2012 Rapid detection of Betacoronavirus 1 from clinical fecal specimens by a novel reverse transcription loop-mediated i

4 Pages·2012·0.81 MB·English

Journal of Veterinary Diagnostic Investigation
24(1) 174–177
© 2012 The Author(s)
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DOI: 10.1177/1040638711425937
http://jvdi.sagepub.com

Betacoronavirus 1 (BCoV-1; order Nidovirales, family Coro-
naviridae, subfamily Coronavirinae, genus Betacoronavirus)
is one of the important agents of neonatal calf diseases1 and
has become a worldwide infectious agent in the cattle indus-
try.1,3 First identified as a diarrheic agent in calves in 1973 in
the United States,10 BCoV-1 can cause diarrhea in calves,2
winter dysentry6 in adult cattle, and respiratory system infec-
tions in various aged cattle,8,14 resulting in serious economic
losses.
Various molecular detection methods, such as semi-nested
reverse transcription polymerase chain reaction (RT-PCR),15
real-time RT-PCR,5 and multiplex RT-PCR,4 have been
developed to detect the BCoV-1 infection. However, most of
these assays require expensive and special instruments, and
therefore may not be readily applicable, particularly in diag-
nostic laboratories in underdeveloped or developing coun-
tries or regions. Thus, it is necessary to develop new assays
for the detection of BCoV-1 in clinical practice. In 2000,
details of a novel nucleic acid amplification method, namely,
the loop-mediated isothermal amplification (LAMP) tech-
nique, were published.11 Compared to other molecular detec-
tion assays (single RT-PCR or nucleic acid hybridization),
the LAMP technique involves reagents that react under iso-
thermal conditions with high specificity, sensitivity, and
rapidity11 and has been used for the detection of different
pathogens.7,13 In the current study, a novel and rapid reverse
transcription LAMP (RT-LAMP) assay was developed to
detect BCoV-1 from clinical samples.
A set of RT-LAMP primers was designed targeting a highly
conserved region located in the nucleocapsid (N) protein gene,

with the region being selected based on alignment analysis.
The analysis involved 24 genomic sequences retrieved from
the GenBank database and aligned using the ClustalV
method.a Two inner primers, forward inner primer (FIP) and
backward inner primer (BIP), and 2 outer primers (F3 and B3)
were designed by using the Primer Explorer version 4 software.b
All primer sequences were analyzed with BLASTnc for speci-
ficity. The primers are shown in Table 1.
Total RNA was extracted from BCoV-1–infected human
rectal tumor (HRT-18G) cells with a commercial RNA kit,d
according to the manufacturer’s instructions. The RNA was
eluted in 20 μl of RNase-free water containing 0.04% sodium
azide and was stored at –80°C until use. The RNA concentra-
tion (ng/μl) was measured with a spectrophotometer.e

The RT-LAMP reaction was carried out in a 25-μl volume
containing 12.5 μl of LAMP buffer (20 mmol Tris–HCl
[pH 8.8], 10 mmol KCl, 8 mmol MgSO4, 10 mmol (NH4)2SO4,
0.1% Triton X-100, 0.8 M betaine, and 1.4 mmol each of
deoxynucleoside triphosphates), 2 μl of primer mixture
(40 pmol each of FIP and BIP, and 5 pmol each of F3 and
B3), 1 μl of Bst DNA polymerasef (8 U), 1 μl of Avian

425937 XXXXXX10.1177/1040638711425937Q
iao et al.Rapid detection of Betacoronavirus 1

From the College of Animal Science and Technology, Shihezi
University, Shihezi City, Xinjiang, China (Qiao, Meng, Chen, Zhang,
Tian), and the State Key Laboratory of Veterinary Etiological Biology,
Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural
Sciences, Lanzhou, Gansu, China (Cai).

1Corresponding Author: Qingling Meng, College of Animal Science
and Technology, Shihezi University, North Street No.4, Shihezi City,
Xinjiang 832003, China.