Development of RT-semi-nested PCR for detection of hepatitis A virus in stool in epidemic conditions.
The purpose of this study was to determine the efficiency of semi-nested PCR in detecting hepatitis A virus (HAV) RNA. During a 2-year period (1990-1991), HAV RNA was searched for in shellfish from the French Brittany coasts using cRNA and vRNA probes. In January 1992, at the time of a hepatitis A outbreak, 28 stool samples were collected from infected patients (18 adults, 10 children) with anti-HAV IgM. Four samples from subjects with negative HAV serology were used as negative controls. Nucleic acid amplification (reverse-transcription-semi-nested PCR) was performed to detect HAV in stool. HAV RNA was purified by phenol-chloroform extraction and converted to cDNA using reverse transcriptase (Mu-MLV). After amplification, PCR products were visualized on an ethidium-bromide-stained gel and confirmed by hybridization with a specific digoxigenin-labelled oligoprobe. Samples were also studied by molecular hybridization with cRNA and vRNA probes. After onset of the illness, HAV RNA was detected over a longer time period by semi-nested PCR (16/28) than by hybridization (0/28). Even though biological diagnosis of hepatitis A will continue to rely on the detection of anti-HAV IgM, PCR should be useful in certain clinical cases (diagnosis of relapse) and for epidemiological and environmental monitoring of viruses.
Apaire-Marchais
V
V
Laboratoire de Virologie, Institut de Biologie, Nantes, France.
Ferre-Aubineau
V
V
Colonna
F
F
Dubois
F
F
Ponge
A
A
Billaudel
S
S
D000293:Adolescent; D000328:Adult; D001483:Base Sequence; D002648:Child; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D005243:Feces / Q000821:virology*; D005260:Female; D005602:France / Q000453:epidemiology; D005784:Gene Amplification; D006506:Hepatitis A / Q000175:diagnosis / Q000453:epidemiology* / Q000235:genetics; D006507:Hepatovirus / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D012680:Sensitivity and Specificity
D004279:DNA, Viral; D012367:RNA, Viral