens of polymicrobial Bloodstream infection (BSI) in patients with neutropenia[29–36]

Summary

Materials and methods

Genomic DNA (gDNA) of E. coli, S. cerevisiae, human serum, DNA oligonucleotides (Tables S1–S3), and chemicals including glutaric anhydride (GA), N-hydroxysuccinimide (NHS), and N,N′-dicyclohexylcarbodiimide (DCC), dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), SSC buffer, and SDS were purchased from Sigma-Aldrich. WhatmanTM qualitative filter paper, DYNAL MyOne Dynabeads Streptavidin C1, 2× PCR SYBR Green master mix, myImageAnalysis software, and Qubit 3.0 fluorometer were purchased from Fisher Scientific. A Qiagen Blood DNA extraction kit and Rotor-Gene 6000 instrument were used. A custom-made magnetic stand (MagRach 16), 2× Kapa PCR readymix (Kapabiosystem), and Techne primer thermal cycler (Techne) were used

Methods

Results and discussion

Conclusions