Loop-mediated isothermal amplification establishment for detection of pseudorabies virus.
A rapid, convenient and reliable pseudorabies virus (PRV) detection system was developed by using the loop-mediated isothermal amplification (LAMP) method. Six special primers were designed successfully based on the PRV DNA-binding protein (DBP) gene. The assay was optimized to amplify PRV DNA by incubation at 63 degrees C for 1h. The LAMP products had a ladder-like pattern of bands from 188 bp when electrophoresed on an agarose gel and its specificity was confirmed by digestion with Hinc II enzyme. Two naked-eye detection methods were developed for use in the field. The detection limit of the LAMP assay was found to be 10 fg DNA sample which was 100-1,000-fold higher than that of PCR. By using DNA (or cDNA) samples extracted from three different PRV strains and six other viruses known to be related genetically to PRV or to cause similar clinical signals in pig, the system was identified to amplify only the PRV DNA. A comparison between the LAMP and PCR assay using five clinical samples showed good correlation.
En
Fang-Xue
FX
School of Life Science, Shanghai University, Shanghai 200444, PR China.
Wei
Xiong
X
Jian
Li
L
Qin
Chen
C
D000818:Animals; D001921:Brain / Q000821:virology; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis / Q000302:isolation & purification; D011558:Herpesvirus 1, Suid / Q000235:genetics / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D011557:Pseudorabies / Q000175:diagnosis / Q000821:virology; D012680:Sensitivity and Specificity; D013552:Swine / Q000821:virology; D013553:Swine Diseases / Q000175:diagnosis / Q000821:virology
D017931:DNA Primers; D004279:DNA, Viral