Screening of Additive Formulations Enables Off-Chip Drop Reverse Transcription Quantitative Polymerase Chain Reaction of Single Influenza A Virus Genomes

dc.contributor.authorLoveday, Emma Kate
dc.contributor.authorZath, Geoffrey K.
dc.contributor.authorBikos, Dimitri A.
dc.contributor.authorJay, Zackary J.
dc.contributor.authorChang, Connie B.
dc.date.accessioned2022-08-04T22:29:52Z
dc.date.available2022-08-04T22:29:52Z
dc.date.issued2021-03
dc.description.abstractThe miniaturization of polymerase chain reaction (PCR) using drop-based microfluidics allows for amplification of single nucleic acids in aqueous picoliter-sized drops. Accurate data collection during PCR requires that drops remain stable to coalescence during thermocycling and drop contents are retained. Following systematic testing of known PCR additives, we identified an optimized formulation of 1% w/v Tween-20, 0.8 μg/μL bovine serum albumin, 1 M betaine in the aqueous phase, and 3 wt % (w/w) of the polyethylene glycol-perfluoropolyether2 surfactant in the oil phase of 50 μm diameter drops that maintains drop stability and prevents dye transport. This formulation enables a method we call off-chip drop reverse transcription quantitative PCR (OCD RT-qPCR) in which drops are thermocycled in a qPCR machine and sampled at various cycle numbers “off-chip”, or outside of a microfluidic chip. qPCR amplification curves constructed from hundreds of individual drops using OCD RT-qPCR and imaged using epifluorescence microscopy correlate with amplification curves of ≈300,000 drops thermocycled using a qPCR machine. To demonstrate the utility of OCD RT-qPCR, influenza A virus (IAV) RNA was detected down to a single viral genome copy per drop, or 0.320 cpd. This work was extended to perform multiplexed detection of IAV M gene RNA and cellular β-actin DNA in drops, and direct amplification of IAV genomes from infected cells without a separate RNA extraction step. The optimized additive formulation and the OCD-qPCR method allow for drop-based RT-qPCR without complex devices and demonstrate the ability to quantify individual or rare nucleic acid species within drops with minimal processing.en_US
dc.identifier.citationLoveday, E. K., Zath, G. K., Bikos, D. A., Jay, Z. J., & Chang, C. B. (2021). Screening of Additive Formulations Enables Off-Chip Drop Reverse Transcription Quantitative Polymerase Chain Reaction of Single Influenza A Virus Genomes. Analytical Chemistry, 93(10), 4365-4373.en_US
dc.identifier.issn0003-2700
dc.identifier.urihttps://scholarworks.montana.edu/handle/1/16998
dc.language.isoen_USen_US
dc.publisherAmerican Chemical Societyen_US
dc.rightscc-by-nc-nden_US
dc.rights.urihttps://creativecommons.org/licenses/by-nc-nd/4.0/en_US
dc.titleScreening of Additive Formulations Enables Off-Chip Drop Reverse Transcription Quantitative Polymerase Chain Reaction of Single Influenza A Virus Genomesen_US
dc.typeArticleen_US
mus.citation.extentfirstpage4365en_US
mus.citation.extentlastpage4373en_US
mus.citation.issue10en_US
mus.citation.journaltitleAnalytical Chemistryen_US
mus.citation.volume93en_US
mus.data.thumbpage2en_US
mus.identifier.doi10.1021/acs.analchem.0c03455en_US
mus.relation.collegeCollege of Engineeringen_US
mus.relation.collegeCollege of Letters & Scienceen_US
mus.relation.departmentCenter for Biofilm Engineering.en_US
mus.relation.departmentChemical & Biological Engineering.en_US
mus.relation.departmentChemistry & Biochemistry.en_US
mus.relation.researchgroupCenter for Biofilm Engineering.en_US
mus.relation.universityMontana State University - Bozemanen_US

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