TY - JOUR
T1 - Direct extraction of Sabin poliovirus genomes from human fecal samples using a guanidine thiocyanate extraction method
AU - Old, Matthew O.
AU - Martinez, Claudia V.
AU - Kwock, Douglas
AU - Garcia, Joaquin
AU - Martin, Gladys
AU - Chan, Christina
AU - Maldonado, Yvonne A.
N1 - Funding Information:
Supported by a grant from the Child Health Research Fund, Lucile Salter Packard Children's Hospital at Stanford. We would like to thank Terry S. Satterfield and Oscar Otanez for their work in developing and testing the guanidine thiocyanate assay and to Dr Meira S. Falkovitz-Halpern for editorial assistance.
PY - 2003/6/30
Y1 - 2003/6/30
N2 - To permit rapid and efficient detection of Sabin poliovirus type 3 from human fecal samples, we developed a guanidine thiocyanate (GuSCN) extraction and reverse transcriptase polymerase chain reaction (RT-PCR) method. Using 10-fold serial dilutions from stock Sabin-Leon 12 a1b poliovirus type 3 at 107 TCID50 per 0.1 ml, genome was detected to a dilution of 103 TCID50 per 0.1 ml. A total of 40 archived fecal samples were examined using this GuSCN extraction method followed by RT-PCR. Fourteen of 20 poliovirus type 3 tissue culture-positive specimens (70%) and two of 20 tissue culture-negative specimens (10%) were detected by GuSCN extraction and RT-PCR. All positive and negative extraction and RT-PCR controls were identified accurately. This GuSCN extraction and RT-PCR technique is rapid, inexpensive, and can be readily adapted to identify genome sequences of other enterovirus types in large numbers of fecal samples. Moreover, the GuSCN technique extracts viral RNA directly from fecal samples, allowing observation of in vivo alterations of genome sequences. Further studies are underway to examine the development of revertant point mutations in the Sabin poliovirus type 3 genome following oral administration of trivalent Sabin Oral Poliovirus Vaccine to humans.
AB - To permit rapid and efficient detection of Sabin poliovirus type 3 from human fecal samples, we developed a guanidine thiocyanate (GuSCN) extraction and reverse transcriptase polymerase chain reaction (RT-PCR) method. Using 10-fold serial dilutions from stock Sabin-Leon 12 a1b poliovirus type 3 at 107 TCID50 per 0.1 ml, genome was detected to a dilution of 103 TCID50 per 0.1 ml. A total of 40 archived fecal samples were examined using this GuSCN extraction method followed by RT-PCR. Fourteen of 20 poliovirus type 3 tissue culture-positive specimens (70%) and two of 20 tissue culture-negative specimens (10%) were detected by GuSCN extraction and RT-PCR. All positive and negative extraction and RT-PCR controls were identified accurately. This GuSCN extraction and RT-PCR technique is rapid, inexpensive, and can be readily adapted to identify genome sequences of other enterovirus types in large numbers of fecal samples. Moreover, the GuSCN technique extracts viral RNA directly from fecal samples, allowing observation of in vivo alterations of genome sequences. Further studies are underway to examine the development of revertant point mutations in the Sabin poliovirus type 3 genome following oral administration of trivalent Sabin Oral Poliovirus Vaccine to humans.
KW - Enteroviruses
KW - Fecal specimens
KW - Guanidine thiocyanate extraction
KW - Rapid diagnosis
KW - Sabin oral poliovirus vaccine type 3
KW - Tissue cultivation
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U2 - 10.1016/S0166-0934(03)00133-2
DO - 10.1016/S0166-0934(03)00133-2
M3 - Article
C2 - 12798248
AN - SCOPUS:0038730775
SN - 0166-0934
VL - 110
SP - 193
EP - 200
JO - Journal of Virological Methods
JF - Journal of Virological Methods
IS - 2
ER -