Use of magnetic beads versus guanidium thiocyanate-phenol-chloroform RNA extraction followed by polymerase chain reaction for the rapid, sensitive detection of enterovirus RNA.
The current study compares the sensitivity of RNA extraction using magnetic beads versus that of a standard extraction method. Streptavadin-coated magnetic beads were labelled with a biotinylated, enterovirus-specific oligonucleotide. RNA was extracted using labelled beads or guanidium thiocyanate-phenol-chloroform from 1, 0.1 and 0.01 TCID50/100 microliters of stock coxsackievirus types A9 and B3, echovirus type 11, enterovirus type 70 and poliovirus type 1. Each strain was tested three times. RNA extraction using magnetic beads was > 50% faster than the standard method. The RNA was amplified using RT-PCR, and the products were detected using agarose gel electrophoresis; 6/15 and 7/15 samples at an initial concentration of 0.01 TCID50/100 microliters were detected using magnetic beads or standard extraction, respectively. Negative-stain electron microscopy was used to determine that 0.01 TCID50/100 microliters of coxsackievirus B3 contained approximately 3 genomes. Thus, use of magnetic beads labelled with an enterovirus-specific oligonucleotide was less toxic, more rapid and as sensitive as the current standard RNA extraction method.
Beaulieux
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F
Laboratoire de Virologie, Faculté de Médecine Lyon Grange Blanche, France.
See
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DM
Leparc-Goffart
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I
Aymard
M
M
Lina
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B
D002725:Chloroform; D004770:Enterovirus / Q000235:genetics*; D019791:Guanidine; D006146:Guanidines; D008280:Magnetics; D008863:Microspheres; D019800:Phenol; D010636:Phenols; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013861:Thiocyanates
D006146:Guanidines; D010636:Phenols; D012367:RNA, Viral; D013861:Thiocyanates; D019800:Phenol; D002725:Chloroform; D019791:Guanidine; C031760:thiocyanate