Determination of a universal nucleic acid extraction procedure for PCR detection of gastroenteritis viruses in faecal specimens.
Four nucleic acid extraction protocols were examined for their suitability for extraction of the ssRNA, dsRNA and dsDNA genomes of gastroenteritis viruses, for PCR detection. Protocol (A), employed specimen lysis with guanidinium thiocyanate, extraction with phenol-chloroform-isoamyl alcohol and nucleic acid purification by size-fractionated silica particles. Protocol (B), utilised specimen lysis with guanidinium thiocyanate and nucleic acid purification by silica, followed by phenol-chloroform-isoamyl alcohol extraction. Protocol (C), employed specimen lysis with guanidinium thiocyanate and nucleic acid purification by RNAID glass powder. Protocol (D), employed specimen lysis with sodium dodecyl sulphate, proteinase K digestion and extraction with phenol-chloroform-isoamyl alcohol. Of the four protocols, (B) appeared to be a suitable candidate 'universal' nucleic acid extraction procedure for PCR detection of different viral agents of gastroenteritis in a single nucleic acid extract of a faecal specimen, irrespective of genome composition. Omission of the phenol-chloroform extraction step did not affect negatively the ability of protocol (B) to allow PCR detection of gastroenteritis viruses in faecal specimens. PCR detection of NLVs, astroviruses, rotaviruses and adenoviruses, in single nucleic acid extracts of faecal specimens obtained from the field, confirmed the universality of the modified protocol (B). We propose the modified protocol (B) as a 'universal' nucleic acid extraction procedure, for monoplex PCR detection of gastroenteritis viruses in single nucleic acid extracts of faecal specimens and for development of multiplex PCR for their simultaneous detection.
Rasool
Nassar B G
NB
Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia. nassar@gene.um.edu.my
Monroe
Stephan S
SS
Glass
Roger I
RI
D000258:Adenovirus Infections, Human / Q000821:virology*; D000260:Adenoviruses, Human / Q000235:genetics / Q000302:isolation & purification; D000818:Animals; D019350:Astroviridae Infections / Q000821:virology*; D002460:Cell Line; D002725:Chloroform; D004279:DNA, Viral / Q000302:isolation & purification*; D003967:Diarrhea / Q000821:virology; D005243:Feces / Q000821:virology*; D005759:Gastroenteritis / Q000821:virology*; D016679:Genome, Viral; D006801:Humans; D008253:Macaca mulatta; D001257:Mamastrovirus / Q000235:genetics / Q000302:isolation & purification; D019800:Phenol; D016133:Polymerase Chain Reaction / Q000379:methods; D012330:RNA, Double-Stranded / Q000302:isolation & purification; D012367:RNA, Viral / Q000302:isolation & purification*; D012401:Rotavirus / Q000235:genetics / Q000302:isolation & purification; D012400:Rotavirus Infections / Q000821:virology*; D014407:Tumor Cells, Cultured
D004279:DNA, Viral; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D019800:Phenol; D002725:Chloroform