Use of PCR-enzyme immunoassay for identification of influenza A virus matrix RNA in clinical samples negative for cultivable virus.
Influenza A virus infections are a major cause of morbidity and mortality worldwide. Standard diagnostic methods either are not efficient in identifying infected individuals in a timely manner or lack sensitivity. We developed a PCR-enzyme immunoassay (PCR-EIA) for the detection of influenza A virus RNA in respiratory secretions. A reverse transcription PCR was performed with oligonucleotide primers directed at a highly conserved area of the influenza A matrix gene. Amplified DNA was identified by hybridization in solution to a nested biotinylated RNA probe and quantitated in an EIA. PCR-EIA detected small quantities of RNA from the three prevalent subtypes of human influenza A virus. Influenza B and C, parainfluenza, measles, mumps, and respiratory syncytial viruses tested negative. The potential efficiency of PCR-EIA for use in clinical diagnosis was determined by testing 90 nasal wash specimens obtained daily over a 10-day period from nine human volunteers infected with influenza A virus. Thirty-seven of the postinfection samples had detectable influenza A virus RNA by PCR-EIA, whereas only 26 postinfection samples were positive by culture. PCR-EIA was particularly efficient for the identification of influenza A virus in samples obtained more than 4 days after infection. Seventeen of 45 such samples were positive, whereas virus was cultivated from 4 samples (P < 0.00005). All preinfection samples from volunteers subsequently infected with influenza A virus were negative by PCR-EIA, as were samples from a volunteer infected with parainfluenza virus type 3. Nucleic acid amplification techniques represent important tools for the timely and sensitive diagnosis of influenza A virus infections and, therefore, their management and control.
Cherian
T
T
Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland.
Bobo
L
L
Steinhoff
M C
MC
Karron
R A
RA
Yolken
R H
RH
D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D005069:Evaluation Studies as Topic; D006801:Humans; D007124:Immunoenzyme Techniques; D009980:Influenza A virus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D007251:Influenza, Human / Q000175:diagnosis* / Q000382:microbiology; D008969:Molecular Sequence Data; D009297:Nasal Mucosa / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012367:RNA, Viral / Q000235:genetics* / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
D017931:DNA Primers; D012367:RNA, Viral