Loop-mediated isothermal amplification assay for the rapid and visual detection of novel porcine circovirus 3

Park, Yu-Ri; Kim, Hye‐Ryung; Kim, Seong‐Hee; Lee, Kyoung-Ki; Lyoo, Young S.; Yeo, Sang-Geon; Park, Choi‐Kyu

doi:10.1016/j.jviromet.2017.12.006

Cited by 26 publications

(16 citation statements)

References 23 publications

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“…In order to improve the specificity and sensitivity of the LAMP reaction, 1 pair of ring primers can be designed between the primers and the internal and external primers. LAMP can achieve rapid amplification of nucleic acids in a simple constant-temperature device, and the amplification products can be detected using a turbidimeter, electrophoresis, and by adding fluorescent dye into the tube reaction (Park et al 2017 ). LAMP detection time is about 60 min, compared with the conventional PCR method; thus it not only reduces reaction time but also increases sensitivity and specificity, with good prospects and development potential.…”

Section: Introductionmentioning

confidence: 99%

Loop-mediated isothermal amplification-lateral-flow dipstick (LAMP-LFD) to detect Mycoplasma ovipneumoniae

Zhang

Cao

Zhu

et al. 2019

World J Microbiol Biotechnol

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In order to establish a rapid detection method for Mycoplasma ovipneumoniae , this study used the loop-mediated isothermal amplification (LAMP) technique to carry out nucleic acid amplification and chromatographic visualization via a lateral flow dipstick (LFD) assay. The M. ovipneumoniae elongation factor TU gene ( EF-TU ) was detected using a set of specific primers designed for the EF-TU gene, and the EF-TU FIP was detected by biotin labeling, which was used in the LAMP amplification reaction. The digoxin-labeled probe specifically hybridized with LAMP products, which were visually detected by LFD. Here, we established the M. ovipneumoniae LAMP-LFD rapid detection method and tested the specificity, sensitivity, and clinical application of this method. Results showed that the optimized LAMP performed at 60 °C for 60 min, and LFD can specifically and visually detect M. ovipneumoniae with a minimum detectable concentration at 1.0 × 10 2 CFU/mL. The sensitivity of LAMP-LFD was 1000 times that of the conventional PCR detection methods, and the clinical lung tissue detection rate was 86% of 50 suspected sheep infected with M. ovipneumoniae . In conclusion, LAMP-LFD was established in this study to detect M. ovipneumoniae , a method that was highly specific, sensitive, and easy to operate, and provides a new method for the prevention and diagnosis of M. ovipneumoniae infection. Electronic supplementary material The online version of this article (10.1007/s11274-019-2601-5) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

Loop-mediated isothermal amplification-lateral-flow dipstick (LAMP-LFD) to detect Mycoplasma ovipneumoniae

Zhang

Cao

Zhu

et al. 2019

World J Microbiol Biotechnol

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“…PCR (Goodwin, Merry, & Sadler, ), real‐time PCR (Goodwin et al., ), LAMP (Park et al., ), and immunohistochemical and blood smear methods (Kong et al., ) have been reported for diagnosis of CyHV‐2, but a more convenient method is still required. Herein, we developed a rapid and efficient assay for the detection of CyHV‐2 using recombinase polymerase amplification (RPA), a type of isothermal amplification, to allow DNA amplification and detection using simple equipment (Jaroenram & Owens, ).…”

Section: Introductionmentioning

confidence: 99%

“…PCR (Goodwin, Merry, & Sadler, 2006), real-time PCR (Goodwin et al, 2009), LAMP (Park et al, 2018), and immunohistochemical and blood smear methods (Kong et al, 2017) have been reported for diagnosis of CyHV-2, but a more convenient method is still required.…”

Section: Introductionmentioning

confidence: 99%

Rapid visual detection of cyprinid herpesvirus 2 by recombinase polymerase amplification combined with a lateral flow dipstick

Wang

Sun

et al. 2018

Journal of Fish Diseases

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Herpesviral haematopoietic necrosis (HVHN), caused by cyprinid herpesvirus 2 (CyHV-2), causes significant losses in crucian carp (Carassius carassius) aquaculture. Rapid and convenient DNA assay detection of CyHV-2 is useful for field diagnosis. Recombinase polymerase amplification (RPA) is a novel isothermal DNA amplification and detection technology that can amplify DNA within 30 min at ~37°C by simulating in vivo DNA recombination. Herein, a rapid and convenient detection assay based on RPA with a lateral flow dipstick (LFD) was developed for detecting CyHV-2. The highly conserved ORF72 of CyHV-2 was targeted by specific and sensitive primers and probes. The optimized assay takes only 15 min at 38°C using a water bath, with analysis of products by 2% agarose gel electrophoresis within 30 min. A simple lateral flow strip based on the unique probe in reaction buffer was developed for visualization. The entire RPA-LFD assay takes 50 min less than the routine PCR method, is 100 times more sensitive and displays no cross-reaction with other aquatic viruses. The combined isothermal RPA and lateral flow assay (RPA-LFD) provides a simple, rapid, reliable method that could improve field diagnosis of CyHV-2 when resources are limited.

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“…This makes it very suitable for point-of-care tests using the colorimetric IMSA assay. Compared with the LAMP assay using hydroxynaphthol blue for PCV3 detection, the color change from red to yellow of the reaction tube in this study is more easily to be distinguished with the naked eye than that of the color change from purple to sky blue (17). Hence, it is very convenient for the observers to read out and judge the results of the colorimetric IMSA.…”

Section: Discussionmentioning

confidence: 95%

The Colorimetric Isothermal Multiple-Self-Matching-Initiated Amplification Using Cresol Red for Rapid and Sensitive Detection of Porcine Circovirus 3

et al. 2020

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In 2016, a novel porcine circovirus (PCV), PCV3, was identified in USA. Subsequently, it was proved to be also epidemic in China, Poland, and Korea. To analyze and control the epidemic situation of PCV3, it is necessary to establish accurate and high-throughput detection methods. In this study, the colorimetric isothermal multiple-self-matching-initiated amplification (IMSA) using cresol red was developed to detect PCV3 for the first time. The reaction can be easily performed by incubating the tube at 63 • C for 60 min. By the addition of pH-sensitive indicator dye cresol red, the initial color of the reaction mixture is red. When PCV3 capsid gene DNA was positive in the sample, the color of the reaction mixture changed from red to yellow after the isothermal incubation at 63 • C, while the negative control maintained the red color. The colorimetric IMSA displayed good specificity in detecting PCV3, PCV2, and PCV1 and 4 porcine DNA pathogens. Moreover, it has a low and repeatable detection limit of 10 copies, which is consistent with TaqMan-based qPCR, but 10 times more sensitive than PCR. In diagnosing 128 clinical specimens, it not only showed 100% agreement with qPCR but also detected 15 positive results more than PCR. The colorimetric IMSA we offered might be a good choice for PCV3 epidemiological investigation and point-of-care testing.

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Loop-mediated isothermal amplification assay for the rapid and visual detection of novel porcine circovirus 3

Cited by 26 publications

References 23 publications

Loop-mediated isothermal amplification-lateral-flow dipstick (LAMP-LFD) to detect Mycoplasma ovipneumoniae

Loop-mediated isothermal amplification-lateral-flow dipstick (LAMP-LFD) to detect Mycoplasma ovipneumoniae

Rapid visual detection of cyprinid herpesvirus 2 by recombinase polymerase amplification combined with a lateral flow dipstick

The Colorimetric Isothermal Multiple-Self-Matching-Initiated Amplification Using Cresol Red for Rapid and Sensitive Detection of Porcine Circovirus 3

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