Triplex Hybridization-Based Nanosystem for the Rapid Screening of Pneumocystis Pneumonia in Clinical Samples

Abstract

Pneumocystis pneumonia (PcP) is a disease produced by the opportunistic infection of the fungus Pneumocystis jirovecii. As delayed or unsuitable treatments increase the risk of mortality, the development of rapid and accurate diagnostic tools for PcP are of great importance. Unfortunately, current standard methods present severe limitations and are far from adequate. In this work, a time-competitive, sensitive and selective biosensor based on DNA-gated nanomaterials for the identification of P. jirovecii is presented. The biosensor consists of a nanoporous anodic alumina (NAA) scaffold which pores are filled with a dye reporter and capped with specific DNA oligonucleotides. In the presence of P. jirovecii genomic DNA, the gated biosensor is open, and the cargo is delivered to the solution where it is monitored through fluorescence spectroscopy. The use of capping oligonucleotides able to form duplex or triplex with P. jirovecii DNA is studied. The final diagnostic tool shows a limit of detection (LOD) of 1 nM of target complementary DNA and does not require previous amplification steps. The method was applied to identify DNA from P. jirovecii in unmodified bronchoalveolar lavage, nasopharyngeal aspirates, and sputum samples in 60 min. This is a promising alternative method for the routinely diagnosis of Pneumocystis pneumonia.

Detection of SARS-CoV-2 Virus by Triplex Enhanced Nucleic Acid Detection Assay (TENADA)

“…The time required for the analysis is less than 1 h. The detection limit, the analysis time, and the requirement of simple instrumentation are strong arguments for further development. In addition, the triplex-assisted assay can be readily adapted for the detection of any pathogen with either DNA or RNA genomes as PPRHs are able to produce strand displacement when targeting duplex DNA [ 15 , 16 , 17 , 18 ] and it only requires the knowledge of the pathogen genome sequence.…”

“…Recently we have described that DNA hairpins that recognize nucleic acid targets by triplex formation can detect Pneumocystis in bronchoalveolar lavage, nasopharyngeal aspirates, and sputum samples using surface plasmon resonance (SPR) [ 15 ] or by using nanoporous anodic alumina scaffolds filled with a fluorescent dye reporter [ 16 ]. These successful strategies make us think that triplex-forming DNA hairpins could be used for the direct detection of viral RNA, concretely the detection of single-stranded SARS-CoV-2 RNA.…”

“…PPRHs have been described as gene silencing tools of several genes mainly involved in cancer with the capacity to produce strand displacement [ 17 ]. Additionally, they have been incorporated as probes in biosensors for the detection of miRNA-145 [ 20 , 21 ], to determine the DNA methylation status of the PAX-5 gene [ 22 ], and for the diagnosis of Pneumocystis pneumonia [ 15 , 16 ].…”

Oligonucleotic Probes and Immunosensors Based on Nanoporous Anodic Alumina for Screening of Diseases

“…Both species have been detected with ultrasensitive molecular gate-based sensing platforms developed by the research group of Martínez-Máñez using appropriate aptamers. [141,142] The same sensing system has been also used for the detection of Pneumocystis jirovecii, [151] the fungus that produces the Pneumocystis pneumonia disease and that can be easily disseminated from person to person producing asymptomatic or severe infections.…”

“…In addition to cocaine, this gated NAA detection system has been successfully used for the monitoring of bacteria ( Staphylococcus aureus , [ 150 ] mycoplasma, [ 143 ] ]) fungus ( Candida auris , [ 141 ] Candida albicans , [ 142 ] Pneumocystis pneumonia [ 151 ] ), biomarkers, [ 152,153 ] and allergenic proteins. [ 154 ]…”