Comparison of real-time reverse transcriptase-polymerase chain reaction and nested or commercial reverse transcriptase-polymerase chain reaction for the detection of hepatitis E virus particle in human serum.
ABSTRACT Hepatitis E virus (HEV) was originally identified as the causative agent of enterically transmitted non-A, non-B hepatitis. The virus is the 7.5-kb single-stranded positive RNA virus and has been classified in the genus Herpevirus [corrected] of the [corrected] Herpeviridae [corrected] Recently, HEVs were identified from several countries worldwide from human and animals including swine. Studies on the genomic analysis of HEV isolates and seroprevalence of anti-HEV antibodies suggested that HEV has been considered as a potent zoonotic agent. The HEV infection has been diagnosed by detection of anti-HEV antibodies or virus by using reverse transcriptase-polymerase chain reaction (RT-PCR) methods in the blood or feces. However, these diagnostic methods were not quantitative and not enough to diagnose small amounts of target molecules. Moreover, these methods were not adequate during the incubation period or early acute phase. To overcome these problems, real-time RT-PCR method was developed with a cloned viral DNA and in vitro transcribed cRNA in this study. The sensitivity of the reaction was 1.68 x 10(1) copies per reaction. Correlation coefficient values of the reactions in the repeated experiments were over 0.99. Ranges of slopes and coefficient variation values were from 3.341 to 3.435 and from 1.20 to 5.98, respectively. In comparison of the real-time PCR with nested or commercial RT-PCR, HEV particles could be detected in the negative samples, which were determined by conventional nested RT-PCR.
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ABSTRACT: The aim of this study was to compare the performance of four TaqMan RT-PCR assays with a commonly used nested RT-PCR and to include the Feline calicivirus (FCV) as an internal control. RNA extracted from 87 swine faecal samples and 103 swine blood samples was subjected to different detection systems. Faecal samples naturally contaminated with Hepatitis E virus (HEV) and negative samples were artificially inoculated with 3.2 x 10(3) PFU of FCV. Detection results obtained on faecal and plasma samples were 35.6% and 4.9% with the nested RT-PCR assay, 8.0% and 0%, 0% and 0%, 13.8% and 0% and 36.8% and 3.9% with TaqMan systems A, B, C and D respectively. The Ct means obtained with the multiplex TaqMan assay were 30.11 and 30.43 for the detection of FCV with HEV contaminated samples and negative samples. The TaqMan system D was more suitable for the detection of swine HEV strains than the three others and FCV was integrated successfully as an internal control. FCV was demonstrated as an efficient control to monitor the RNA extraction process and HEV amplification procedure in a multiplex HEV/FCV TaqMan assay. This control would be helpful in limiting false negative results.Journal of Applied Microbiology 02/2009; 106(4):1360-9. DOI:10.1111/j.1365-2672.2008.04104.x · 2.39 Impact Factor
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ABSTRACT: In der vorliegenden Arbeit wurde analysiert, wie sich die Defizienz von Syndecan-4, einem Heparansulfat Proteoglykan, auf den Verlauf der renalen Pathogenese nach Anwendung von zwei unterschiedlichen Nierenerkrankungsmodellen auswirkt. Syndecan-4 wird im Vergleich zu den anderen drei Mitgliedern der Syndecan-Familie sehr stark in der Niere exprimiert. Nach einer unilateralen Nephrektomie (UNX) von 60 Tagen zeigte sich ausschließlich in den männlichen Syndecan-4-defizienten Mäusen eine glomerulosklerotische Entwicklung. Diese Glomerulosklerose war gekennzeichnet durch eine erhöhte Mesangialexpansion sowie TGF-1 Expression und die Hochregulation von Syndecan-2 und mehreren fibrotischen Markerproteinen. Weibliche Syndecan-4-defiziente Mäuse sowie Wildtypmäuse waren nicht betroffen. Nach einer unilateralen Ureterligation konnten dagegen keine sichtbaren Unterschiede in der fibrotischen Reaktion zwischen Wildtyp- und Syndecan-4-defizienten Mäusen festgestellt werden.
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ABSTRACT: To compare the specificity and sensitivity of a real-time fluorescent RT-PCR assay with conventional RT-PCR, sera from 110 healthy blood donors, 120 patients with a clinical diagnosis of chronic hepatitis B, and 416 patients with non-A-C acute hepatitis, as well as serial dilutions of HEV genotypes 1 and 4, were tested with both assays. All samples from healthy blood donors and patients with chronic hepatitis B were negative by both assays. Real-time RT-PCR could detect the same final dilution of genotype 1 as conventional RT-PCR but could detect a 10-fold lower concentration of genotype 4 than conventional RT-PCR. Of 416 samples from patients with a clinical diagnosis of non-A-C acute hepatitis, 127 (30.5%) and 83 (20.0%) were positive for HEV by real-time and conventional RT-PCR, respectively. The concordance of real-time and conventional RT-PCR was 80.8%. Furthermore, 96 and 57 of 171 samples were positive for anti-HEV IgM by real-time and conventional RT-PCR, respectively, and 31 and 26 of 245 samples negative for anti-HEV IgM, were positive by real-time and conventional RT-PCR, respectively. All amplicons positive by conventional RT-PCR were sequenced. Of 83 isolates, 7 and 76 belonged to genotypes 1 and 4, respectively. Thus, both assays have a high specificity, but the real-time RT-PCR assay is more sensitive than conventional RT-PCR. Furthermore, HEV genotype 4 is responsible for most sporadic cases of hepatitis E in the north of China. J. Med. Virol. 79:1966-1973, 2007.Journal of Medical Virology 01/2007; · 2.22 Impact Factor