Detection of swine transmissible gastroenteritis coronavirus using loop-mediated isothermal amplification.
A conserved nucleic acid fragment of the nucleocapsid gene of Swine Transmissible Gastroenteritis Coronavirus (TGEV) was chosen as the target, six special primers were designed successfully. Loop-mediated isothermal amplification (LAMP) was developed to detect the TGEV by incubation at 60°C for 1 h and the product specificity was confirmed by HphI digestion. Standard curves with high accuracy for TGEV quantization was constructed by adding 1 × SYBR greenI in the LAMP reaction. The assay established in this study was found to detect only the TGEV and no cross-reaction with other viruses, demonstrating its high specificity. By using serial sample dilutions as templates, the detection limit of LAMP was about 10 pg RNA, 10 times more sensitive than that of PCR and could be comparable to the nest-PCR.
Chen
Qin
Q
School of Life Science, Shanghai University, Shanghai 200444, China. chenqincc@yahoo.com.cn
Li
Jian
J
Fang
Xue-En
XE
Xiong
Wei
W
D000818:Animals; D052160:Benzothiazoles; D017124:Conserved Sequence; D017931:DNA Primers / Q000235:genetics; D003959:Diamines; D005761:Gastroenteritis, Transmissible, of Swine / Q000175:diagnosis* / Q000821:virology*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D019251:Nucleocapsid / Q000235:genetics; D009930:Organic Chemicals / Q000378:metabolism; D011804:Quinolines; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D013552:Swine; D013696:Temperature; D005760:Transmissible gastroenteritis virus / Q000302:isolation & purification*; D014773:Virology / Q000379:methods*
D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I
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