Advanced loop-mediated isothermal amplification method for sensitive and specific detection of Tomato chlorosis virus using a uracil DNA glycosylase to control carry-over contamination

Kil, Eui-Joon; Kim, Sunhoo; Lee, Yeji; Kang, Eun-Ha; Lee, Minji; Cho, Sang-Ho; Kim, Mi-Kyeong; Lee, Kyeong-Yeoll; Heo, Noh-Youl; Choi, Hongsoo; Kwon, Suk‐Tae; Lee, Sukchan

doi:10.1016/j.jviromet.2014.10.020

Cited by 59 publications

(37 citation statements)

References 35 publications

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“…Therefore, they are not ideal approaches for controlling carryover contamination in LAMP reaction33. Compared with the aforementioned methods, the UDG-mediated digestion of dUTP-incorporated contaminant DNA is simple, inexpensive, and does not require substantial modification of existing protocols42.…”

Section: Discussionmentioning

confidence: 99%

Advanced uracil DNA glycosylase-supplemented real-time reverse transcription loop-mediated isothermal amplification (UDG-rRT-LAMP) method for universal and specific detection of Tembusu virus

Tang

Chen

Diao

2016

Sci Rep

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Tembusu virus (TMUV) is a mosquito-borne flavivirus which threatens both poultry production and public health. In this study we developed a complete open reading frame alignment-based rRT-LAMP method for the universal detection of TUMV. To prevent false-positive results, the reaction was supplemented with uracil DNA glycosylase (UDG) to eliminate carryover contamination. The detection limit of the newly developed UDG-rRT-LAMP for TMUV was as low as 100 copies/reaction of viral RNA and 1 × 100.89 − 1 × 101.55 tissue culture infectious dose/100 μL of viruses. There were no cross-reactions with other viruses, and the reproducibility of the assay was confirmed by intra- and inter-assay tests with variability ranging from 0.22–3.33%. The new UDG-rRT-LAMP method for TMUV produced the same results as viral isolation combined with RT-PCR as the “gold standard” in 96.88% of cases for 81 clinical samples from subjects with suspected TMUV infection. The addition of UDG can eliminate as much as 1 × 10−16 g/reaction of contaminants, which can significantly reduce the likelihood of false-positive results during the rRT-LAMP reaction. Our result indicated that our UDG-rRT-LAMP is a rapid, sensitive, specific, and reliable method that can effectively prevent carryover contamination in the detection of TMUV.

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Section: Discussionmentioning

confidence: 99%

Advanced uracil DNA glycosylase-supplemented real-time reverse transcription loop-mediated isothermal amplification (UDG-rRT-LAMP) method for universal and specific detection of Tembusu virus

Tang

Chen

Diao

2016

Sci Rep

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“…This decreases the Mg 2+ ion concentration, which is indicated by a colour change from violet to blue in the presence of HNB (Goto et al 2009). LAMP detection assays have been developed for several pathosystems, including oomycetes, fungi, bacteria and viruses (Harper et al 2010;Dai et al 2012;B€ uhlmann et al 2013;Chen et al 2013;Moradi et al 2013;Duan et al 2014;Kil et al 2015;Thiessen et al 2015). A LAMP assay for the detection of P. infestans, an organism which significantly impacts potato and tomato production globally, has not yet been developed.…”

Section: Introductionmentioning

confidence: 99%

Loop‐mediated isothermal amplification for detection of the tomato and potato late blight pathogen, P hytophthora infestans

et al. 2016

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“…Therefore, precautions should be taken such as careful opening and closing of reaction tubes, frequent changing the gloves, and separating the progress of pre- and post-RPA amplification. Furthermore, replacement of dTTP with dUTP may help prevent such carryover contamination as demonstrated in PCR and LAMP assays [35, 36]. Our results showed that the developed RPA assay could tolerate base mismatch in some degree as the primes could not completely match with the two PCV2 strains used in our work based on sequence alignment, and it may detect PCV-2 strains in some degree of variability.…”

Section: Discussionmentioning

confidence: 81%

Development of Isothermal Recombinase Polymerase Amplification Assay for Rapid Detection of Porcine Circovirus Type 2

Yang

Qin

Sun

et al. 2017

BioMed Research International

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Porcine circovirus virus type II (PCV2) is the etiology of postweaning multisystemic wasting syndrome (PMWS), porcine dermatitis, nephropathy syndrome (PDNS), and necrotizing pneumonia. Rapid diagnosis tool for detection of PCV2 plays an important role in the disease control and eradication program. Recombinase polymerase amplification (RPA) assays using a real-time fluorescent detection (PCV2 real-time RPA assay) and RPA combined with lateral flow dipstick (PCV2 RPA LFD assay) were developed targeting the PCV2 ORF2 gene. The results showed that the sensitivity of the PCV2 real-time RPA assay was 102 copies per reaction within 20 min at 37°C and the PCV2 RPA LFD assay had a detection limit of 102 copies per reaction in less than 20 min at 37°C. Both assays were highly specific for PCV2, with no cross-reactions with porcine circovirus virus type 1, foot-and-mouth disease virus, pseudorabies virus, porcine parvovirus, porcine reproductive and respiratory syndrome virus, and classical swine fever virus. Therefore, the RPA assays provide a novel alternative for simple, sensitive, and specific identification of PCV2.

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Advanced loop-mediated isothermal amplification method for sensitive and specific detection of Tomato chlorosis virus using a uracil DNA glycosylase to control carry-over contamination

Cited by 59 publications

References 35 publications

Advanced uracil DNA glycosylase-supplemented real-time reverse transcription loop-mediated isothermal amplification (UDG-rRT-LAMP) method for universal and specific detection of Tembusu virus

Advanced uracil DNA glycosylase-supplemented real-time reverse transcription loop-mediated isothermal amplification (UDG-rRT-LAMP) method for universal and specific detection of Tembusu virus

Loop‐mediated isothermal amplification for detection of the tomato and potato late blight pathogen, P hytophthora infestans

Development of Isothermal Recombinase Polymerase Amplification Assay for Rapid Detection of Porcine Circovirus Type 2

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