Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Aug:246:27-33.
doi: 10.1016/j.jviromet.2017.04.006. Epub 2017 Apr 21.

Evaluation of a rapid detection for Coxsackievirus B3 using one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP)

A Monazah  1 , M Zeinoddini  2 , A R Saeeidinia  1
Affiliations

Evaluation of a rapid detection for Coxsackievirus B3 using one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP)

A Monazah et al. J Virol Methods. 2017 Aug.

Abstract

Coxsackievirus B3 (CVB3) is a member of the genus Enterovirus within the family Picornaviridae and is an important pathogen of viral myocarditis, which accounts for more than 50% viral myocarditis cases. VP1 is major capsid protein that this region has a low homology in both amino acid and nucleotide sequences among Enteroviruses. Therefore we have chosen this region for designed a set of RT-LAMP primers for CVB3 detection. For this the total RNA was extracted from 24-h post infected-HeLa cells with complete cytopathic effect (CPE), and applied to a one-step reverse transcription loop-mediated isothermal amplification reaction (RT-LAMP) using CVB3-specific primers. The optimization of RT-LAMP reaction was carried out with three variables factors including MgSO4 concentration, temperature and time of incubation. Amplification was analyzed by using 2% agarose gel electrophoresis and ethidium bromide and SYBR Green staining. Our results were shown the ladder-like pattern of the VP1 gene amplification. The LAMP reaction mix was optimized and the best result observed at 4mM MgSO4 and 60°C for 90min incubation. RT-LAMP had high sensitivity and specificity for detection of CVB3 infection. This method can be used as a rapid and easy diagnostic test for detection of CVB3 in clinical laboratories.

Keywords: Coxsackievirus B3; Detection; Optimization; RT-LAMP.

PubMed Disclaimer

Figures

None
Graphical abstract
Fig. 1
Fig. 1
Locations of primer-binding sequences (CVB3 strain Nancy, GenBank accession no. JX312064).
Fig. 2
Fig. 2
Analysis of RT-PCR product using 1% agarose gel electrophoresis. Lane 1: RT-PCR product using primers B3/F3, lane 2: RT-PCR product using primers BIP/FIP, lane M: 100 bp DNA leader.
Fig. 3
Fig. 3
Effect of MgSO4 concentrations on the RT-LAMP reaction: Lane 1: 2 mM, lane 2: 3 mM, lane 3: 4 mM, lane 4: 5 mM, lane 5: 6 mM, lane 6: 7 mM, lane 7: 8 mM, lane 8: 9 mM, lane 9: 10 mM, lane 10: 11 mM and lane 11: 12 mM MgSO4, lane 12: negative control and lane M: 100 bp DNA leader.
Fig. 4
Fig. 4
A) Effect of temperature on the RT-LAMP reaction: Lane 1: 56 °C, lane 2: 58 °C, lane 3: 60 °C, lane 4: 62 °C, lane 5: 64 °C and lane M: 100 bp DNA marker. B) Effect of incubation time on the RT-LAMP amplification: Lane 1: 30 min, lane 2: 60 min, lane 3: 90 min, lane 4: 120 min and lane M: 100 bp DNA leader.
Fig. 5
Fig. 5
The specificity of RT-LAMP (A) and RT-PCR (B) was determined using some serotypes of genus Enteroviruses. Lane 1: Coxsackievirus A16, lane 2: Echovirus, lane 3: Rhinovirus, Lane 4: Coxsackievirus B3 (positive control), lane 5: without template (negative control) and lane M: 100 bp DNA leader.
Fig. 6
Fig. 6
Sensitivity of RT-LAMP (A) and RT-PCR (B) assays for CVB3 detection. 7 different concentrations of total RNA were prepared and used as template. Lane 1: 10 μg, lane 2: 100 ng, lane 3: 1 ng, lane 4: 10 pg, lane 5: 100 fg, lane 6: 1 fg and lane 7: 10 ag of total RNA, and lane M: 100 bp DNA leader.
Fig. 7
Fig. 7
Sensitivity of RT-LAMP according to TCID50. 11 different TCID50/ml from CVB3 were used for RNA extraction and RT-LAMP assay. Lane 1: 106, lane 2: 105, lane 3:10 4, lane 4: 103, lane 5: 102, lane 6: 101, lane 7: 1, lane 8: 10−1, lane 9: 10−2, lane 10: 10−3 and lane 11: 10−4 TCID50/ml, lane M: 100 bp DNA leader.
Fig. 8
Fig. 8
RT-PCR (A) and RT-LAMP (B) detections for heart of infected mice (lane 1), un-infected mice (lane 2), spleen of infected mice (lane 3), un-infected mice (lane 4), feces of infected mice (lane 5), un-infected mice (lane 6), and blood of infected mice (lane 7), un-infected mice (lane 8).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Arita M., Ling H., Yan D., Nishimura Y., Yoshida H., Wakita T., Shimizu H. Development of a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) system for a highly sensitive detection of enterovirus in the stool samples of acute flaccid paralysis cases. BMC Infect. Dis. 2009;9(1) (p.1) - PMC - PubMed
    1. Bedard K.M., Semler B.L. Regulation of Picornavirus gene expression. Microbes Infect. 2004;6(7):702–713. - PubMed
    1. Caipang C.M., Haraguchi I., Ohira T., Hirono I., Aoki T. Rapid detection of a fish iridovirus using loop-mediated isothermal amplification (LAMP) J. Virol. Methods. 2004;121:155–161. - PubMed
    1. Chang G.H., Lin L., Luo Y.J., Cai L.J., Wu X.Y., Xu H.M., Zhu Q.Y. Sequence analysis of six enterovirus 71 strains with different virulences in humans. Virus Res. 2010;151(1):66–73. - PubMed
    1. Chen Q., Hu Z., Zhang Q., Yu M. Development and evaluation of a real-time method of simultaneous amplification and testing of enterovirus 71 incorporating a RNA internal control system. J. Virol. Methods. 2014;196:139–144. - PubMed

Publication types

MeSH terms