One-step real-time loop-mediated isothermal amplification (RT-LAMP): evaluation and its application for the detection of foot-and-mouth-disease virus and its serotypes
Availability of a rapid and sensitive diagnostic technique is key in successful prevention and control of infectious diseases like foot-and-mouth disease (FMD). Existing conventional diagnostic tests for FMD are laborious and time-consuming with low sensitivity and specificity. Molecular-based techniques are costly and difficult, involving refined apparatus like a thermal cycler. In the present study, the technique of real-time loop-mediated isothermal amplification (RT-LAMP) was standardized for diagnosing FMDV and its serotypes, evaluated using field samples, and compared with the existing real-time PCR in Pakistan. RT-LAMP amplified the target 3D gene using specific primers at 65 °C for 60 min and the VP1 gene using serotype specific primers at 63 °C for 60 min. A total of 38 samples out of 50 were positive by RT-LAMP and identified serotypes were A (n = 15), O (n = 15), and Asia-1 (n = 8). The efficiency of RT-LAMP in this study was highest for serotype Asia-1 (80.9%) followed by serotype A (73.4%) and O (62.35).
___
- Brooksby J. Portraits of viruses: foot-and-mouth disease virus.
Intervirology 1982; 18:
1-23.
- Grubman MJ, Baxt B. Foot-and-mouth disease. Clin Microbiol
Rev 2004; 17:
465-493.
- Alexandersen S, Zhang Z, Donaldson A, Garland A. The
pathogenesis and diagnosis of foot-and-mouth disease. J
Comp Pathol 2003; 129:
1-36.
- Verma A, Uzun O, Hu Y, Hu Y, Han HS, Watson N, Chen
S, Irvine DJ, Stellacci F. Surface-structure-regulated cell
membrane penetration by monolayer-protected nanoparticles.
Nat Mater 2008; 7: 588-595.
- Cao Y, Lu Z, Li D, Fan P, Sun P, Bao H, Fu Y, Li P, Bai X, Chen
Y. Evaluation of cross-protection against three topotypes of
serotype O foot-and-mouth disease virus in pigs vaccinated
with multi-epitope protein vaccine incorporated with poly
(I:C). Vet Microbiol 2014; 168: 294-301.
- Yamazaki W, Mioulet V, Murray L, Madi M, Haga T, Misawa
N, Horii Y, King DP. Development and evaluation of multiplex
RT-LAMP assay for rapid and sensitive detection of foot-and-
mouth disease virus. J Virol Methods 2013; 192: 18-24.
- Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe
K, Amino N, Hase T.
Loop-mediated isothermal amplification
of DNA. Nucleic Acids Res 2000; 28:
e63-e63.
- Farooq U, Latif A, Irshad H, Ullah A, Zahur A, Naeem K,
Khan SH, Ahmed Z, Rodriguez L, Smoliga G.
Loop-mediated
isothermal amplification (RT-LAMP): a new approach for the
detection of foot-and-mouth disease virus and its serotypes in
Pakistan. Iran J Vet Res 2015; 16:
331.
- Madhanmohan M, Nagendrakumar S, Manikumar K, Yuvaraj
S, Parida S, Srinivasan V.
Development and evaluation of a
real-time reverse transcription-loop-mediated isothermal
amplification assay for rapid serotyping of foot-and-mouth
disease virus. J Virol Methods 2013; 187: 195-202.
- Reid SM, Ferris NP, Hutchings GH, Zhang Z, Belsham GJ,
Alexandersen S. Detection of all seven serotypes of foot-
and-mouth disease virus by real-time, fluorogenic reverse
transcription polymerase chain reaction assay. J Virol Methods
2002; 105: 67-80.
- Callahan JD, Brown F, Osorio FA, Sur JH, Kramer E, Long GW,
Lubroth J, Ellis SJ, Shoulars KS, Gaffney KL.
Use of a portable
real-time reverse transcriptase polymerase chain reaction assay
for rapid detection of foot-and-mouth disease virus. J Am Vet
Med Assoc 2002; 220: 1636-1642.
- Shaw AE, Reid SM, Ebert K, Hutchings GH, Ferris NP, King
D P.
Implementation of a one-step real-time RT-PCR protocol
for diagnosis of foot-and-mouth disease. J Virol Methods 2007;
143: 81-85.
- Reid SM, Ebert K, Bachanek-Bankowska K, Batten C, Sanders
A, Wright C, Shaw AE, Ryan ED, Hutchings GH, Ferris NP.
The performance of real-time reverse transcription polymerase
chain reaction for the detection of foot-and-mouth disease
virus during field outbreaks in the United Kingdom in 2007. J
Vet Diagn Invest 2009; 21: 321-330.
- Dukes J, King D, Alexandersen S.
Novel reverse transcription
loop-mediated isothermal amplification for rapid detection
of foot-and-mouth disease virus. Arch Virol 2006; 151: 1093-
1106.
- Chen HT, Zhang J, Liu YS, Liu XT.
Detection of foot-and-
mouth disease virus RNA by reverse transcription loop-
mediated isothermal amplification. Virol J 2011; 8: 1-3.
- Ding YZ, Zhou JH, Ma LN, Qi YN, Wei G, Zhang J, Zhang YG.
A
reverse transcription loop-mediated isothermal amplification
assay to rapidly diagnose foot-and-mouth disease virus C. J Vet
Sci 2014; 15: 423-426.
- Fukuta S, Iida T, Mizukami Y, Ishida A, Ueda J, Kanbe M,
Ishimoto Y.
Detection of Japanese yam mosaic virus by RT-
LAMP. Arch Virol 2003; 148: 1713-1720.
- Nie K, Qi SX, Zhang Y, Luo L, Xie Y, Yang MJ, Zhang Y, Li
J, Shen H, Li Q. Evaluation of a direct reverse transcription
loop-mediated isothermal amplification method without
RNA extraction for the detection of human enterovirus 71
subgenotype C4 in nasopharyngeal swab specimens. PLoS one
2012; 7: 52486.