A sensitive and robust method for measles RNA detection.
The aim of this study was to compare measles RNA amplification methods and to develop and select the most rapid, sensitive and robust procedure. The use of hybrid capture for measles RNA isolation was evaluated, and three RNA amplification detection techniques were compared. These were: (a) reverse transcription followed by nested polymerase chain reaction (RT-PCR) with MMLV reverse transcriptase and Taq polymerase; (b) a combined RT-PCR reaction using rTth polymerase; and (c) NASBA. An internal positive control was also developed. The sensitivities of the detection methods were quantified by using a dilution series of a known amount of total RNA from measles-infected Vero cells or by calculation of the number of transcript molecules (produced from a recombinant plasmid containing an insert measles nucleoprotein DNA) present in each amplification reaction, respectively. The results indicated that hybrid capture followed by combined RT-PCR with rTth polymerase was the most reproducibly robust and sensitive protocol and could detect as few as 10(4) synthetic measles RNA transcripts added to tissue homogenates. However, NASBA proved to be the most sensitive method for measles RNA detection in water.
Chadwick
N
N
Inflammatory Bowel Disease Study Group, Royal Free Hospital School of Medicine, Hampstead, London, UK.
Bruce
I
I
Davies
M
M
van Gemen
B
B
Schukkink
R
R
Khan
K
K
Pounder
R
R
Wakefield
A
A
D000818:Animals; D015139:Blotting, Southern; D002522:Chlorocebus aethiops; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel; D005188:False Negative Reactions; D006801:Humans; D008459:Measles virus / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D012194:RNA-Directed DNA Polymerase; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014709:Vero Cells
D017931:DNA Primers; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase