Development of a recombinase-aided amplification assay for rapid and sensitive detection of porcine circovirus 3

Yu, Zhaorong; Jiao, Suli; Liu, Yuhui; Ni, Hongxia; Wang, Yong

doi:10.1016/j.jviromet.2020.113904

Cited by 20 publications

(16 citation statements)

References 22 publications

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“…Moreover, a practical pathogen detection method not only needs to be rapid, specific, sensitive, and simple but also to be economical. Compared to many testing methods, P-S-MCDA has desirable economical features such as lower reagent cost compared to RAA (26) and less equipment requirements than droplet digital PCR (27).…”

Section: Discussionmentioning

confidence: 99%

Single Multiple Cross Displacement Amplification for Rapid and Real-Time Detection of Porcine Circovirus 3

et al. 2021

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Since 2016, a novel porcine circovirus, PCV3, has been infecting pigs, causing significant economic losses to the pig industry. In recent years, the infection rate of PCV3 has been increasing, and thus rapid and accurate detection methods for PCV3 are essential. In this study, we established a novel probe-based single multiple cross displacement amplification (P-S-MCDA) method for PCV3. The method was termed as P-S-MCDA. The P-S-MCDA uses seven primers to amplify the capsid gene, and the assay can be performed at 60°C for 30 min, greatly shortening the reaction time. The results of P-S-MCDA can not only be monitored in real time through fluorescence signals but also be determined by observing the fluorescence of the reaction tubes using a smartphone-based cassette. This method demonstrated good specificity and the same sensitivity as qPCR, with a minimum detection limit of 10 copies. In 139 clinical samples, the coincidence rate with qPCR was 100%. The P-S-MCDA can be widely applied in PCV3 detection in laboratories or in rural areas.

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Section: Discussionmentioning

confidence: 99%

Single Multiple Cross Displacement Amplification for Rapid and Real-Time Detection of Porcine Circovirus 3

et al. 2021

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“…Each 20-µL reaction mixture contained 0.4 pmol of forward and reverse primers, 0.8 pmol of probe, 10 µL of 2 × One Step RT-PCR buffer, 2 µL of template, 0.4 µL of TaKaRa Ex Taq HS, 0.4 µL of PrimeScript RT Enzyme Mix II, and RNase Free dH 2 O added to make the volume 20 µL. The primer pairs were COG2R and QNIF2d (Loisy and Atmar et al, 2005). The size of the ampli cation product was 89 bp.…”

Section: Rt-qpcr Assaymentioning

confidence: 99%

“…The reaction is typically completed in approximately 30 min at 37-42 °C. RAA has been successfully applied in the detection of pathogens (Bai and Ma et al, 2020;Li and Yu et al, 2020;Wang and Cui et al, 2020; and single nucleotide polymorphisms (SNPs) (Duan and Li et al, 2018). Owing to its speed, low-cost, and high sensitivity, RAA is highly suitable for clinical applications, and is a potential assay for the point-of-care testing (POCT) for foodborne pathogens.…”

Section: Introductionmentioning

confidence: 99%

Development of a Recombinase-Aided Amplification Assay for Rapid Detection of Human Norovirus GII.4

Qin

Xue

Cai

et al. 2020

Preprint

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Background: Human noroviruses are one of the main causes of foodborne illnesses and represent a serious public health concern. Rapid and sensitive assays for human norovirus detection are undoubtedly necessary for clinical diagnosis, especially in regions without more sophisticated equipment.Method: The recombinase-aided amplification (RT-RAA) is a fast, robust and isothermal nucleic acid detection method based on enzyme reaction. This method can complete the sample detection at 39°C in 30 minutes. In this study, we successfully established a rapid reverse transcription recombinase-aided amplification (RT-RAA) assay for the detection of human norovirus GII.4 and applied this assay to clinical samples, as well as comparison with commercial RT-qPCR.Results: An analytical sensitivity detection of the RT-RAA at a 95% probability of 3.425 log10 genomic copies (LGC)/reaction. Moreover, no cross-reaction was observed with other norovirus genogroups and other common foodborne viruses. Stool samples were examined by RT-RAA and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Compared with RT-qPCR, kappa values for human norovirus detection with RT-RAA were 0.894 (p < 0.001), indicating that both assays were in agreement.Conclusion: This RT-RAA assay provides a rapid, specific, and sensitive assay for human norovirus detection and is suitable for clinical testing.

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“…As a novel isothermal nucleic acid amplification technology, recombinase‐aided amplification (RAA) does not require a sophisticated thermal cycler and instead can be performed at a constant temperature by a simple water bath or heating block. The reaction process of RAA primarily relies on three essential enzymes: i) a recombinase, which pairs the specific primers to the template DNA; ii) a single‐stranded DNA‐binding protein (SSB), which is applied to form single‐stranded DNA without the need for heating; and iii) a strand‐displacing DNA polymerase, which is responsible for the amplification and extension (Chen et al., 2018; Li et al., 2020; Xue et al., 2020). Once the RAA reaction was initiated, an exponential DNA amplification of the template progresses rapidly and generates a detectable level in less than 20 min.…”

Section: Introductionmentioning

confidence: 99%

“…ii) a single-stranded DNA-binding protein (SSB), which is applied to form single-stranded DNA without the need for heating; and iii) a strand-displacing DNA polymerase, which is responsible for the amplification and extension (Chen et al, 2018;Li et al, 2020;Xue et al, 2020). Once the RAA reaction was initiated, an exponential DNA amplification of the template progresses rapidly and generates a detectable level in less than 20 min.…”

mentioning

confidence: 99%

Development of a fluorescent probe‐based real‐time reverse transcription recombinase‐aided amplification assay for the rapid detection of classical swine fever virus

Yang

et al. 2020

Transbound Emerg Dis

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Classical swine fever (CSF), formerly known as "hog cholera", is a highly contagious infectious disease that affects both domestic pigs and wild boars, with high morbidity and mortality. Due to its tremendous impact on the swine industry, CSF is therefore notifiable to the World Organization for Animal Health (OIE) (Zhou, 2019). Classical swine fever virus (CSFV), the causative agent of CSF, is classified in the genus Pestivirus within the family Flaviviridae. Currently, the genus Pestivirus includes eleven recognized species, such as CSFV, bovine viral diarrhoea viruses 1 and 2 (BVDV-1 and BVDV-2), and border disease virus. CSFV has a linear, non-segmented, single-stranded, positive-sense RNA genome of approximately 12.3 kb (Edwards et al., 2000). The genome consists

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Development of a recombinase-aided amplification assay for rapid and sensitive detection of porcine circovirus 3

Cited by 20 publications

References 22 publications

Single Multiple Cross Displacement Amplification for Rapid and Real-Time Detection of Porcine Circovirus 3

Single Multiple Cross Displacement Amplification for Rapid and Real-Time Detection of Porcine Circovirus 3

Development of a Recombinase-Aided Amplification Assay for Rapid Detection of Human Norovirus GII.4

Development of a fluorescent probe‐based real‐time reverse transcription recombinase‐aided amplification assay for the rapid detection of classical swine fever virus

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