TY - JOUR
T1 - Development of a loop-mediated isothermal amplification method for rapid detection of caprine arthritis-encephalitis virus proviral DNA
AU - Huang, Jinhai
AU - Sun, Yuehui
AU - Liu, Yebing
AU - Xiao, Huazhi
AU - Zhuang, Shiwen
N1 - Funding Information:
This work was supported by the National Natural Science Foundation of China (No. 30771613) and the Application Basement & Advanced Technology Project of Tianjin Science and Technology Committee (08JCZDJC22600) in Tianjin, China.
PY - 2012/8
Y1 - 2012/8
N2 - A rapid detection assay based on loop-mediated isothermal amplification (LAMP) has been developed for detecting caprine arthritis-encephalitis (CAEV) proviral DNA. The LAMP assay utilized a set of five primers designed against highly conserved sequences located within the p25 gene region. The assay successfully detected CAEV proviral DNA in total DNA extracts originating from cell culture, whole blood samples and separated PBMCs. There was no cross-reaction with the negative control. Amplification was monitored using a Loopamp real-time turbidimeter; turbidity and the corresponding time were recorded. Amplification from CAEV-Shanxi DNA was detected as early as 17 min, with a maximum sensitivity of 0. 0001 TCID 50, reached at 32 min. Sixty-eight animal blood samples were tested using AGID, PCR and LAMP assay, and the positive rates were 30.9 %, 33.8 % and 47.1 %, respectively. Whole blood can be used directly, eliminating the need for separation of PBMCs and nucleic acid extraction, reducing the overall procedure time to approximately 80 min. Therefore, the LAMP assay provides a specific and sensitive means for detecting CAEV proviral DNA in a simple, fast, and cost-effective manner and should be useful in eradication programs and epidemiological studies. Furthermore, the LAMP assay can be performed in less-well-equipped laboratories as well as in the field.
AB - A rapid detection assay based on loop-mediated isothermal amplification (LAMP) has been developed for detecting caprine arthritis-encephalitis (CAEV) proviral DNA. The LAMP assay utilized a set of five primers designed against highly conserved sequences located within the p25 gene region. The assay successfully detected CAEV proviral DNA in total DNA extracts originating from cell culture, whole blood samples and separated PBMCs. There was no cross-reaction with the negative control. Amplification was monitored using a Loopamp real-time turbidimeter; turbidity and the corresponding time were recorded. Amplification from CAEV-Shanxi DNA was detected as early as 17 min, with a maximum sensitivity of 0. 0001 TCID 50, reached at 32 min. Sixty-eight animal blood samples were tested using AGID, PCR and LAMP assay, and the positive rates were 30.9 %, 33.8 % and 47.1 %, respectively. Whole blood can be used directly, eliminating the need for separation of PBMCs and nucleic acid extraction, reducing the overall procedure time to approximately 80 min. Therefore, the LAMP assay provides a specific and sensitive means for detecting CAEV proviral DNA in a simple, fast, and cost-effective manner and should be useful in eradication programs and epidemiological studies. Furthermore, the LAMP assay can be performed in less-well-equipped laboratories as well as in the field.
UR - http://www.scopus.com/inward/record.url?scp=84864476655&partnerID=8YFLogxK
U2 - 10.1007/s00705-012-1322-y
DO - 10.1007/s00705-012-1322-y
M3 - Article
C2 - 22566005
AN - SCOPUS:84864476655
SN - 0304-8608
VL - 157
SP - 1463
EP - 1469
JO - Archives of Virology
JF - Archives of Virology
IS - 8
ER -