Detection and quantitation of hepatitis E virus in human faeces by real-time quantitative PCR.
Hepatitis E Virus (HEV) is the causative agent of an acute and self-limited form of hepatitis. The virus is transmitted by the faecal-oral route and is a major cause of viral hepatitis in much of the developing world where it causes sporadic infections and large-scale epidemics. A simple and rapid protocol for the measurement of HEV faecal shedding by a real-time polymerase chain reaction (PCR) with the SYBR Green method on a LightCycler instrument, is described. After only 3h the real-time quantitative PCR method detected 10 molecules of HEV cDNA fragment per reaction tube and showed a high linear dynamic range of quantitation (10-10(6) molecules of cDNA/reaction) with a good correlation (r = -1.00). Its specificity was confirmed by assay in human faecal samples.
Orrù
Germano
G
Dipartimento di Scienze Odontostomatologiche, Universita' degli Studi di Cagliari, Via Binaghi No. 4, 09121 Cagliari, Italy. orru@unica.it
Masia
Giuseppina
G
Orrù
Ginevra
G
Romanò
Luisa
L
Piras
Vincenzo
V
Coppola
Rosa Cristina
RC
D052160:Benzothiazoles; D018076:DNA, Complementary / Q000235:genetics; D003959:Diamines; D005243:Feces / Q000821:virology*; D016751:Hepatitis E / Q000821:virology*; D016752:Hepatitis E virus / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D009930:Organic Chemicals / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D011804:Quinolines; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D052160:Benzothiazoles; D018076:DNA, Complementary; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I