Rapid Detection of Shrimp White Spot Syndrome Virus by Real Time, Isothermal Recombinase Polymerase Amplification Assay

Xia, Xiaoming; Yu, Yongxin; Weidmann, Manfred; Pan, Yingjie; Yan, Shuling; Wang, Yongjie

doi:10.1371/journal.pone.0104667

Cited by 89 publications

(52 citation statements)

References 36 publications

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“…This assay demonstrated a similar performance to RT-PCR with a detection limit of 14 pg/l of purified RNA and no cross-reactivity with a closely related potyvirus. Previous studies concerning the development of RPA assays for the detection of several pathogens, such as the zoonotic pathogen Francisella tularensis (Euler et al, 2012), biothreat agents (Euler et al, 2013), dengue virus (Teoh et al, 2015) and White spot syndrome virus (Xia et al, 2014), also reported comparable sensitivities between RPA and PCR-based methods. However, the time and man-power required to perform a RPA assay is considerably reduced.…”

Section: Discussionmentioning

confidence: 85%

Rapid and specific detection of Yam mosaic virus by reverse-transcription recombinase polymerase amplification

Silva

Bömer

Nkere

et al. 2015

Journal of Virological Methods

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28Yam mosaic virus (YMV; genus Potyvirus) is considered to cause the most economically important 29 viral disease of yams (Dioscorea spp.) in West Africa which is the dominant region for yam 30 production globally. 31Yams are a vegetatively propagated crop and the use of virus-free planting material forms an essential 32 component of disease control. Current serological and PCR-based diagnostic methods for YMV are 33 time consuming involving a succession of target detection steps. 34In this study, a novel assay for specific YMV detection is described that is based on isothermal 35 reverse transcription-recombinase polymerase amplification (RT-exoRPA). This test has been shown 36 to be reproducible and able to detect as little as 14 pg/μl of purified RNA obtained from an YMV-37 infected plant, a sensitivity equivalent to that obtained with the reverse transcription-polymerase chain 38 reaction (RT-PCR) in current general use. The RT-exoRPA assay has, however, several advantages 39 over the RT-PCR; positive samples can be detected in less than 30 min, and amplification only 40 requires a single incubation temperature (optimum 37°C). These features make the RT-exoRPA assay 41 a promising candidate for adapting into a field test format to be used by yam breeding programmes or 42 certification laboratories.

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Section: Discussionmentioning

confidence: 85%

Rapid and specific detection of Yam mosaic virus by reverse-transcription recombinase polymerase amplification

Silva

Bömer

Nkere

et al. 2015

Journal of Virological Methods

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“…The time required to amplify the DNA to detectable levels inherently depends on the number of starting DNA copies, but 20 min are usually adequate, although amplification times of as low as 3e4 min have been observed [15]. Long incubation times are unlikely to be beneficial in most applications, as for solution phase RPA the recombinase consumes all the available ATP within 25 min.…”

Section: Incubation Timementioning

confidence: 99%

Recombinase polymerase amplification: Basics, applications and recent advances

Lobato

O’Sullivan

2018

TrAC Trends in Analytical Chemistry

727

522

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a b s t r a c tRecombinase polymerase amplification (RPA) is a highly sensitive and selective isothermal amplification technique, operating at 37e42 C, with minimal sample preparation and capable of amplifying as low as 1e10 DNA target copies in less than 20 min. It has been used to amplify diverse targets, including RNA, miRNA, ssDNA and dsDNA from a wide variety of organisms and samples. An ever increasing number of publications detailing the use of RPA are appearing and amplification has been carried out in solution phase, solid phase as well as in a bridge amplification format. Furthermore, RPA has been successfully integrated with different detection strategies, from end-point lateral flow strips to real-time fluorescent detection amongst others. This review focuses on the different methodologies and advances related to RPA technology, as well as highlighting some of the advantages and drawbacks of the technique.

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“…Protocols for the preparation of plasmid standards used in the qPCR assay were described in our previous work (Xia et al, ). Briefly, the target 16S rRNA gene sequences were amplified in a 50 μl final volume containing 100 ng of DNA, 0.4 μM each of primers (Table ), 2.5 μl of 10× PCR buffer, 0.2 mM dNTP mix and 1.5 U of Taq polymerase (TaKaRa).…”

Section: Methodsmentioning

confidence: 99%

Common specific indigenous bacteria reside in the intestinal tract of Chinese mitten crab ( Eriocheir sinensis )

et al. 2020

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In our previous study, five bacterial phylotypes of Mollicutes group 1, Mollicutes group 2, Bacteroides, Meniscus and Marinifilum were found to be dominant (abundance > 0.5%) in the intestine of Chinese mitten crab (CMC, Eriocheir sinensis) farmed in Lake Tai, China. To shed light on whether these five bacterial lineages are common specific indigenous intestinal bacteria, samples of adult CMCs collected from eight geographically separated farms in China, juvenile crabs, farming water and crab feed are subjected to analysing by using 16S rRNA gene sequencing and phylotype‐specific, real‐time quantitative PCR (qPCR). Four phylotypes of the Mollicutes group 1, the Mollicutes group 2, Bacteroides and Meniscus are detected, with relatively higher number, in all crab samples, including adults and juveniles. The Mollicutes group 1 and 2 are more dominant, with the group 1 more abundant in midgut while the group 2 in hindgut. The Marinifilum phylotype is almost undetectable in crab samples but abundant in the farming water and feed. By contrast, phylotypes of the Mollicutes group 1, the Mollicutes group 2, Bacteroides and Meniscus are neither detectable in the water nor in feed. These four lineages appear to be the common specific indigenous intestinal bacteria in the entire CMC species, with the Mollicutes group 1 and 2 likely serving as the major symbiotic players in CMCs. Their beneficial contributions to CMC host await future deep investigation.

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Rapid Detection of Shrimp White Spot Syndrome Virus by Real Time, Isothermal Recombinase Polymerase Amplification Assay

Cited by 89 publications

References 36 publications

Rapid and specific detection of Yam mosaic virus by reverse-transcription recombinase polymerase amplification

Rapid and specific detection of Yam mosaic virus by reverse-transcription recombinase polymerase amplification

Recombinase polymerase amplification: Basics, applications and recent advances

Common specific indigenous bacteria reside in the intestinal tract of Chinese mitten crab ( Eriocheir sinensis )

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