Targeted Molecular Detection
Techniques such as Polymerase Chain Reaction (PCR) and Loop-mediated isothermal AMPlification (LAMP) have been used for many years to selectively amplify and detect specific sequences of DNA or RNA. Both rely on short DNA sequences to recognize and bind with known target sequences, and both use polymerase enzymes to exponentially replicate the target sequences. PCR is considered the gold standard for detection of known pathogens, among many other uses, and LAMP has grown in popularity due to its versatility.
Exhaled Breath Condensate (EBC) is an excellent sample matrix for use in molecular amplification techniques such as PCR and LAMP. Because EBC consists primarily of water distilled from breath, it is extremely clean and typically free of inhibitors. Many PCR and LAMP assays operate uninhibited with a composition of 50% or more EBC. This allows large volumes of sample to be added to the assay without purification steps.
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Molecular amplification techniques are also well suited to EBC samples due to their high level of sensitivity. Many PCR assays reliably detect and quantify as little as a single copy of a target nucleic acid. This sensitivity enables accurate detection of pathogens or other sources of nucleic acids with a short, comfortable breath sample [3]. LAMP methods also frequently detect within single digit counts of nucleic acid targets and may give results within 10 minutes. This rapid turnaround time complements the speed and ease of VosBio’s breath collection technology, allowing testing from sample to result to be completed in under 15 minutes.
LAMP
Polymerase chain reaction (PCR)
3. Lane, G., Zhou, G., Hultquist, J. F., Simons, L. M., Redondo, R. L.-, Ozer, E. A., McCarthy, D. M., Ison, M. G., Achenbach, C. J., Wang, X., Wai, C. M., Wyatt, E., Aalsburg, A., Yang, Q., Noto, T., Alisoltani, A., Ysselstein, D., Awatramani, R., Murphy, R., … Zelano, C. (2024). Quantity of SARS-CoV-2 RNA copies exhaled per minute during natural breathing over the course of COVID-19 infection. https://doi.org/10.7554/eLife.91686.1
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Whole-Sample Molecular Profiling with Untargeted Sequencing
Researchers have detected a wide range of nucleic acids in Exhaled Breath Condensate (EBC). DNA and RNA can derive from infectious pathogens, benign microbes in the lung microbiome, extracellular vesicles and other sources. While targeted approaches such as PCR and LAMP can sensitively and rapidly detect known targets, they are not suitable for discovering unknown nucleic acid sequences.
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Several strategies are suitable to detect and identify unknown targets in EBC, generally relying on sequencing any present nucleic acids. Metagenomic and transcriptomic approaches have been used to identify DNA and mRNA from breath, providing insights into the microbiome of the lungs as well as gene regulation. Further applications of these techniques will continue to drive discoveries in pulmonary health.
9. Glendinning, L., Wright, S., Tennant, P., Gill, A. C., Collie, D., & McLachlan, G. (2017). Microbiota in exhaled breath condensate and the lung. Applied and Environmental Microbiology, 83(12), e00515-17. https://doi.org/10.1128/AEM.00515-17
Mass Spectrometry
Exhaled Breath Condensate (EBC) is introduced into the mass spectrometer, where molecules are ionized and separated based on their mass-to-charge ratio, allowing for identification and quantification of specific biomarkers.
Mass spectrometry involves the measurement of the mass-to-charge ratio of ions. The technique transforms molecules from a liquid phase to the gas phase and then ionizes the molecules. The generated ions are focused into a beam and directed to a detector where their mass can be determined. The masses of the ions and/or their fragments are mass analyzed based on their mass to charge ratio (m/z). This process identifies the molecules present in Exhaled Breath Condensate (EBC), to determine biomarkers for various lung diseases. Essentially, mass spectrometry provides the chemical fingerprint of someone's breath to diagnose potential health issues. Collecting EBC through breathing into a collection device is a non-invasive method compared to traditional lung biopsies or bronchoscopy, making it more patient friendly.
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Researchers can identify specific molecules including proteins, peptides, and metabolites that may be elevated in certain diseases, potentially serving as biomarkers for research, diagnosis and disease monitoring. Small molecules such as acetone, urea and others also can be detected. EBC analysis with MS has been explored for diagnosing conditions like asthma, cystic fibrosis, lung cancer, and even inflammatory lung diseases.
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Some challenges and considerations of this approach include the possibility that the composition of EBC can be influenced by factors like hydration level, breathing patterns, and environmental conditions. This may require careful sample collection and standardization techniques. EBC may contain a complex mixture of molecules, necessitating advanced MS techniques like liquid chromatography – mass spectrometry (LC-MS) or gas chromatography – mass spectrometry (GC-MS) to separate and identify individual components.
ELISA
Enzyme-Linked Immunosorbent Assay (ELISA) is a powerful and sensitive tool for detecting and quantifying specific proteins and biomarkers in biological samples. When applied to Exhaled Breath Condensate (EBC), ELISA offers a non-invasive approach for assessing airway inflammation and other respiratory conditions by measuring key mediators such as cytokines, chemokines, and leukotrienes. This method has been successfully used in studies to monitor respiratory diseases like asthma and Chronic Obstructive Pulmonary Disease (COPD), providing valuable insights into disease mechanisms and treatment responses. As a highly adaptable technique, ELISA continues to demonstrate its potential for expanding diagnostic capabilities in EBC research.
