Quantification of Viral Inactivation by Photochemical Treatment with Amotosalen and UV A Light, Using a Novel Polymerase Chain Reaction Inhibition Method with Preamplification

Allain, Jean‐Pierre; Hsu, Jocelyn; Pranmeth, Manisha; Hanson, Deborah; Stassinopoulos, Adonis; Fischetti, Lucia; Corash, Laurence; Lin, Lily

doi:10.1086/509260

Cited by 25 publications

(27 citation statements)

References 23 publications

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“…A similar approach was described for determining the inactivation of hepatitis B virus with amotosalen and UV in blood platelets (Allain et al . ). In this study, reduction in PCR signal was related to results obtained with an ELISA assay.…”

Section: Discussionmentioning

confidence: 97%

Long-range quantitative PCR for determining inactivation of adenovirus 2 by ultraviolet light

Rodríguez¹,

Bounty

Linden

2013

J Appl Microbiol

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Aims: An extra-long-range quantitative PCR (LR-qPCR) method was developed for estimating genome damage to adenovirus 2 caused by UV irradiation. The objective was to use LR-qPCR as a rapid method to determine adenovirus UV inactivation. Methods: The LR-qPCR consisted of two steps: a long-range PCR (up to 10 kb fragment) and a real-time, quantitative (q) PCR for quantifying the products of the first PCR. We evaluated LR-qPCR with adenovirus irradiated with medium-pressure (MP, polychromatic emission) and low-pressure (LP, 254 nm) mercury vapour lamps and compared results with cell culture infectivity. Results: Using LR-qPCR, a fragment of 6 kb estimated DNA damage in a linear relationship to doses between 0 and 20 mJ cm À2 , and a 1-kb fragment related linearly to doses between 20 and 100 mJ cm À2 . The LR-qPCR results for the 6-kb fragment were similar to infectivity assays results for adenovirus exposed to MP UV. For adenovirus irradiated with LP lamps, LR-qPCR results for the shorter fragment size (1 kb) were similar to reduction in viral infectivity. No difference was observed between 10 and 6 kb LR-qPCR results. Conclusion:The LR-qPCR can be used as a tool for estimating DNA damage caused by UV in adenovirus. The LR-qPCR results were related to reduction in viral infectivity. Significance and Impact of the Study: The use of LR-qPCR to determine DNA damage and estimate inactivation of adenovirus 2 from UV disinfection allows for same-day results compared with >7 days required for cell culture. This accelerates adenovirus inactivation results for the water industry where adenovirus is used as a representative virus for crediting UV systems. This PCR approach provides a framework that can be used for other viral viability assays using the inhibition of amplification of viral nucleic acid after pretreatments, such as propidium monoazide, and for cellular biology studies of DNA damage.

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Section: Discussionmentioning

confidence: 97%

Long-range quantitative PCR for determining inactivation of adenovirus 2 by ultraviolet light

Rodríguez¹,

Bounty

Linden

2013

J Appl Microbiol

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“…In parallel, more than 1000‐fold inhibition of PCR amplification of a 242‐bp fragment in the HLA‐DQα locus was observed. Additionally, PCR inhibition studies measuring viral targets showed approximately 1 log reduction in amplification of short fragments (244 bp for parvovirus B19; 495 bp for HBV) after amotosalen and UVA treatment . For mtDNA, the adduct density after INTERCEPT treatment of leukoreduced PLT concentrates was calculated at one adduct per 268 bp based on liquid scintillation, and PCR amplification of a 166‐bp mtDNA fragment was unaffected, although quantitation was not performed .…”

Section: Discussionmentioning

confidence: 99%

Assessment of nucleic acid modification induced by amotosalen and ultraviolet A light treatment of platelets and plasma using real‐time polymerase chain reaction amplification of variable length fragments of mitochondrial DNA

et al. 2015

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BACKGROUND Pathogen inactivation methods are increasingly used to reduce the risk of infections after transfusion of blood products. Photochemical treatment (PCT) of platelets (PLTs) and plasma with amotosalen and ultraviolet A (UVA) light inactivates pathogens and white blood cells through formation of adducts between amotosalen and nucleic acid that block replication, transcription, and translation. The same adducts block the amplification of nucleic acids using polymerase chain reaction (PCR) in a manner that correlates with the number of adducts formed, providing a direct quality control (QC). Current QC measures for PCT rely on indirect methods that measure the delivered UVA dose or percent residual amotosalen after illumination, rather than directly measuring nucleic acid modification. STUDY DESIGN AND METHODS Endogenous mitochondrial DNA (mtDNA), which is detectable in PLT and plasma units, was chosen as a target for the quantification of photochemically induced modifications. DNA was extracted from untreated or amotosalen and UVA–treated PLTs or plasma, and mtDNA fragments of variable lengths were quantified using a real‐time PCR inhibition assay. RESULTS PCT induced increasing real‐time PCR inhibition of mtDNA amplification for larger amplicon sizes. Amplification was unaffected by treatment with amotosalen or UVA alone, whereas up to 3 log inhibition was observed after PCT. Blinded PCR testing of a panel of 110 samples each, from PLT or plasma components prepared for routine use within a blood center, allowed 100% discrimination between untreated and treated units. CONCLUSION Our initial findings indicate that an adequately sensitive, quantitative real‐time PCR inhibition assay targeting mtDNA could provide a valuable tool to confirm and monitor PCT.

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“…Additionally, they induce the cross-linking of pyrimidines only following UV exposure (5,6). This feature of psoralens has made them attractive in transfusion medicine for pathogen inactivation (1,7,11), particularly the use of amotosalen as an alternative to traditional leukocyte reduction methods for the prevention of parvovirus (1) and cytomegalovirus (10) transmission.…”

Section: Discussionmentioning

confidence: 99%

Immunogenicity of a Psoralen-Inactivated Dengue Virus Type 1 Vaccine Candidate in Mice

Maves

Oré

Porter

et al. 2010

Clin Vaccine Immunol

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Quantification of Viral Inactivation by Photochemical Treatment with Amotosalen and UV A Light, Using a Novel Polymerase Chain Reaction Inhibition Method with Preamplification

Cited by 25 publications

References 23 publications

Long-range quantitative PCR for determining inactivation of adenovirus 2 by ultraviolet light

Long-range quantitative PCR for determining inactivation of adenovirus 2 by ultraviolet light

Assessment of nucleic acid modification induced by amotosalen and ultraviolet A light treatment of platelets and plasma using real‐time polymerase chain reaction amplification of variable length fragments of mitochondrial DNA

Immunogenicity of a Psoralen-Inactivated Dengue Virus Type 1 Vaccine Candidate in Mice

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