Direct extraction of Sabin poliovirus genomes from human fecal samples using a guanidine thiocyanate extraction method.
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 10(7) TCID(50) per 0.1 ml, genome was detected to a dilution of 10(3) TCID(50) 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.
Old
Matthew O
MO
Department of Pediatrics, School of Medicine, Stanford University, MC 5208, 300 Pasteur Drive, Stanford, CA 94305, USA.
Martinez
Claudia V
CV
Kwock
Douglas
D
Garcia
Joaquin
J
Martin
Gladys
G
Chan
Christina
C
Maldonado
Yvonne A
YA
D000818:Animals; D002460:Cell Line; D017931:DNA Primers; D005243:Feces / Q000821:virology*; D016679:Genome, Viral; D006146:Guanidines; D006801:Humans; D011051:Poliomyelitis / Q000821:virology; D017955:Poliovirus / Q000235:genetics / Q000302:isolation & purification*; D011055:Poliovirus Vaccine, Oral; D012367:RNA, Viral / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013861:Thiocyanates; D013997:Time Factors; D014776:Virus Cultivation
D017931:DNA Primers; D006146:Guanidines; D011055:Poliovirus Vaccine, Oral; D012367:RNA, Viral; D013861:Thiocyanates; C054436:guanidine thiocyanate