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Evaluating the Performance of a Magnetic Nanoparticle-Based Detection Method Using Circle-to-Circle Amplification.

ABSTRACT: This work explores several issues of importance for the development of a diagnostic method based on circle-to-circle amplification (C2CA) and oligonucleotide-functionalized magnetic nanoparticles. Firstly, the performance of the detection method was evaluated in terms of sensitivity and speed. Synthetic target sequences for Newcastle disease virus and Salmonella were used as model sequences. The sensitivity of the C2CA assay resulted in detection of 1 amol of starting DNA target with a total amplification time of 40 min for both target sequences. Secondly, the functionalization of the nanoparticles was evaluated in terms of robustness and stability. The functionalization was shown to be very robust, and the stability test showed that 92% of the oligos were still attached on the particle surface after three months of storage at 4 °C. Altogether, the results obtained in this study provide a strong foundation for the development of a quick and sensitive diagnostic assay.

SUBMITTER: Sanchez Martin D

PROVIDER: S-EPMC8226732 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature

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Evaluating the Performance of a Magnetic Nanoparticle-Based Detection Method Using Circle-to-Circle Amplification.

Sánchez Martín Darío D Oropesa-Nuñez Reinier R Zardán Gómez de la Torre Teresa T

Biosensors 20210528 6

This work explores several issues of importance for the development of a diagnostic method based on circle-to-circle amplification (C2CA) and oligonucleotide-functionalized magnetic nanoparticles. Firstly, the performance of the detection method was evaluated in terms of sensitivity and speed. Synthetic target sequences for Newcastle disease virus and Salmonella were used as model sequences. The sensitivity of the C2CA assay resulted in detection of 1 amol of starting DNA target with a total amp ...[more]

PMID: 34071179

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Project description:BackgroundIntrahepatic hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is the original template for HBV replication. The persistence of cccDNA is responsible for the recurrence of HBV infection. The detection of cccDNA can help the development of new antiviral drugs against HBV replication links, and reduce the resistance and recurrence as well as to discover extrahepatic HBV infection. In situ polymerase chain reaction (IS-PCR) can be used to determine the distribution and localization of cccDNA in liver tissues, but it is hampered by its low sensitivity and specificity. We developed a novel method to detect HBV cccDNA using rolling circle amplification (RCA) combined with IS-PCR.MethodsBiopsy liver tissues were obtained from 26 patients with HBV infection, including 10 chronic hepatitis B (CHB), 6 liver cirrhosis (LC) and 10 hepatocellular carcinoma (HCC) patients. Four pairs of primers were designed to mediating RCA for the first round amplification of HBV cccDNA specifically. The liver tissue sections from patients were treated by plasmid-safe ATP-dependent DNase (PSAD) prior to RCA. After RCA, HBV cccDNA was further amplified by a pair of selective primers labeled digoxigenin that target the gap region between the two direct repeat regions (DR1 and DR2) of the virus via IS-PCR.ResultsHBVcccDNA was expressed and located in hepatocyte nucleus in 19 patients (73.07%). Compared with the IS-PCR, the introduction of RCA increase the limit of detection. RCA combined with IS-PCR yielded strong positive signals in HCC liver tissue in spite of low copy number cccDNA (2 copies of target sequence per cell), meanwhile, no positive signal was detected via negative control.ConclusionsRCA combined with IS-PCR is an effective and practicable method which could detect the presence of low copy number of cccDNA sensitively and specifically, and reflect the relationship between cccDNA expression level and liver tissue pathological characteristics.

Dataset Information

Evaluating the Performance of a Magnetic Nanoparticle-Based Detection Method Using Circle-to-Circle Amplification.

Publications

Evaluating the Performance of a Magnetic Nanoparticle-Based Detection Method Using Circle-to-Circle Amplification.

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