Development of a novel droplet digital PCR assay for the sensitive detection of carp edema virus

Wang, Na; Zhang, Zhou; Jing, Hongli; Zhang, Min; Wu, Shaoqiang; Lin, Xiaoyang

doi:10.1016/j.aquaculture.2021.737162

Cited by 10 publications

(20 citation statements)

References 33 publications

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“…Each droplet contained one or more target gene copies and was measured as positive droplet; and therefore, the actual number of DNA copies was higher than that measured. The same appearance was also be mentioned previously, and the upper limit of the sample concentration for ddPCR was 10 5 copies/μl (Pinheiro et al, 2012; Wang et al, 2021; Yan et al, 2016). Therefore, the high‐abundance samples need to be diluted before test.…”

Section: Discussionsupporting

confidence: 66%

“…The ddPCR is also a promising tool for aquatic virus detection, especially detection of low‐level virus genomic copies in host and even in water. Wang et al (2021) developed a sensitive ddPCR assay for carp edema virus diagnosis, the detection limit was 2.2 ± 0.26 copies per reaction. Although ddPCR reaction is less susceptible to PCR inhibitors, mismatch between primer and template (Salipante & Jerome, 2020), the annealing temperature could affect ddPCR reaction.…”

Section: Discussionmentioning

confidence: 99%

“…Nevertheless, ddPCR is an easy‐to‐use technology for nucleic acids detection and quantification, because of the high sensitivity and without of the use of calibration curve. Moreover, ‘overloading’ droplets were all tested positive, which would not affect the qualitatively diagnosis of the positive of samples (Wang et al, 2021).…”

Section: Discussionmentioning

confidence: 99%

“…The droplet digital PCR (ddPCR) is a direct absolute quantitative PCR that has been broadly used in pathogen detection, especially in low‐level quantitation of virus (Henrich et al, 2012; Liu et al, 2017; Wang et al, 2021). However, the ddPCR assay for LMBRaV have not been established.…”

Section: Introductionmentioning

confidence: 99%

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Development of a droplet digital PCR method for the sensitive detection and quantification of largemouth bass ranavirus

Jiang

Shen

Zhou

et al. 2022

Journal of Fish Diseases

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Section: Discussionsupporting

confidence: 66%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Development of a droplet digital PCR method for the sensitive detection and quantification of largemouth bass ranavirus

Jiang

Shen

Zhou

et al. 2022

Journal of Fish Diseases

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“…The ddPCR detection procedures for CEV and GSIV referenced the published works by Wang [ 30 ] and Liu [ 31 ], respectively. Their primers are shown in Table 1 .…”

Section: Methodsmentioning

confidence: 99%

Rapid Nucleic Acid Extraction for Aquatic Animal DNA Virus Determination Using Chelex 100 Resin via Conventional PCR and Digital Droplet PCR Detection

Huang

Jiang

et al. 2022

Animals

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Molecular diagnostic testing for viral pathogens is crucial in aquaculture. The efficient and convenient preparation of pathogenic microbial nucleic acids is the basis of molecular diagnosis. Here, we developed a simplified deoxyribonucleic acid (DNA) extraction method from aquatic animal DNA viruses using the Chelex 100 resin. The nucleic acid was extracted from infected tissues and cell culture for the detection of three common aquatic viral pathogens (CEV, CyHV-2, and GSIV). We compared the extraction effects of a current commercial kit extraction method and the Chelex 100 resin extraction method according to nucleic acid concentration, conventional polymerase chain reaction (PCR), and digital droplet PCR (ddPCR). The results indicated that both extraction procedures could obtain high-quality nucleotide samples. Extracting DNA using the Chelex 100 resin led to better detective efficiency for ddPCR molecular diagnostic testing. The whole process took less than 20 min, and only Chelex 100 resin solution was added to the tissues or cells without multiple tubes being transferred several times. The extracted DNA concentration and the detection sensitivity were high. These results indicated that the Chelex 100 resin solution has the advantages of speed, efficiency, and economy compared to the commercial kit. In addition, the higher pH value (10–11) of the Chelex 100 resin solution markedly improved the detection sensitivity compared to a lower pH value (9–10). In conclusion, the comparison of the Chelex 100 Resin and commercial viral DNA extraction kits revealed the good performance of the Chelex 100 resin solution at pH 10–11 in DNA extraction for PCR amplification from aquatic animal viral samples of tissues and cells in molecular diagnostic testing. It is both rapid and cost-effective.

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Infectious Diseases of Warmwater Fish in Fresh Water

Shinn¹,

Dong²,

Vinh³

et al. 2023

Climate Change on Diseases and Disorders of Finfish in Cage Culture

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Development of a novel droplet digital PCR assay for the sensitive detection of carp edema virus

Cited by 10 publications

References 33 publications

Development of a droplet digital PCR method for the sensitive detection and quantification of largemouth bass ranavirus

Development of a droplet digital PCR method for the sensitive detection and quantification of largemouth bass ranavirus

Rapid Nucleic Acid Extraction for Aquatic Animal DNA Virus Determination Using Chelex 100 Resin via Conventional PCR and Digital Droplet PCR Detection

Infectious Diseases of Warmwater Fish in Fresh Water

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