Accurate Quantification and Characterization of Adeno-Associated Viral Vectors

Dobnik, David; Kogovšek, Polona; Jakomin, Tjaša; Košir, Nejc; Žnidarič, Magda Tušek; Leskovec, Maja; Kaminsky, Stephen M.; Mostrom, Janet; Lee, Hyunmi; Ravnikar, Maja

doi:10.3389/fmicb.2019.01570

Cited by 86 publications

(97 citation statements)

References 30 publications

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“…However, when Ct values were between 34 and 38, the viral load of samples with the same Ct value was significantly different, indicating that the Ct value of RT-PCR may not sensitively reflect the level of viral load when the viral load is low. This result is consistent with previous reports [15,16]. In 67 single-gene-positive samples and 4 RT-PCR-negative samples, ddPCR gave positive results with low viral load, suggesting that RT-PCR was unstable in the detection of low-viral-load samples.…”

Section: Discussionsupporting

confidence: 92%

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Quantitative Detection and Viral Load Analysis of SARS-CoV-2 in Infected Patients

Yan

Wang

et al. 2020

Clinical Infectious Diseases

663

664

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Background Coronavirus disease 2019 (COVID-19) has become a public health emergency. The widely used reverse transcription–polymerase chain reaction (RT-PCR) method has limitations for clinical diagnosis and treatment. Methods A total of 323 samples from 76 COVID-19–confirmed patients were analyzed by droplet digital PCR (ddPCR) and RT-PCR based 2 target genes (ORF1ab and N). Nasal swabs, throat swabs, sputum, blood, and urine were collected. Clinical and imaging data were obtained for clinical staging. Results In 95 samples that tested positive by both methods, the cycle threshold (Ct) of RT-PCR was highly correlated with the copy number of ddPCR (ORF1ab gene, R2 = 0.83; N gene, R2 = 0.87). Four (4/161) negative and 41 (41/67) single-gene positive samples tested by RT-PCR were positive according to ddPCR with viral loads ranging from 11.1 to 123.2 copies/test. The viral load of respiratory samples was then compared and the average viral load in sputum (17 429 ± 6920 copies/test) was found to be significantly higher than in throat swabs (2552 ± 1965 copies/test, P < .001) and nasal swabs (651 ± 501 copies/test, P < .001). Furthermore, the viral loads in the early and progressive stages were significantly higher than that in the recovery stage (46 800 ± 17 272 vs 1252 ± 1027, P < .001) analyzed by sputum samples. Conclusions Quantitative monitoring of viral load in lower respiratory tract samples helps to evaluate disease progression, especially in cases of low viral load.

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Section: Discussionsupporting

confidence: 92%

“…Seven patients were of uncertain stage because their chest imaging was normal. The average days from symptom onset to the early, progressive, and recovery phases were 4 (range, 2-6), 12 (range, [7][8][9][10][11][12][13][14][15][16][17][18][19], and 20 (range, 10-33) days after disease onset, respectively.…”

Section: Patient Characteristicsmentioning

confidence: 99%

Quantitative Detection and Viral Load Analysis of SARS-CoV-2 in Infected Patients

Yan

Wang

et al. 2020

Clinical Infectious Diseases

663

664

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“…Due to the existence of empty capsids that do not possess therapeutic benefits, titration by viral genome is preferred for clinical dosing, manufacturing, and analytical testing. 2 , 3 , 4 Traditionally, AAV genome titer is determined by qPCR using a plasmid DNA standard. For the past two decades, extensive efforts have been made in improving qPCR standard preparation, sample treatment, and primer/probe design, etc.…”

Section: Introductionmentioning

confidence: 99%

“… 8 Currently, the field is quickly moving toward droplet digital PCR (ddPCR), a new PCR technology that can achieve superior accuracy and precision, without the need of standard curves or special sample preparation. 2 , 9 However, ddPCR has its own shortcomings. Different from qPCR, ddPCR cannot be scaled up from 96 to 384 wells, and each well must be read individually, which limits throughput and prolongs assay time.…”

Section: Introductionmentioning

confidence: 99%

A qPCR Method for AAV Genome Titer with ddPCR-Level of Accuracy and Precision

Wang

Menon

Shen

et al. 2020

Molecular Therapy - Methods & Clinical Development

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Recombinant adeno-associated virus (rAAV) is one of the main vectors used in gene therapy. An accurate genome titer is not only critical for clinical dosing, but also a prerequisite for many analytical assays for AAV product characterization. AAV genome titer is traditionally determined by qPCR; however, assay precision is not optimal despite extensive efforts. More recently, droplet digital PCR (ddPCR) emerged as a powerful alternative that offers excellent accuracy and precision. However, currently ddPCR is not as widely available as qPCR and operates at a lower throughput and a higher cost. In this paper, we introduce an improved qPCR method with two major optimizations: (1) using an AAV reference material as qPCR standard instead of plasmid DNA and (2) implementing a “digestion-free” method by adding 5% Tween 20 to standard and sample preparations. The new method has been extensively tested with AAV of different serotypes, purification status, and transgenes encapsidated and was found to be highly accurate, precise, and robust. This significantly improved and simplified assay can be easily adopted by researchers in the gene therapy field and further automated for high-throughput applications.

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“…ddPCR is an endpoint PCR approach with the capability of measuring absolute number of DNA targets. Unlike qPCR, ddPCR is independent of reference materials and is less sensitive to inhibitors of PCR reactions, making it more accurate for measuring AAV titers 28 . However, ddPCR titration method is not widely used.…”

Section: Introductionmentioning

confidence: 99%

Quantification of Adeno-Associated Virus with Safe Nucleic Acid Dyes

Xu¹,

DeVries²,

Zhu³

2020

Preprint

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AbstractAdeno-associated virus (AAV) is the most commonly used viral vector for both biological and gene therapeutic applications1. Although many methods have been developed to measure quantity attributes of AAV, they are often technically challenging and time consuming. Here we report a method to titer AAV with GelGreen® dye, a safe green fluorescence nucleic acid dye recently engineered by Biotium company (Fremont, CA). This method, hereinafter referred to as GelGreen method, provides a fast (~ 30 minutes) and reliable strategy for AAV titration. To validate GelGreen method, we measured genome titer of an AAV reference material AAV8RSM and compared our titration results with those determined by Reference Material Working Group (ARMWG). We showed that GelGreen results and capsid Elisa results are comparable to each other. We also showed that GelRed® dye, a red fluorescence dye from Biotium, can be used to directly “visualize” AAV genome titer on a conventional gel imager, presenting an especially direct approach to estimate viral quantity. In summary, we described a technique to titer AAV by using new generation of safe DNA dyes. This technique is simple, safe, reliable and cost-efficient. It has potential to be broadly applied for quantifying and normalizing AAV viral vectors.

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Accurate Quantification and Characterization of Adeno-Associated Viral Vectors

Cited by 86 publications

References 30 publications

Quantitative Detection and Viral Load Analysis of SARS-CoV-2 in Infected Patients

Quantitative Detection and Viral Load Analysis of SARS-CoV-2 in Infected Patients

A qPCR Method for AAV Genome Titer with ddPCR-Level of Accuracy and Precision

Quantification of Adeno-Associated Virus with Safe Nucleic Acid Dyes

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