Rapid and visual detection of milk vetch dwarf virus using recombinase polymerase amplification combined with lateral flow strips

Cao, Yuhao; Yan, Dankan; Wu, Xinyang; Chen, Ziqiang; Lai, Yuchao; Lv, Lanqing; Yan, Fei; Chen, Jianping; Zheng, Hongying; Song, Xuemei

doi:10.1186/s12985-020-01371-5

Cited by 19 publications

(24 citation statements)

References 27 publications

(32 reference statements)

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“…First, all these tests were performed with the following commercial RPA kits: TwistDx nfo [27,84,[208][209][210][211], TwistDx Basic [212][213][214][215], TwistDx Exo [51], and Amplifier (Agdia) [27,92,[216][217][218]. The main criterion for the selection of a gene as a target for RPA was maximal specificity for the species of interest.…”

Section: Rpa-based Testsmentioning

confidence: 99%

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The Potential Use of Isothermal Amplification Assays for In-Field Diagnostics of Plant Pathogens

Иванов

Safenkova

Zherdev

et al. 2021

Plants

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Rapid, sensitive, and timely diagnostics are essential for protecting plants from pathogens. Commonly, PCR techniques are used in laboratories for highly sensitive detection of DNA/RNA from viral, viroid, bacterial, and fungal pathogens of plants. However, using PCR-based methods for in-field diagnostics is a challenge and sometimes nearly impossible. With the advent of isothermal amplification methods, which provide amplification of nucleic acids at a certain temperature and do not require thermocyclic equipment, going beyond the laboratory has become a reality for molecular diagnostics. The amplification stage ceases to be limited by time and instruments. Challenges to solve involve finding suitable approaches for rapid and user-friendly plant preparation and detection of amplicons after amplification. Here, we summarize approaches for in-field diagnostics of phytopathogens based on different types of isothermal amplification and discuss their advantages and disadvantages. In this review, we consider a combination of isothermal amplification methods with extraction and detection methods compatible with in-field phytodiagnostics. Molecular diagnostics in out-of-lab conditions are of particular importance for protecting against viral, bacterial, and fungal phytopathogens in order to quickly prevent and control the spread of disease. We believe that the development of rapid, sensitive, and equipment-free nucleic acid detection methods is the future of phytodiagnostics, and its benefits are already visible.

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Section: Rpa-based Testsmentioning

confidence: 99%

“…The kit is similar to the Nfo kit, using the ExoIII nuclease instead of the Nfo one. Other papers have proposed different approaches [212,215]. The authors used TwistDx basic kit without cleavage and no complex DNA probe.…”

Section: Rpa-based Testsmentioning

confidence: 99%

The Potential Use of Isothermal Amplification Assays for In-Field Diagnostics of Plant Pathogens

Иванов

Safenkova

Zherdev

et al. 2021

Plants

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“…The reactions can be completed in 5-20 min depending on the starting copy number of the targeted template and the size of the amplicon. The RPA assay has been reported in diagnosis of several plant viruses, including those with DNA genomes such as wheat dwarf virus (Glais and Jacquot 2015), banana bunchy top virus (Kapoor et al 2017), citrus yellow mosaic virus (Kumar et al 2018), bean golden yellow mosaic virus, tomato mottle virus, tomato yellow leaf curl virus (Londoño et al 2016;Wang and Yang 2019), milk vetch dwarf virus (Cao et al 2020), and piper yellow mottle virus (Mohandas and Bhat 2020); and with RNAbased genomes such as little cherry virus-2 (Mekuria et al 2014), plum pox virus (Zhang et al 2014;Babujee et al 2019), rose rosette virus (Babu et al 2017), rice blackstreaked dwarf virus (Zhao et al 2019), cucumber mosaic virus (Srivastava et al 2019), maize chlorotic mottle virus (Jiao et al 2019a), apple stem pitting virus (Kim et al 2019), cucumber green mottle mosaic virus (Jiao et al 2019b;Zeng et al 2019), cucurbit yellow stunting disorder virus (Kalischuk et al 2020), and peach latent mosaic viroid (Lee et al 2020). RPA has also been successfully applied for detection of the potyviruses plum pox virus (Zhang et al 2014), yam mosaic virus and yam mild mosaic virus (Silva et al 2015), potato virus Y (Glais and Jacquot 2015;Babujee et al 2019), and chilli veinal mottle virus (Jiao et al 2020).…”

Section: Introductionmentioning

confidence: 99%

Development of a recombinase polymerase amplification combined with lateral flow dipstick assay for rapid and sensitive detection of bean common mosaic virus

et al. 2021

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Bean common mosaic virus (BCMV) is one of the most widespread and damaging viruses of cultivated legumes in the world. In addition to serious yield reduction and germplasm decline, BCMV infection also makes legumes more vulnerable to other pathogens. Early diagnosis of the virus is particularly important in limiting its spread. Recombinase polymerase amplification (RPA) is a novel isothermic amplification technology. The whole reaction can be done outside the laboratory environment after the nucleic acid sample is obtained. In this study, we established a rapid and sensitive RPA combined with the lateral flow dipstick (LFD) assay for detection of BCMV, based on the conserved BCMV coat protein (CP) gene sequence. Specific primers and a probe were designed, which amplify ~ 150 bp CP fragments from BCMV-infected samples under a constant temperature of 37 °C for 20 min. The end-labeled amplification products were detected by high-affinity LFD within 5 min. Sensitivity of this RPA-LFD assay was 1000 times greater than that of the conventional polymerase chain reaction (PCR) assay. Furthermore, when the primers/probe were used against related potyviruses including soybean mosaic virus (SMV), bean yellow mosaic virus (BYMV), and turnip mosaic virus (TuMV), the three potyviruses were not detected, indicating that the assay was BCMV species-specific. The RPA-LFD assay was also successfully applied for the detection of seed-borne BCMV in beans. The RPA-LFD assay has great potential application in the rapid diagnosis of BCMV in the field.

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“…In recent years, RPA has been developed rapidly in the detection of plant viruses [21]. Since the emergence of application of RPA to detect little cherry virus 2 (LChV2) [22], more and more plant viruses have been detected by RPA, especially in 2020, such as chilli veinal mottle virus (ChiVMV), milk vetch dwarf virus (MDV), piper yellow mottle virus (PYMoV), cardamom vein clearing virus (CdVCV), barley yellow dwarf virus (BYDV), cucurbit yellow stunting disorder virus (CYSDV) [23][24][25][26][27][28]. In this study, pepper leaves with suspected symptoms of TSWV were collected from Tieling city of Liaoning Province in 2020.…”

Section: Introductionmentioning

confidence: 99%

Characterization and Establishment of a Recombinase Polymerase Amplification Assay for Rapid Detection of Tomato Spotted Wilt Virus in Pepper

Hao

Yang

et al. 2021

Preprint

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Tomato spotted wilt virus (TSWV) is one of the most economically destructive and scientifically challenging plant viruses, which has seriously affected the production of commercial crops. At present, there is no effective strategy to control this virus. Therefore, there is an urgent need for a rapid and simple method to detect TSWV, which is of great significance to prevent its spread. In this study, an isolate of TSWV (TSWV-LNTL) infecting pepper from Liaoning Province of northeast China was obtained. A phylogenetic tree based on neighbor-joining using coat protein (CP) gene was established. A rapid method for detecting TSWV by recombinase polymerase amplification (RPA) was established. The phylogenetic tree based on the nucleotide sequences of coat protein (CP) genes of different TSWV isolates showed that the genetic relationship of TSWV-LNTL was most closely related to that of TSWV-LX-Lettuce-12 (Yunnan) and TSWV-TSHL (Shandong) isolates in China. It can be finished at 39 °C for 20 min and then purified by heating at 65 °C for 10 min. The RPA primers were highly specific and no cross-reactivity was detected with other selected viruses infecting pepper. The results of sensitivity test revealed that the detection limit of RPA is 1.0 × 103 copies/μL, which was tenfold lower than that of PCR method. In addition, the RPA method was successfully applied to detect TSWV in field samples. These results reported the occurrence of TSWV on crop in Liaoning Province of northeast China and demonstrated that the established RPA assay provided an effective molecular diagnostic tool for the accurate and rapid detection of TSWV to prevent its spread.

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Rapid and visual detection of milk vetch dwarf virus using recombinase polymerase amplification combined with lateral flow strips

Cited by 19 publications

References 27 publications

The Potential Use of Isothermal Amplification Assays for In-Field Diagnostics of Plant Pathogens

The Potential Use of Isothermal Amplification Assays for In-Field Diagnostics of Plant Pathogens

Development of a recombinase polymerase amplification combined with lateral flow dipstick assay for rapid and sensitive detection of bean common mosaic virus

Characterization and Establishment of a Recombinase Polymerase Amplification Assay for Rapid Detection of Tomato Spotted Wilt Virus in Pepper

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